Streptozotocin-induced Diabetes and the Neurochemistry of Vagal Afferent Neurons

Brain Research. May, 2002  |  Pubmed ID: 12031529

To assess whether diabetes alters the content and/or expression of neuroactive agents and protooncogenes in afferent neurons of the vagus nerve, the nodose ganglia of streptozotocin (STZ)-induced diabetic rats were studied at 8, 16, and 24 weeks after induction of diabetes. Neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), the immediate early gene c-Jun, vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP) content and expression were measured in nodose ganglia of control, diabetic, and diabetic+insulin-treated rats using immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The numbers of nNOS-immunoreactive (ir) neurons were increased in the nodose ganglion of diabetic compared to control rats at the 8- and 16-week time points. However, no change was noted in the nNOS mRNA content of the diabetic nodose ganglion at either time point. Moreover, no alterations in the numbers of vagal efferent NOS-containing neurons (labeled with NADPH-diaphorase histochemistry) were noted in the dorsal motor nucleus of the vagus (DMV) or the nucleus ambiguous (NA) of control, diabetic, and diabetic+insulin-treated rats at any time point. Neither the numbers of TH-ir neurons nor the content of TH mRNA was altered in the diabetic rats at the 8- and 16-week time points. However, 24 weeks of diabetes resulted in a reduction in the numbers of TH-ir neurons in the diabetic nodose ganglia when compared to control, an effect not seen in diabetic rats receiving insulin. The number of nodose ganglion neurons labeled for the protooncogene, c-Jun, was small yet slightly increased in the diabetic nodose ganglia at the 8-week time point and was reversed with insulin treatment. The increase in c-Jun-ir neurons was not found at 16 or 24 weeks of diabetes. VIP-ir and CGRP-ir were unchanged at any of the time points. These data show that diabetes affects the content of some, but not all, neuroactive agents in the nodose ganglion and may reflect a modest level of diabetes-induced damage and/or alterations in axonal transport in the vagus nerve.

Streptozotocin-induced Diabetes Reduces Retrograde Axonal Transport in the Afferent and Efferent Vagus Nerve

Brain Research. Jun, 2002  |  Pubmed ID: 12031555

Diabetes-induced alterations in nerve function include reductions in the retrograde axonal transport of neurotrophins. A decreased axonal accumulation of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of streptozotocin (STZ)-induced diabetic rats was previously shown. In the current study, no changes in the NGF and NT-3 protein or mRNA levels in the stomach or atrium, two vagally innervated organs, were noted after 16 or 24 weeks of diabetes. Moreover, the amounts of neurotrophin receptor (p75, TrkA, TrkC) mRNAs in the vagus nerve and vagal afferent nodose ganglion were not reduced in diabetic rats. These data suggest that neither diminished access to target-derived neurotrophins nor the loss of relevant neurotrophin receptors accounts for the diabetes-induced alteration in the retrograde axonal transport of neurotrophins. To assess whether diabetes causes a defect in axonal transport that may not be specific to neurotrophin transport, we studied the ability of a neuronal tracer (FluoroGold, FG) to be retrogradely transported by vagal neurons of control and diabetic rats. After vagal target tissue (stomach) injections of FG, the numbers of FG-labeled afferent and efferent vagal neurons were counted in the nodose ganglion and in the dorsal motor nucleus of the vagus, respectively. After 24 weeks of diabetes, FG was retrogradely transported to more than 50% fewer afferent and efferent vagal neurons in the STZ-diabetic compared to control rats. The diabetes-induced deficit in retrograde axonal transport of FG is likely to reflect alterations in basic axonal transport mechanisms in both the afferent and efferent vagus nerve that contribute to the previously observed reductions in neurotrophin transport.

Abnormal PI3 Kinase/Akt Signal Pathway in Vagal Afferent Neurons and Vagus Nerve of Streptozotocin-diabetic Rats

Brain Research. Molecular Brain Research. Feb, 2003  |  Pubmed ID: 12591159

The PI3 (phosphatidylinositol-3) kinase/Akt (protein kinase B) signal pathway is involved in the molecular signaling that regulates retrograde axonal transport of neurotrophins in the nervous system. Previous work showed that a reduced retrograde axonal transport of endogenous nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the vagus nerve of diabetic rats occurred in the presence of normal production of neurotrophins and neurotrophin receptors. To assess the potential involvement of an impaired PI3 kinase/Akt signal pathway in the diabetes-induced reduction in retrograde axonal transport of neurotrophins in the vagus nerve, we characterized diabetes-induced changes in the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Control and streptozotocin (STZ)-induced diabetic rats with a duration of 16 weeks, kinase assays, Western blotting, and immunocytochemistry were used to show that diabetes resulted in alterations in activity and protein expression of the PI3 kinase/Akt signal pathway in the vagus nerve and vagal afferent neurons. Diabetes caused a significant decrease in enzymatic activity of PI3 kinase and Akt (52 and 36% of control, respectively) in the vagus nerve. The reduced enzymatic activity was not associated with decreased protein expression of the p85 subunit of PI3 kinase, Akt and phosphorylation of Akt (ser473). In contrast, there was a significant increase in the phosphorylation of p70s6 kinase (thr421/ser424) along with a normal protein expression of p70s6 kinase in the vagus nerve of diabetic rats. However, diabetes induced an overall decrease in immunoreactivity of the p85 subunit of PI3 kinase, phospho-Akt (ser473) and phospho-p70s6/p85s6 kinase (thr421/ser424) in vagal afferent neurons. Thus, impaired PI3 kinase/Akt signal pathway may partly account for the reduced retrograde axonal transport of neurotrophins in the vagus nerve of STZ-induced diabetic rats.

Analysis of a Repressor Region in the Human Neuropeptide Y Gene That Binds Oct-1 and Pbx-1 in GT1-7 Neurons

Biochemical and Biophysical Research Communications. Aug, 2003  |  Pubmed ID: 12878188

The mechanisms dictating the developmental expression of individual neuropeptides within the hypothalamus have not yet been elucidated. In this paper we have studied the cis-acting elements involved in the repression of neuropeptide Y (NPY) gene expression in a gonadotropin-releasing hormone (GnRH) neuronal cell model, GT1-7 cells. Using transient transfection of the human NPY 5(') regulatory region into the GT1-7 neurons, we have found a repressor region located between -867 and -1078. DNase I footprint analysis of this region revealed three specific protein binding elements. Further analysis of the region between -942 and -922bp using electrophoretic mobility shift assays revealed that four different transcription factor-DNA complexes form with GT1-7 nuclear proteins, whereas only three complexes are detected using baby hamster kidney (BHK) cell nuclear extract. Mutation of the consensus binding sequence abolishes all complex formation on the -924/-922 oligonucleotide. Antibody supershift assays revealed that Oct-1 and Pbx-1 antibodies were able to eliminate the appearance of two specific complexes. Therefore we suggest that this region may be important for transcriptional repression of the NPY gene in a heterologous cell model, through complex, coordinate protein-protein interactions.

Expression of Circadian Rhythm Genes in Gonadotropin-releasing Hormone-secreting GT1-7 Neurons

Endocrinology. Dec, 2003  |  Pubmed ID: 12960008

The center for circadian rhythms in mammals is the suprachiasmatic nucleus (SCN) of the hypothalamus, composed of single cell circadian oscillators driven by a transcriptional/translational feedback loop where clock proteins drive clock gene expression. These genes are expressed in peripheral tissues and several brain areas outside the SCN. It is likely that some peripheral oscillators are synchronized by the SCN. The pineal hormone melatonin plays an important role in the entrainment of circadian rhythms through feedback to the SCN. Melatonin also plays a role in reproduction, including direct effects on GnRH-secreting GT1-7 neurons. The intrinsic rhythmicity of GnRH neurons suggests that these neurons may express the components of the circadian oscillator. Using the GT1-7 cell line, we demonstrate expression of the circadian rhythm genes, clock, BMAL1,timeless (tim), period1,period2, cryptochrome1, andcryptochrome2. Furthermore, semiquantitative RT-PCR demonstrates that BMAL1, period1, andperiod2 as well as GnRH mRNAs are expressed with a circadian-like rhythm after synchronization over 54 h. With available antibodies, we demonstrated CLOCK, BMAL1, and PERIOD1 protein expression in these cells, with BMAL1 protein levels showing a rhythmic expression pattern. In addition, receptors for melatonin, mt1 and MT2, also show a circadian expression pattern in the GT1-7 cells, and their expression is down-regulated by melatonin treatment. These findings suggest that the components of the clock machinery in mammals may play a role in GnRH neuronal function.

Alpha-lipoic Acid Treatment Prevents the Diabetes-induced Attenuation of the Afferent Limb of the Baroreceptor Reflex in Rats

Autonomic Neuroscience : Basic & Clinical. Oct, 2003  |  Pubmed ID: 14614962

Autonomic neuropathies, common complications of prolonged diabetes, may result from diabetes-induced increased oxidative stress. Recently, we found that the afferent component of the baroreceptor reflex is attenuated in streptozotocin-induced diabetic rats. This study sought to determine the influence of the anti-oxidant, alpha-lipoic acid on the diabetes-induced deficits of the afferent limb of the baroreceptor reflex and on plasma malondialdehyde (a measure of lipid peroxidation). The number of c-Fos-ir neurons in the nucleus tractus solitarius in response to phenylephrine-induced baroreceptor activation was used as an index of the integrity of the afferent limb of the baroreceptor reflex. Groups of streptozotocin-induced diabetic and non-diabetic control rats, maintained from 8 to 16 weeks, were treated with alpha-lipoic acid (100 mg kg(-1) IP, 5x/week), or vehicle for the last 4 weeks prior to the experimental procedure. Vehicle-treated diabetic rats had elevated plasma malondialdehyde levels when compared to non-diabetic rats. alpha-Lipoic acid-treated diabetic rats had plasma malondialdehyde levels similar to those seen in non-diabetic rats and less than those of vehicle-treated diabetic rats at both the 8- and 16-week time points.alpha-Lipoic acid treatment did not affect the baseline (absence of baroreceptor activation) presence of c-Fos-ir in the nucleus tractus solitarius. In response to phenylephrine and regardless of treatment, the diabetic and control rats displayed increases in blood pressure and reflex bradycardia. As previously reported, phenylephrine-induced baroreceptor activation resulted in significantly fewer c-Fos-ir neurons in the nucleus tractus solitarius (commissural and caudal subpostremal regions) of diabetic rats when compared to non-diabetic rats at both 8- and 16-week time points. Four weeks of alpha-lipoic acid treatment reversed the diabetes-induced decrement in the numbers of c-Fos-ir neurons in the nucleus tractus solitarius in response to baroreceptor activation. alpha-Lipoic acid-treated diabetic rats showed the same phenylephrine-induced c-Fos response in the nucleus tractus solitarius as those of alpha-lipoic-acid- and vehicle-treated control rats at both 8- and 16-week time points. These data suggest that diabetes-induced oxidative stress plays a role in diabetes-induced baroreceptor dysfunction and that the alpha-lipoic acid may have a beneficial effect in treatment of diabetic autonomic neuropathy.

Uptake of Botulinum Neurotoxin into Cultured Neurons

Biochemistry. Jan, 2004  |  Pubmed ID: 14717608

Botulinum neurotoxins (BoNTs) act within the synaptic terminal to block neurotransmitter release. The toxin enters the neuron by binding to neuronal membrane receptor(s), being taken up into an endosome-like compartment, and penetrating the endosome membrane via a pH-dependent translocation process. Once within the synaptic cytoplasm, BoNT serotypes A and E cleave separate sites on the C-terminus of the neuronal protein SNAP-25, one of the SNARE proteins required for synaptic vesicle fusion. In this study, we measured the effect of brief toxin exposure on SNAP-25 proteolysis in neuronal cell cultures as an indicator of toxin translocation. The results indicate that (1) uptake of both BoNT-A and -E is enhanced with synaptic activity induced by K+ depolarization in the presence of Ca2+ and (2) translocation of BoNT-A from the acidic endosomal compartment is slow relative to that of BoNT-E. Polyclonal antisera against each toxin protect cells when applied with the toxin during stimulation but has no effect when added immediately after toxin exposure, indicating that toxin endocytosis occurs with synaptic activity. Both serotypes cleave SNAP-25 at concentrations between 50 pM and 4 nM. IC50 values for SNAP-25 cleavage are approximately 0.5 nM for both serotypes. Inhibition of the pH-dependent translocation process by pretreating cultures with concanamycin A (Con A) prevents cleavage of SNAP-25 with IC50 values of approximately 25 nM. Addition of Con A at times up to 15 min after toxin exposure abrogated BoNT-A action; however, addition of Con A after 40 min was no longer protective. In contrast, Con A inhibited, but did not prevent, translocation of BoNT-E even when added immediately after toxin exposure, indicating that pH-dependent translocation of BoNT-E is rapid relative to that of BoNT-A. This study demonstrates that uptake of both BoNT-A and -E is enhanced with synaptic activity and that translocation of the toxin catalytic moiety into the cytosol occurs at different rates for these two serotypes.

Day/night Rhythms in Gene Expression of the Normal Murine Heart

Journal of Molecular Medicine (Berlin, Germany). Apr, 2004  |  Pubmed ID: 14985853

Molecular circadian oscillators have recently been identified in heart and many other peripheral organs; however, little is known about the physiologic significance of circadian gene cycling in the periphery. While general temporal profiles of gene expression in the heart have been described under constant lighting conditions, patterns under normal day/night conditions may be distinctly different. To understand how gene expression contributes to cardiac function, especially in human beings, it is crucial to examine these patterns in 24-h light and dark environments. High-density oligonucleotide microarrays were used to assess myocardial expression of 12,488 murine genes at 3-h intervals under the normal conditions of light and dark cycling. Variation in genetic activity was considerable, as 1,634 genes (approximately 13% of genes analyzed) exhibited statistically significant changes across the 24-h cycle. Some genes exhibited rhythmic expression, others showed abrupt change at light-to-dark and dark-to-light transitions. Importantly, genes that exhibited significant cycling rhythms mapped to key biological pathways, including for example cardiac cellular growth and remodeling, as well as transcription, translation, mitochondrial respiration, and signaling pathways. Gene expression in the heart is remarkably different in the day versus the night. Some gene cycling may be driven by the central circadian pacemaker, while other changes appear to be responses to light and dark. This has important implications regarding our understanding of how the molecular physiology of the heart is controlled, including temporal patterns of organ growth, renewal, and disease, comparative gene expression, and the most appropriate times for administration of therapy.

A New Locus for Autosomal Dominant Charcot-Marie-Tooth Disease Type 2 (CMT2L) Maps to Chromosome 12q24

Human Genetics. May, 2004  |  Pubmed ID: 15021985

Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neurological disorders with a prevalence estimated at 1/2500. The axonal form of this disorder is referred to as Charcot-Marie-Tooth type 2 disease (CMT2). Recently, a large Chinese family with CMT2 was found in the Hunan and Hubei provinces of China. The known loci for CMT1A, CMT2D, CMT1B (the same locus is also responsible for CMT2I and CMT2J), CMT2A, CMT2E, and CMT2F were excluded in this family by linkage analysis. A genome-wide screening was then carried out, and the results revealed linkage of CMT2 to a locus at chromosome 12q24. Haplotype construction and analyses localized this novel locus to a 6.8-cM interval between microsatellite markers D12S366 and D12S1611. The maximal two-point LOD score of 6.35 and multipoint LOD score of 8.08 for marker D12S76 at a recombination fraction (theta) of 0 strongly supported linkage to this locus. Thus, CMT2 neuropathy in this family represents a novel genetic entity that we have designated as CMT2L.

A Novel Mutation in KCNQ2 Gene Causes Benign Familial Neonatal Convulsions in a Chinese Family

Journal of the Neurological Sciences. Jun, 2004  |  Pubmed ID: 15178210

Benign familial neonatal convulsions (BFNC) are a rare autosomal dominant inherited epilepsy syndrome. Two voltage-gated potassium channel genes, KCNQ2 on chromosome 20q13.3 and KCNQ3 on chromosome 8q24, have been identified as the genes responsible for benign familial neonatal convulsions. By linkage analysis and mutation analysis of KCNQ2 gene, we found a novel frameshift mutation of KCNQ2 gene, 1931delG, in a large Chinese family with benign familial neonatal convulsions. This mutation is located in the C-terminus of KCNQ2, in codon 644 predicting the replacement of the last 201 amino acids with a stretch of 257 amino acids showing a completely different sequence. An unusual clinical feature of this family is that the seizures of every patient did not remit until 12 to 18 months. This is the first report of KCNQ2 gene mutation in China.

The Combination of Suicide Gene Therapy and Radiation Enhances the Killing of Nasopharyngeal Carcinoma Xenographs

Journal of Radiation Research. Jun, 2004  |  Pubmed ID: 15304972

Nasopharyngeal carcinoma (NPC) is very common in Southern China and Southeast Asian countries. To explore a novel and more effective approach to NPC therapy, a combined strategy of suicide genes and radiation was designed in this study. Five suicide gene expression cassettes, yeast CD, yeast CD/UPRT, and yeast CDglyTK gene controlled by CMV, and Egr-1 and a synthetic CMV-enhanced Egr-1 promoter (CE) were constructed in an expression vector p11MS. The expression of suicide genes in NPC CNE-2 cells were detected by RT-PCR and Western blot. The cytotoxicity of suicide gene therapy and radiation were analyzed by MTT assay. An animal study in which yeast CD/UPRT-expressing CNE-2 tumors in nude mice were treated with 5-FC and radiation was also developed. Our results revealed that p11MSCEyCD/UPRT and p11MSCEyCDglyTK are superior over three other constructs in the killing of NPC cells in vitro. We combined suicide gene-expressing tumors, 5-FC treatment, and radiation in vivo and found that the tumors greatly regressed, some disappeared completely in 3 nude mice in the yCD/UPRT group, and a significant difference of tumor volumes was observed between this group and the other four groups (p < 0.05). Our results indicated that suicide gene therapy and radiation have a synergic effect on NPC therapy, and the combined strategy of radiogene therapy is of great potential as a substitute for the traditional method, radiation alone, in NPC therapies.

A Novel Fusion Suicide Gene Yeast CDglyTK Plays a Role in Radio-gene Therapy of Nasopharyngeal Carcinoma

Cancer Gene Therapy. Dec, 2004  |  Pubmed ID: 15499380

To investigate a novel suicide gene for nasopharyngeal carcinoma (NPC) therapy, the yCDglyTK gene was constructed by fusing yeast cytosine deaminase (CD) and herpes simplex type 1 thymidine kinase. The expression of the yCDglyTK gene was detected by RT-PCR and Western blotting, and its bioactivity was demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. An animal study was carried out in which BALB/C nude mice bearing yCDglyTK gene-modified tumors were treated with prodrugs and radiation. Our results revealed that the yCDglyTK gene could be expressed in CNE-2 cells in vitro. In MTT analysis, at the transfection rate of 10%, 66% cells were killed. The synergistic effect of CD and TK showed 91% of yCDglyTK-transfected cells were killed with the treatment of 5-fluorocytosine (5-FC) alone, 60% killed with ganciclovir (GCV) alone, and 75% killed with 5-FC and GCV together. In vivo, the tumor volume in all of the four prodrugs and/or radiation-treated groups were significantly different from that in the PBS-controlled group (P<.01); also yCDglyTK+prodrug+radiation group was different from the other three groups (P<.05). Our findings suggested there was a synergistic antitumor effect when combining suicide gene therapy and radiation, and yCDglyTK has potent antitumor efficacy and may be a candidate suicide gene for cancer therapy.

Generation of a Phenotypic Array of Hypothalamic Neuronal Cell Models to Study Complex Neuroendocrine Disorders

Endocrinology. Jan, 2004  |  Pubmed ID: 14551229

Knowledge of how the brain achieves its diverse central control of basic physiology is severely limited by the virtual absence of appropriate cell models. Isolation of clonal populations of unique peptidergic neurons from the hypothalamus will facilitate these studies. Herein we describe the mass immortalization of mouse primary hypothalamic cells in monolayer culture, resulting in the generation of a vast representation of hypothalamic cell types. Subcloning of the heterogeneous cell populations resulted in the establishment of 38 representative clonal neuronal cell lines, of which 16 have been further characterized by analysis of 28 neuroendocrine markers. These cell lines represent the first available models to study the regulation of neuropeptides associated with the control of feeding behavior, including neuropeptide Y, ghrelin, urocortin, proopiomelanocortin, melanin-concentrating hormone, neurotensin, proglucagon, and GHRH. Importantly, a representative cell line responds appropriately to leptin stimulation and results in the repression of neuropeptide Y gene expression. These cell models can be used for detailed molecular analysis of neuropeptide gene regulation and signal transduction events involved in the direct hormonal control of unique hypothalamic neurons, not yet possible in the whole brain. Such studies may contribute information necessary for the strategic design of therapeutic interventions for complex neuroendocrine disorders, such as obesity.

[Genetic Linkage Analysis in Localizing a Gene of Autosomal Dominant Familial Dilated Cardiomyopathy with Conduction Defect]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Oct, 2005  |  Pubmed ID: 16320577

To localize the gene of autosomal dominant familial dilated cardiomyopathy with conduction defect.

[Mutation Analysis of DJ1 Gene in Patients with Autosomal Recessive Early-onset Parkinsonism]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Dec, 2005  |  Pubmed ID: 16331561

To investigate the mutation characteristics of DJ1 gene in Chinese patients with autosomal recessive early-onset Parkinsonism (AR-EP).

Confirmation and Refinement of a Genetic Locus of Congenital Motor Nystagmus in Xq26.3-q27.1 in a Chinese Family

Human Genetics. Jan, 2005  |  Pubmed ID: 15517395

Congenital motor nystagmus (CMN), a subtype of nystagmus, may reduce vision or be associated with other, more serious, conditions that limit vision. The genetic basis for CMN is still unknown. To identify a locus for CMN, genotyping and linkage analysis were performed in 22 individuals from a Chinese family with X-linked CMN using markers from X chromosome. The maximum LOD score obtained for microsatellite maker DXS1192 linked the CMN locus in this family to Xq. By haplotype construction the locus for CMN was finally localized to an approximately 4.4-cM region at chromosome Xq26.3-q27.1. The SLC9A6 and FGF13 genes in this region, were selected and screened for mutation in this family, but no mutation was detected.

Small Heat-shock Protein 22 Mutated in Autosomal Dominant Charcot-Marie-Tooth Disease Type 2L

Human Genetics. Feb, 2005  |  Pubmed ID: 15565283

Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. We have previously described a large Chinese CMT family and assigned the locus underlying the disease (CMT2L; OMIM 608673) to chromosome 12q24. Here, we report a novel c.423G-->T (Lys141Asn) missense mutation of small heat-shock protein 22-kDa protein 8 (encoded by HSPB8), which is also responsible for distal hereditary motor neuropathy type (dHMN) II. No disease-causing mutations have been identified in another 114 CMT families.

Mutation Screening of Cx32 in Han Chinese Patients with Charcot-Marie-Tooth Disease

Beijing Da Xue Xue Bao. Yi Xue Ban = Journal of Peking University. Health Sciences. Feb, 2005  |  Pubmed ID: 15719046

To investigate the Cx32 mutation features and the clinical manifestations of Chinese patients with Charcot-Marie-Tooth disease(CMT).

[Phenotype Positioning on Chromosomes in a Patient with the Syndrome of Partial Trisomy 7p21.2-->pter]

Yi Chuan Xue Bao = Acta Genetica Sinica. Feb, 2005  |  Pubmed ID: 15759858

By using the techniques of human chromosome G-banding, high resolution banding and fluorescence in situ hybridization (FISH), we investigated a patient with the karyotype of partial trisomy 7p21.2-->pter. Combining with the comparative review of the clinical data in 14 cases with partial trisomy 7p syndrome from reported literatures,we searched for the correlation between the karyotype and the phenotype and between the karyotype and the associated gene in the patients with partial trisomy 7p syndrome. The results indicated that the 7p21.2-->p22 is the critical segment of partial trisomy 7p syndrome. The phenotypes of the genital malformation and the dislocation of hip joint are associated with the duplication of 7p15. The cardiac anomalies is resulted from the dysfunctions of several genes on the long arm of chromosome 7. The gene associated with the craniosynostosis may locate on the region of 7p21.2-->p21.3.

Identification of the Alternative Promoters of the KChIP4 Subfamily

Acta Biochimica Et Biophysica Sinica. Apr, 2005  |  Pubmed ID: 15806290

The subfamily of voltage-dependent potassium (Kv) channel interacting protein 4 (KChIP4) is made up of the auxiliary interacting protein of voltage-dependent potassium channels. In this study, the structure of four splicing variants of the human KChIP4 gene was analyzed. Three of the four isoforms of the KChIP4 gene, KChIP4.1, KChIP4.2 and KChIP4.4, were amplified from mouse and human fetal brain tissues by reverse transcription-polymerase chain reaction and then identified. Based on the bioinformatics analysis of the genomic sequences of the gene, we cloned and characterized two promoter fragments from the KChIP4 gene. One was a 325 bp fragment upstream of the 5' end of the KChIP4.1 mRNA sequence and the other was an 818 bp fragment located immediately at the 5' end of the KChIP4.4 variant. Both of them can initiate the transcription of the reporter gene in HT1080 cells and Sprague-Dawley (SD) rat fetal brain neurons, and they contain CG islands, except typical TATA boxes and CAAT boxes. This shows that the KChIP4 gene expression is regulated by an alternative promoter.

Research on Screening and Identification of Proteins Interacting with Ataxin-3

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Jun, 2005  |  Pubmed ID: 15952105

This study sought to isolate and identify the proteins that interact with ataxin-3, to confirm the interacted domain, and to provide new clues for exploring the function of ataxin-3 and the pathogenesis of spinocerebellar ataxia type 3 and Machado-Joseph disease (SCA3/MJD).

[Effect of IP3 on BK Channels of Porcine Coronary Artery Smooth Muscle Cells]

Sheng Li Xue Bao : [Acta Physiologica Sinica]. Jun, 2005  |  Pubmed ID: 15968424

D-myo-inositol 1,4,5-trisphosphate (IP(3)) plays an important role in signal transduction. It releases Ca(2+) from intracellular sites, which activates the Ca(2+)-dependent channels such as large-conductance Ca(2+)-activated potassium channels (BK channels). The present study was therefore designed to determine if the activity of BK channels in porcine coronary artery smooth muscle cells was increased by IP(3). Using the inside-out patch-clamp technique, the activity of single BK channels was recorded in porcine coronary artery smooth muscle cells. In excised inside-out membrane patches, IP(3) (10-50 micromol/L) enhanced the open probability (Po) of BK channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. The open-state probability of the BK channels increased from a control level of 0.0402+/-0.0152 to 0.1365+/-0.0212 (20 micromol/L IP(3)) and 0.1865+/-0.0175 (30 micromol/L IP(3)). IP(3) decreased the mean close time markedly, but had no effect on the amplitude of BK channels. The activation of IP(3) on BK channels did not decline. The metabolite of IP(3) had no obvious effect on BK channels. This study provides evidence that IP(3) activates BK channels in porcine coronary artery smooth muscle cells in a dose-dependence manner.

Spinocerebellar Ataxia Type 6 in Mainland China: Molecular and Clinical Features in Four Families

Journal of the Neurological Sciences. Sep, 2005  |  Pubmed ID: 15979648

The hereditary spinocerebellar ataxias (SCAs) are a clinically and genetically heterogeneous group of neurodegenerative disorders. The genes causing 11 of these diseases have been identified. To date, there is no report of SCA type 6 (SCA6) in Mainland Chinese. Using a molecular approach, we investigated SCA6 as well as other SCA subtype in 120 Mainland Chinese families with dominantly inherited ataxias and in 60 Mainland Chinese patients with sporadic ataxias. Clinical and molecular features of SCA6 were further characterized in 13 patients from 4 families. We found that SCA3/MJD was the most common type of autosomal dominant SCA in Mainland Chinese, accounting for 83 patients from 59 families (49.2%), followed by SCA2 (8 [6.7%]), SCA1 (7 [5.8%]), SCA6 (4 [3.3%]), SCA7 (1 [0.8%]), SCA8 (0%), SCA10 (0%), SCA12 (1 [0.8%]), SCA14 (0%), SCA17 (0%) and DRPLA (0%). The genes responsible for 40 (33.3%) of dominantly inherited SCA families remain to be determined. Among the 60 patients with sporadic ataxias in the present series, 3 (5.0%) were found to harbor SCA3 mutations, whereas none were found to harbor SCA6 mutations. In the 4 families with SCA6, we found significant anticipation in the absence of genetic instability on transmission. This is the first report of geographic cluster of families with SCA6 subtype in Mainland China.

Cx31 is Assembled and Trafficked to Cell Surface by ER-Golgi Pathway and Degraded by Proteasomal or Lysosomal Pathways

Cell Research. Jun, 2005  |  Pubmed ID: 15987604

Gap junctions, consisting of connexins, allow the exchange of small molecules (less than 1 KD) between adjacent cells, thus providing a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Connexin 31 is a member of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearing impairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseases are still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in HeLa cells stably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells were treated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or a-glycyrrhetinic acid, suggesting that Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin 31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, a-glycyrrhetinic acid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assay revealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstrated that in HeLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cells were treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed, and possibly degraded by both proteasomal and lysosomal pathways.

[Stable Suppression of Beta-catenin Expression in Prostate Cancer Cell Line by Retrovirus Mediated RNAi]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Jun, 2005  |  Pubmed ID: 16045007

To set up a prostate cancer cell line in which beta-catenin expression is stably suppressed and to investigate the role of Wnt/beta-catenin signaling pathway in prostate tumorgenesis.

Intracellular Distribution, Assembly and Effect of Disease-associated Connexin 31 Mutants in HeLa Cells

Acta Biochimica Et Biophysica Sinica. Aug, 2005  |  Pubmed ID: 16077902

Mutations in connexin 31 (Cx31) are associated with erythrokeratodermia variabilis (EKV), hearing impairment and peripheral neuropathy; however, the pathological mechanism of Cx31 mutants remains unknown. This study analyzed 11 disease-associated Cx31 variants and one non-disease-associated Cx31 variant and compared their intracellular distribution and assembly in HeLa cells and their effect on these cells. The fluorescent localization assay showed no gap junction plaque formation in the cells expressing the recessive EKV-associated mutant (L34P) and four hearing impairment-associated mutants (66delD, 141delI, R180X and E183K), significantly reduced plaque formation in the cells with five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) and no obvious change in the cells with two other mutants (I141V and 652del12). Immunoblotting analysis showed that 12 mutated Cx31s, like WT-Cx31, are able to form the Triton X-100 insoluble complex; however, the quantity of Triton X-100 insoluble complex in the transfected HeLa cells varied among different Cx31 mutants. Additionally, the expression of five EKV-associated dominant mutants (G12R, G12D, R42P, C86S and F137L) caused cell death in HeLa cells. However, the five hearing impairment-associated mutants did not induce cell death. The above results suggest that disease-associated mutants gain deleterious functions differentially. In summary, disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels, and the diseases associated with Cx31 mutations may result from the abnormal assembly, trafficking and metabolism of the Cx31 mutants.

Mutation Analysis of Small Heat-shock Protein 22 Gene in Chinese Patients with Charcot-Marie-Tooth Disease

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Aug, 2005  |  Pubmed ID: 16086267

To study the characteristics of the mutation of small heat-shock protein 22 (HSP22) gene in Chinese patients with Charcot-Marie-Tooth (CMT) disease.

Molecular Analysis of SLC26A4 Gene in a Chinese Deafness Family

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Aug, 2005  |  Pubmed ID: 16086271

To identify the pathogenic gene for a non-syndromic hearing loss family.

Mutation Analysis of the Small Heat Shock Protein 27 Gene in Chinese Patients with Charcot-Marie-Tooth Disease

Archives of Neurology. Aug, 2005  |  Pubmed ID: 16087758

Charcot-Marie-Tooth (CMT) disease, the most common hereditary peripheral neuropathy, is highly clinically and genetically heterogeneous, and mutations in at least 18 genes have been identified. Recently, mutations in small heat shock protein 27 (Hsp27) were reported to cause CMT disease type 2F and distal hereditary motor neuropathy.

[Mutation Analysis of PINK1 Gene in Chinese Patients with Autosomal Recessive Early-onset Parkinsonism Type 6]

Zhonghua Yi Xue Za Zhi. Jun, 2005  |  Pubmed ID: 16179113

To detect PINK1 gene mutations and study the clinical features in Chinese patients with autosomal recessive early-onset parkinsonism (AREP) type 6.

[Prenatal Diagnosis of Prelingual Deafness by Determination of SLC26A4 Gene Mutation]

Zhonghua Fu Chan Ke Za Zhi. Sep, 2005  |  Pubmed ID: 16202311

To identify deafness related gene and provide its prenatal diagnosis to avoid deaf fetus delivery.

[Mutation Analysis of Small Heat Shock Protein 27 Gene in Chinese Patients with Charcot-Marie-Tooth Disease]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Oct, 2005  |  Pubmed ID: 16215937

To investigate the features of small heat shock protein 27 (HSP27) gene mutation in Chinese patients with Charcot-Marie-Tooth disease (CMT).

[Detection of Duplications or Deletions of the PMP22 Gene Using Real-time Quantitative PCR]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Oct, 2005  |  Pubmed ID: 16215943

To detect the duplication or deletion of peripheral myelin protein 22(PMP22) gene in Chinese patients with Charcot-Marie-Tooth disease(CMT) or hereditary neuropathy with liability to pressure palsies(HNPP) using real-time quantitative polymerase chain reaction.

Anorexigenic Hormones Leptin, Insulin, and Alpha-melanocyte-stimulating Hormone Directly Induce Neurotensin (NT) Gene Expression in Novel NT-expressing Cell Models

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2005  |  Pubmed ID: 16221860

Neurotensin (NT) is implicated in the regulation of energy homeostasis, in addition to its many described physiological functions. NT is postulated to mediate, in part, the effects of leptin in the hypothalamus. We generated clonal, immortalized hypothalamic cell lines, N-39 and N-36/1, which are the first representative NT-expressing cell models available for the investigation of NT gene regulation and control mechanisms. The cell lines express the Ob-Rb leptin receptor neuropeptide Y (NPY)-Y1, Y2, Y4, Y5 receptors, melanocortin 4 receptor, insulin receptor, and the NT receptor. NT mRNA levels are induced by approximately 1.5-fold to twofold with leptin, insulin, and alpha-melanocyte stimulating hormone treatments but not by NPY. Leptin-mediated induction of NT gene expression was biphasic at 10(-11) and 10(-7) M. The leptin responsive region was localized to within -381 to -250 bp of the 5' regulatory region of the NT gene. Furthermore, we demonstrated direct leptin-mediated signal transducers and activators of transcription (STAT) binding to this region at 10(-11) m, but not 10(-7) m leptin, in chromatin precipitation assays. Leptin-induced NT regulation was attenuated by dominant-negative STAT3 protein expression. These data support the hypothesis that NT may have a direct role in the neuroendocrine control of feeding and energy homeostasis.

[The Characteristics of Gene Mutations in Chinese Patients with Charcot-Marie-Tooth Disease]

Zhonghua Yi Xue Za Zhi. Jul, 2005  |  Pubmed ID: 16253183

To study the characteristics of gene mutations in Chinese patients with Charcot-Marie-Tooth disease (CMT).

[Construction of the Eukaryotic Expression Vector of MJD1 and Its Expression in SH-SY5Y Cells]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Dec, 2005  |  Pubmed ID: 16708800

To construct the eukaryotic expression vector of MJD1 with normal copies of CAG trinucleotide repetition and MJD1 with CAG trinucleotide repetition expansion mutation respectively, and to determine whether the polyglutamine expansion in ataxin-3 could lead to the formation of intranuclear aggregation.

Essential Role of Pten in Body Size Determination and Pancreatic Beta-cell Homeostasis in Vivo

Molecular and Cellular Biology. Jun, 2006  |  Pubmed ID: 16738317

PTEN (phosphatase with tensin homology) is a potent negative regulator of phosphoinositide 3-kinase (PI3K)/Akt signaling, an evolutionarily conserved pathway that signals downstream of growth factors, including insulin and insulin-like growth factor 1. In lower organisms, this pathway participates in fuel metabolism and body size regulation and insulin-like proteins are produced primarily by neuronal structures, whereas in mammals, the major source of insulin is the pancreatic beta cells. Recently, rodent insulin transcription was also shown in the brain, particularly the hypothalamus. The specific regulatory elements of the PI3K pathway in these insulin-expressing tissues that contribute to growth and metabolism in higher organisms are unknown. Here, we report PTEN as a critical determinant of body size and glucose metabolism when targeting is driven by the rat insulin promoter in mice. The partial deletion of PTEN in the hypothalamus resulted in significant whole-body growth restriction and increased insulin sensitivity. Efficient PTEN deletion in beta cells led to increased islet mass without compromise of beta-cell function. Parallel enhancement in PI3K signaling was found in PTEN-deficient hypothalamus and beta cells. Together, we have shown that PTEN in insulin-transcribing cells may play an integrative role in regulating growth and metabolism in vivo.

[Exon Rearrangement Analysis of Parkin Gene in Patients with Isolated Early-onset Parkinsonism Using Semi-quantitative PCR]

Zhonghua Yi Xue Za Zhi. Jun, 2006  |  Pubmed ID: 16842693

To investigate the value of fluorescence semi-quantitative PCR in analysis of the exon rearrangement of parkin gene in Chinese patients with isolated early-onset parkinsonism (EOP).

Comparison of Extracellular and Intracellular Potency of Botulinum Neurotoxins

Infection and Immunity. Oct, 2006  |  Pubmed ID: 16988237

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When naïve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.

[Functional Interaction of the C-terminal of Nogo Protein with Connexin 26 and the Expression of Nogo's MRNA in the Murine Inner Ear]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Oct, 2006  |  Pubmed ID: 17029193

To screen and identify the proteins that interact with connexin 26 (CX26) and to analyze the expressions of these proteins in cochlea so as to explore the proteins that relate to the trafficking, assembly, localizing and gap junction functions of CX26.

Ubiquitin-proteasome Pathway Mediates Degradation of APH-1

Journal of Neurochemistry. Dec, 2006  |  Pubmed ID: 17059559

Gamma-secretase catalyzes intramembraneous proteolysis of several type I transmembrane proteins, including beta-amyloid precursor protein (APP), to generate amyloid beta protein (Abeta), a key player in the pathogenesis of Alzheimer's disease (AD). The critical components of the gamma-secretase complex include presenilin (PS), nicastrin (NCT), presenilin enhancer-2 (PEN-2) and anterior pharynx defective-1 (APH-1). Abnormalities of the ubiquitin-proteasome pathway have been implicated in the pathogenesis of AD; while PS and PEN-2 turnover is regulated by this pathway, it is unknown whether the ubiquitin-proteasome pathway is also involved in the degradation of APH-1 protein. In this study, we found that the expression of endogenous and exogenous APH-1 significantly increased in cells treated with proteasome-specific inhibitors. The effect of the proteasome inhibitors on APH-1 was dose- and time-dependent. APH-1 protein was ubiquitinated. Pulse-chase metabolic labeling experiments showed that the degradation of newly synthesized radiolabeled APH-1 proteins was inhibited by lactacystin. Disruption of the PS1 and PS2 genes did not affect the degradation of APH-1 by the ubiquitin-proteasome pathway. Furthermore, over-expression of APH-1 and inhibition of proteasomal APH-1 degradation facilitated gamma-secretase cleavage of APP to generate Abeta. These results demonstrate that the degradation of APH-1 protein is mediated by the ubiquitin-proteasome pathway.

Leptin Signaling in Neurotensin Neurons Involves STAT, MAP Kinases ERK1/2, and P38 Through C-Fos and ATF1

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Dec, 2006  |  Pubmed ID: 17077290

The adipokine leptin signals energy status to the hypothalamus, which triggers a network of neuropeptide responses. Each hypothalamic cell type expresses a unique complement of neuropeptides, receptors, and second messengers; thus each likely responds specifically to peripheral hormones. We describe here the analysis of leptin signaling in a clonal population of mouse neurotensin (NT) -expressing hypothalamic neurons, N-39. Leptin induced phosphorylation of STAT3 and MAPK ERK1/2, but not the downstream effector of PI3K, Akt, and also induced c-Fos protein. We found activation of p38 MAPK by leptin, accompanied by phosphorylation of its downstream effector ATF-1. Phosphorylation of ATF-1 is blocked by the p38 MAPK inhibitor SB 203580. We linked this signaling directly to NT transcription. Protein binding analysis indicates that both ATF-1 and c-Fos are capable of binding to the mouse NT/N gene predominantly at physiological or high concentrations of leptin. The evidence indicates activation of distinct leptin signal transduction pathways that directly result in changes in NT gene expression and links these specific neurons to the control of energy homeostasis.

Hypoxia Facilitates Alzheimer's Disease Pathogenesis by Up-regulating BACE1 Gene Expression

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2006  |  Pubmed ID: 17121991

The molecular mechanism underlying the pathogenesis of the majority of cases of sporadic Alzheimer's disease (AD) is unknown. A history of stroke was found to be associated with development of some AD cases, especially in the presence of vascular risk factors. Reduced cerebral perfusion is a common vascular component among AD risk factors, and hypoxia is a direct consequence of hypoperfusion. Previously we showed that expression of the beta-site beta-amyloid precursor protein (APP) cleavage enzyme 1 (BACE1) gene BACE1 is tightly controlled at both the transcriptional and translational levels and that increased BACE1 maturation contributes to the AD pathogenesis in Down's syndrome. Here we have identified a functional hypoxia-responsive element in the BACE1 gene promoter. Hypoxia up-regulated beta-secretase cleavage of APP and amyloid-beta protein (Abeta) production by increasing BACE1 gene transcription and expression both in vitro and in vivo. Hypoxia treatment markedly increased Abeta deposition and neuritic plaque formation and potentiated the memory deficit in Swedish mutant APP transgenic mice. Taken together, our results clearly demonstrate that hypoxia can facilitate AD pathogenesis, and they provide a molecular mechanism linking vascular factors to AD. Our study suggests that interventions to improve cerebral perfusion may benefit AD patients.

Carbohydrate Mimicry of Campylobacter Jejuni Lipooligosaccharide is Critical for the Induction of Anti-GM1 Antibody and Neuropathy

Muscle & Nerve. Feb, 2006  |  Pubmed ID: 16270308

The expression of ganglioside-mimicking structures of Campylobacter jejuni lipooligosaccharides (LOS) is considered essential for the induction of antiganglioside antibodies that lead to Guillain-Barré syndrome (GBS). The galE gene in C. jejuni is involved in the biosynthesis of the LOS outer-core oligosaccharide structures. We have demonstrated that the C. jejuni HB9313 (HS:19) parental strain expresses a LOS structure containing GM1-like epitopes, and the C. jejuni knockout mutant of the galE gene expresses a truncated LOS structure without GM1-like epitopes. To clarify whether the ganglioside-like structures in Campylobacteri LOS are crucial for induction of antiganglioside antibody responses and neuropathy, we performed immunization experiments in guinea pig models using the parental strain HB9313 and its galE mutant derivative. The anti-GM1 IgG antibody responses in immunized animals were measured by enzyme-linked immunosorbent assay. Sciatic nerve specimens were evaluated pathologically. High levels of the anti-GM1 IgG antibody were induced in guinea pigs immunized with HB9313, but not in those immunized with the galE mutant. The mean percentage of abnormality of sciatic-nerve teased fibers from animals sensitized with C. jejuni HB9313 was significantly higher than from animals immunized with the galE mutant. Furthermore, significant changes were found in semithin sections of the sciatic nerve from animals inoculated with C. jejuni HB9313. The major pathological finding was axonal degeneration; no significant morphological findings, except for occasional demyelination, were observed in animals immunized with the galE mutant. These results indicate that ganglioside-mimicry structures in C. jejuni LOS are necessary for induction of antiganglioside antibody response and neuropathy.

Mutation Analysis of the ATM Gene in Two Chinese Patients with Ataxia Telangiectasia

Journal of the Neurological Sciences. Feb, 2006  |  Pubmed ID: 16380133

Ataxia telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia, telangiectasia, immunodeficiency, elevated alpha-fetoprotein level, chromosomal instability, predisposition to cancer, and radiation sensitivity. Although a lot of mutations in the ATM gene have been described, there is still no report about ATM mutations in Chinese population. Using a molecular approach, we screened for ATM mutations in two patients from two unrelated Chinese families. 100 normal controls were analyzed to exclude possibility of polymorphism. Two novel mutations in the ATM gene were identified. The first one is a novel, homozygous, 1346G>C (Gly449Ala) missense mutation. The second one is a compound heterozygous mutation, which consists of a novel, 610G>T (Gly204Stop) nonsense mutation, combined with a previously reported, 6679C>T (Arg2227Cys) missense mutation. The transversions 1346G>C (Gly449Ala) and 610G>T (Gly204Stop) are not localized either in the conserved PI-3 kinase domain or in the other domains of the ATM protein. The phenotypic features were characterized by progressive cerebellar ataxia, ocular telangiectasia, elevated alpha-fetoprotein level, immunodeficiency (agammaglo-bulinemia and T-cell defect), and rearrangements of chromosomes 7 and 14; brain MRI showed cerebellar atrophy, brain SPECT showed cerebellar regional cerebral blood flow (rCBF) hypoperfusion. To our knowledge, this is the first report of ATM mutations in Mainland China, in which the transversions 1346G>C (Gly449Ala) and 610G>T (Gly204Stop) are two novel, disease-causing mutations.

A Father and Son with Mental Retardation, a Characteristic Face, Inv(12), and Insertion Trisomy 12p12.3-p11.2

American Journal of Medical Genetics. Part A. Feb, 2006  |  Pubmed ID: 16411213

A male patient with mental retardation (MR) and mild facial features was shown by high-resolution G-banding to have pericentric inversion of chromosome 12 with an unknown segment inserted into the long arm of the inverted chromosome [46,XY,inv(12)(pter-->p11.2::q14.1-->p11.2::?::q14.1-->qter)]. Both the inverted chromosome 12 and clinical manifestations were transmitted to his son. Karyotypes of the propositus' parents were normal. Studies with fluorescence in situ hybridization (FISH) in both the propositus and his son revealed that the extra segment was derived from 12p. Further FISH mapping and the genome-wide copy number detection by GeneChip Mapping 100K Array showed that an 11-Mb segment of 12p between two BAC clones, RP11-22H10 and RP11-977P2, was inserted at one of the reunion points in the long arm of the inv(12) chromosome. Analysis of parent-child transmissions of duplicated alleles using microsatellite markers defined the maternal origin of the chromosomal anomaly in the propositus and suggested a mechanism of its formation through a sister-chromatid rearrangement (SCR), that is, mismatched pairing and unequal crossover between sister chromatids as well as three break rearrangements including a U type rearrangement. Karyotypes of the propositus and his son were thus inv(12)(pter-->p11.22::q14.1-->p12.3::q14.1-->qter). This is the first report of "pure" proximal 12p-trisomy including p12.3-p11.22 region.

[Genotype and Phenotype Analyses of Three Families with Autosomal Recessive Juvenile Parkinsonism]

Zhonghua Yi Xue Yi Chuan Xue Za Zhi = Zhonghua Yixue Yichuanxue Zazhi = Chinese Journal of Medical Genetics. Feb, 2006  |  Pubmed ID: 16456791

To investigate the gene mutations and the clinical features of Chinese patients with autosomal recessive juvenile parkinsonism(AR-JP).

[Effects of Tetramethylpyrazine on Large-conductance Ca(2+)-activated Potassium Channels in Porcine Coronary Artery Smooth Muscle Cells.]

Sheng Li Xue Bao : [Acta Physiologica Sinica]. Feb, 2006  |  Pubmed ID: 16489409

The aim of the present study was to examine the effects of tetramethylpyrazine (TMP) on large-conductance Ca(2+)-activated potassium channels (BK(Ca) channels) in porcine coronary artery smooth muscle cells, in order to provide the experimental evidence for expounding the mechanism of TMP in dilating coronary artery. Cell-attached and inside-out single channel recording techniques were used to observe the effects of TMP on BK(Ca) channels as well as the effects after the cells were treated by protein kinase A (PKA) inhibitor or protein kinase G (PKG) inhibitor. In inside-out patch, TMP activated BK(Ca) channels by increasing open-state probability (N(Po)) and decreasing close time (Tc) in a concentration-dependent manner. TMP (0.73~8.07 mmol/L) in the bath solution increased N(Po) from (0.01+/-0.003) to (0.03+/-0.01)~(1.21+/-0.18) (P<0.01, n=10)ìand decreased Tc from (732.33+/-90.67) ms to (359.67+/-41.30) ~ (2.96+/-0.52) ms (P<0.01, n=10). These actions of TMP occurred even when the free Ca(2+) concentration in the bath was reduced to ~ 0 mmol/L. The specific inhibitors of PKA (H-89, 3 mumol/L) and PKG (KT-5823, 1 mumol/L) had no influence on the activation of TMP on BK(Ca) channels. These findings suggest that TMP can directly activate BK(Ca) channels in coronary artery smooth muscle, which probably is an important mechanism in dilating coronary artery.

Mapping and Identification of Disease Responsible Genes Based on Genetic Resource Reservation and Bioinformatics Approach

Beijing Da Xue Xue Bao. Yi Xue Ban = Journal of Peking University. Health Sciences. Feb, 2006  |  Pubmed ID: 16514726

[Identification and Clone of Human Alzheimer's Disease Related Gene Nicastrin Promoter]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Feb, 2006  |  Pubmed ID: 16562666

To identify the promoter of human nicastrin (NCT) gene, a major component of gamma-secretase which is closely related with pathogenesis of Alzheimer's disease.

The Sialic Acid Residue is a Crucial Component of C. Jejuni Lipooligosaccharide Ganglioside Mimicry in the Induction Guillain-Barré Syndrome

Journal of Neuroimmunology. May, 2006  |  Pubmed ID: 16567003

Guillain-Barré syndrome (GBS) is an autoimmune neuropathy that often follows C. jejuni infection. Sialic acid (N-acetylneuraminic acid, NANA) is a common constituent of lipooligosaccharide (LOS). The molecular mimicry between C. jejuni LOS and human peripheral nerve gangliosides is believed to play an important role in the pathogenesis of GBS. The neuB1 encodes NANA synthetase, required for the synthesis of NANA of C. jejuni LOS. A neuB1 mutant was constructed from a C. jejuni HS:19 wild strain. Mutant LOS could not bind the cholera toxin B subunit, failed to induce anti-GM1 antibodies, and did not cause pathological changes in the peripheral nerves. These data suggest that the NANA residue in LOS is a crucial epitope in realization of ganglioside molecular mimicry.

Coordinate Regulation of Neuropeptide Y and Agouti-related Peptide Gene Expression by Estrogen Depends on the Ratio of Estrogen Receptor (ER) Alpha to ERbeta in Clonal Hypothalamic Neurons

Molecular Endocrinology (Baltimore, Md.). Sep, 2006  |  Pubmed ID: 16675543

Neuropeptide Y (NPY) and agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. We hypothesize that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, we demonstrate that 17beta-estradiol differentially regulates estrogen receptor (ER)alpha and ERbeta levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ERalpha and ERbeta or ERalpha alone, respectively, whereas the induction of NPY and AgRP in N-38 neurons is strictly ERbeta dependent, as assessed by ER-specific agonists and small interfering RNA knockdown of ERalpha or ERbeta. Through transient transfection analysis in N-38 neurons, we have mapped the estrogen-mediated repression of NPY to within -1078 of the 5' regulatory region of the NPY gene. Our results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERbeta to ERalpha. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction.

Role of Calcium Mobilization in the Regulation of Spontaneous Transient Outward Currents in Porcine Coronary Artery Myocytes

Science in China. Series C, Life Sciences / Chinese Academy of Sciences. Oct, 2007  |  Pubmed ID: 17879066

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca(2+)-activated-K(+) (BK(Ca)) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK(Ca) channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca(2+) concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca(2+) abolished STOCs activity. Ca(2+) ionophore A23187 (10 micromol/L) increased STOCs activity significantly. Verapamil (20 micromol/L) and CdCl(2) (200 micromol/L), two kinds of organic L-type voltage-dependent Ca(2+) channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 micromol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) by 2APB (40 micromol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK(Ca) channels. Extracellular Ca(2+) is essential for STOCs activity, while Ca(2+) entry through L-VDCCs has little effect on STOCs. Intracellular Ca(2+) release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.

Characteristic of Spontaneous Transient Outward Potassium Currents in Vascular Smooth Muscle Cells of Porcine Coronary Artery

Sheng Li Xue Bao : [Acta Physiologica Sinica]. Feb, 2007  |  Pubmed ID: 17294039

Spontaneous transient outward currents (STOCs) play an important role in the myogenic regulation of small artery tone, such as coronary artery. In the present study, we investigated the electrophysiological properties and the regulation of STOCs in vascular smooth muscle cells (VSMCs) of porcine coronary artery by perforated patch-clamp technique. Our data showed that STOCs were dependent on voltage and extracellular calcium and they were highly variable in amplitudes and frequencies. STOCs superimposed stochastically onto whole-cell K(+) currents induced by step and ramp protocols. STOCs were completely abolished by ChTX [inhibitor of large-conductance Ca(2+)-activated potassium (BK(Ca)) channels], removal of extracellular Ca(2+), or addition of ryanodine (50 mumol/L) respectively. In contrast, CdCl2 and verapamil, inhibitors of voltage-dependent L-type Ca(2+) channels, had little effect on STOCs. Caffeine (5 mmol/L) transiently increased STOCs (hump), followed by a temporary inhibition. Ca(2+) ionophore A23187 increased both amplitude and frequency of STOCs. Na(+) ionophore monensin increased the frequency of STOCs. STOCs were strongly inhibited by KB-R7943, a selective inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger. Based on these observations, we conclude that STOCs are mediated by BK(Ca) channels. The generation and activation of STOCs depend upon Ca(2+) influx through Na(+)/Ca(2+) exchange and release of Ca(2+) from sarcoplasmic reticulum (SR) via ryanodine receptors. This suggests that Na(+)/Ca(2+) exchange determines calcium store refilling. Recycling of entering Ca(2+) from superficial SR may locally elevate Ca(2+) concentration at the plasma membrane, thereby activating BK(Ca) channels and then initiating STOCs.

Degradation of Nicastrin Involves Both Proteasome and Lysosome

Journal of Neurochemistry. May, 2007  |  Pubmed ID: 17326768

The glycoprotein nicastrin (NCT) is an essential component of the gamma-secretase complex, a high molecular weight complex which also contains the presenilin proteins, Aph-1 and Pen-2. The gamma-secretase complex is not only involved in APP processing but also in the processing of an increasing number of other type I integral membrane proteins. As the largest subunit of the gamma-secretase complex, NCT plays a crucial role in its activation. Considerable information exists on the distribution, structure and function of NCT; however, little is known of its proteolysis. The present study is aimed at exploring the molecular mechanism of NCT degradation. We found that either proteasomal or lysosomal inhibition can significantly increase the levels of both endogenous and exogenous NCT in various cell lines, and the effect of these inhibitions on NCT was time- and dose-dependent. Immunofluorescent microscopic analysis revealed that NCT accumulates in the ER and Golgi apparatus after proteasomal inhibition, while lysosomal inhibition leads to the accumulation of NCT in the lysosomal apparatus. Co-immunoprecipitation can pull down both NCT and ubiquitin. Taken together, our results demonstrate that NCT degradation involves both the proteasome and the lysosome.

Glucose Regulates AMP-activated Protein Kinase Activity and Gene Expression in Clonal, Hypothalamic Neurons Expressing Proopiomelanocortin: Additive Effects of Leptin or Insulin

The Journal of Endocrinology. Mar, 2007  |  Pubmed ID: 17332528

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

[Azoospermia Factor Microdeletion on Y Chromosome in Patients with Idiopathic Azoospermia or Severe Oligozoospermia]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Apr, 2007  |  Pubmed ID: 17478930

To investigate the correlation between male infertility and Y chromosome microdeletions of azoospermia factor (AZF) regions, and to establish a reliable genetic diagnosis in idiopathic infertile male patients with azoospermia or severe oligozoospermia.

[Identification of the Origin of Marker Chromosome by Comparative Genomic Hybridization]

Zhong Nan Da Xue Xue Bao. Yi Xue Ban = Journal of Central South University. Medical Sciences. Apr, 2007  |  Pubmed ID: 17478934

To identify the origin of the marker chromosome in a patient with chromosome aberration, and to provide the precise genetic diagnosis.

SP1 Regulates a Human SNAP-25 Gene Expression

Journal of Neurochemistry. Apr, 2008  |  Pubmed ID: 18194215

The synaptosomal-associated protein of 25 kDa (SNAP-25) is a pre-synaptic plasma membrane protein. SNAP-25 plays an important role in synaptic vesicle membrane docking and fusion, which is involved in the regulation of neurotransmitter release. SNAP-25 has been implicated in the pathogenesis of neuropsychiatric disorders including Schizophrenia, attention-deficit hyperactivity disorder and Alzheimer's disease. We cloned a 1584 bp segment of the 5' flanking region of the human SNAP-25 gene. A series of nested deletions of the 5' flanking region fragment were subcloned into the pGL3-basic luciferase reporter plasmid. N2A cells were transfected with the SNAP-25 promoter constructs and luciferase activity was measured as an indication of promoter activity. We identified a 188 bp fragment containing the transcription initiation site as the minimal region necessary for promoter activity. Several putative cis-acting elements including SP1, hypoxia inducible factor (HIF), cAMP-response element binding protein, T-cell factor/lymphocyte enhancer factor 1 (TCF/LEF1), AP1 and the signal transducer and activator of transcription-6 (STAT6) are found in the 5' flanking region of SNAP-25 gene. Transcriptional activation and gel shift assays showed that the human SNAP-25 gene promoter contains functional SP1 response elements. Over-expression of SP1 increased SNAP-25 gene expression and inhibition of SP1-mediated transcriptional activation reduced SNAP-25 gene expression. These results suggest that SP1 plays an important role in regulation of the human SNAP-25 gene expression.

Efficient Protein Expression from the Endogenous RNA Polymerase I Promoter Using a Human Ribosomal DNA Targeting Vector

Biochemical and Biophysical Research Communications. Mar, 2008  |  Pubmed ID: 18194663

Vector systems to deliver, integrate and express therapeutic genes in host cells are essential for gene therapy. In the present study, we investigated a novel vector system for integration and expression of a transgene. In this system, the transgene expression was driven by an endogenous RNA polymerase I (Pol I) promoter after being integrated into the ribosomal DNA (rDNA) locus. Human coagulation factor IX coding sequence (FIX), with an internal ribosome entry sites element at its leader region, was targeted into the 18S rDNA locus via homologous recombination. FIX protein expression, which was under the control of the endogenous Pol I promoter, was found to be similar to that of a moderate Pol II promoter. The average FIX expression level of the rDNA recombinants was additionally enhanced to that from a strong Pol II promoter as a result of elimination of position effects. Our data suggest the possibility of applying this system in gene therapy for hereditary diseases.

East Learns from West: Asiatic Honeybees Can Understand Dance Language of European Honeybees

PloS One. , 2008  |  Pubmed ID: 18523550

The honeybee waggle dance, through which foragers advertise the existence and location of a food source to their hive mates, is acknowledged as the only known form of symbolic communication in an invertebrate. However, the suggestion, that different species of honeybee might possess distinct 'dialects' of the waggle dance, remains controversial. Furthermore, it remains unclear whether different species of honeybee can learn from and communicate with each other. This study reports experiments using a mixed-species colony that is composed of the Asiatic bee Apis cerana cerana (Acc), and the European bee Apis mellifera ligustica (Aml). Using video recordings made at an observation hive, we first confirm that Acc and Aml have significantly different dance dialects, even when made to forage in identical environments. When reared in the same colony, these two species are able to communicate with each other: Acc foragers could decode the dances of Aml to successfully locate an indicated food source. We believe that this is the first report of successful symbolic communication between two honeybee species; our study hints at the possibility of social learning between the two honeybee species, and at the existence of a learning component in the honeybee dance language.

Estrogen Facilitates Both Phosphatidylinositol 3-kinase/Akt and ERK1/2 Mitogen-activated Protein Kinase Membrane Signaling Required for Long-term Neuropeptide Y Transcriptional Regulation in Clonal, Immortalized Neurons

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jun, 2008  |  Pubmed ID: 18562618

It is established that increases in neuropeptide Y (NPY) expression are associated with hyperphagia and obesity. These effects can be reversed by estrogen, a recognized anorexigen. We found that 17beta-estradiol (E(2)) regulates biphasic NPY gene expression in a clonal, immortalized hypothalamic cell line, N-38, through estrogen receptor (ER) action at the level of the NPY promoter. However, rapid, nongenomic actions of estrogen, linked to the phosphatidylinositol 3-kinase (PI3-K)/Akt and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, may also play a role. We therefore examined the changes in the phosphorylation status of Akt, ERK1/2, and cAMP response element-binding protein (CREB) after treatment with 10 nm E(2) in the N-38 neurons and found activation of these signaling proteins within 5-30 min. We also demonstrated possible cross talk between the estrogen-activated PI3-K/Akt and MAPK/extracellular signal-regulated kinase pathways using pharmacological inhibitors. We find that only ERalpha is involved in the early signaling events using the ERalpha agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. Furthermore, we can detect colocalization of ERalpha and caveolin-1, a membrane-associated signaling protein. Remarkably, we find that the membrane-mediated events are critical for the long-term estrogen-mediated repression of NPY gene expression that can be mapped to within -97 bp of the NPY promoter. To link the early signaling events to downstream effectors, we detected induction of c-fos and inactivation of MSK-1 by estrogen and binding of CREB to this minimal promoter region. These observations suggest that rapid estrogen-mediated signaling is mediated by ERalpha, and the signal transduction events potentiate the genomic actions of estrogen on NPY gene expression in the N-38 NPY neurons.

Activation of High Conductance Ca(2+)-activated K(+) Channels by Sodium TanshinoneII-A Sulfonate (DS-201) in Porcine Coronary Artery Smooth Muscle Cells

European Journal of Pharmacology. Nov, 2008  |  Pubmed ID: 18831973

High conductance Ca(2+) activated K(+) channels (BK(Ca)) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca(2+) influx through voltage gated Ca(2+) channels. Agents that alter the activity of Ca(2+) channels or BK(Ca) thus affect the vascular tone in both physiological and pathological conditions. Danshen, the dried root of Salvia miltiorrhiza, is a commonly used traditional Chinese medicine and is widely used as an effective remedy for cardiovascular and cerebral vascular diseases partly by its vasodilatation. Sodium tanshinoneII-A sulfonate (DS-201) is a water-soluble derivative of Tanshinone IIA, the main active component of Danshen. The purpose of this study was to explore possible mechanisms of vasodilative effects of DS-201 using porcine coronary artery smooth muscle. DS-201 induced relaxation of the coronary smooth muscle which had been contracted with 30 mM KCl, and the relaxation was inhibited by 100 nM iberiotoxin (IbTX), a specific BK(Ca) channel blocker. Using perforated whole-cell recordings and single channel recordings, effects of DS-201 on BK(Ca) were examined. The results showed that DS-201 activated BK(Ca). Extracellular application of DS-201 at 40, 80 microM under the whole-cell configuration induced increases of the BK(Ca) macroscopic currents by 43.6%, 42.1% respectively, and the spontaneous transient outward K(+) currents (STOCs) by 48.7%, 47.4% respectively. In inside-out patches, bath application of 20-150 muM of DS-201 activated BK(Ca) by 5.4-173.2 fold. These results indicate that the vasodilatation by DS-201 is related to activation of BK(Ca).

Oncogenicity of DNA in Vivo: Tumor Induction with Expression Plasmids for Activated H-ras and C-myc

Biologicals : Journal of the International Association of Biological Standardization. May, 2008  |  Pubmed ID: 18218323

All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.

[UTP Regulates Spontaneous Transient Outward Currents in Porcine Coronary Artery Smooth Muscle Cells Through PLC-IP(3) Signaling Pathway.]

Sheng Li Xue Bao : [Acta Physiologica Sinica]. Feb, 2008  |  Pubmed ID: 18288360

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm x 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 degrees C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54 +/- 5.34)% and in frequency by (77.46 +/- 8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 mumol/L) remarkably reduced STOC amplitude by (31.04 +/- 7.46)% and frequency by (41.65 +/- 16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 mumol/L) and CdCl2 (200 mumol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 mumol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44 +/- 24.66)% and frequency by (61.35 +/- 21.47)% (P<0.01, n=12); UTP (40 mumol/L), applied in the presence of 1 mumol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 mumol/L) abolished STOC activity. (5) In the presence of UTP (40 mumol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 mumol/L) reduced STOC amplitude by (24.08 +/- 3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 mumol/L) significantly reduced STOC amplitude by (31.43 +/- 6.34)% and frequency by (40.59 +/- 19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 mumol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 mumol/L) or ryanodine (50 mumol/L), UTP (40 mumol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

Valproic Acid Inhibits Abeta Production, Neuritic Plaque Formation, and Behavioral Deficits in Alzheimer's Disease Mouse Models

The Journal of Experimental Medicine. Nov, 2008  |  Pubmed ID: 18955571

Neuritic plaques in the brains are one of the pathological hallmarks of Alzheimer's disease (AD). Amyloid beta-protein (Abeta), the central component of neuritic plaques, is derived from beta-amyloid precursor protein (APP) after beta- and gamma-secretase cleavage. The molecular mechanism underlying the pathogenesis of AD is not yet well defined, and there has been no effective treatment for AD. Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for treating epilepsy and bipolar disorder. We found that VPA decreased Abeta production by inhibiting GSK-3beta-mediated gamma-secretase cleavage of APP both in vitro and in vivo. VPA treatment significantly reduced neuritic plaque formation and improved memory deficits in transgenic AD model mice. We also found that early application of VPA was important for alleviating memory deficits of AD model mice. Our study suggests that VPA may be beneficial in the prevention and treatment of AD.

Structure of SF-1 Bound by Different Phospholipids: Evidence for Regulatory Ligands

Molecular Endocrinology (Baltimore, Md.). Jan, 2009  |  Pubmed ID: 18988706

Despite the fact that many nuclear receptors are ligand dependent, the existence of obligate regulatory ligands is debated for some receptors, including steroidogenic factor 1 (SF-1). Although fortuitously bound bacterial phospholipids were discovered in the structures of the SF-1 ligand-binding domain (LBD), these lipids might serve merely as structural ligands. Thus, we examined whether exogenously added phospholipids would exchange for these bacterial lipids and bind to SF-1. Here, we report the first crystal structure of the SF-1 LBD bound by the exchanged phosphatidylcholine. Although the bound phosphatidylcholine phospholipid mimics the conformation of bound bacterial phosphoplipids, two surface loops, L2-3 and L11-12, surrounding the entrance to the pocket vary significantly between different SF-1 LBD structures. Based on this observation, we hypothesized that a bound ligand might control the conformations of loops L2-3 and L11-12, and that conserved residues in these dynamic loops could influence ligand binding and the receptor function. Consistent with this hypothesis, impaired phospholipid exchange and diminished transcriptional activity were observed for loop L11-12 SF-1 mutants and for the loop L2-3 human mutant R255L. The endocrine disease associated with this L2-3 mutation coupled with our cellular and biochemical data suggest that critical residues at the mouth of the ligand-binding pocket have evolved for efficient binding of phospholipid ligands and for achieving optimal SF-1 activity.

Intestinal Trefoil Factor Maybe Useful in Prophylaxis of Acute Graft-versus-host Disease

Medical Hypotheses. Oct, 2009  |  Pubmed ID: 19564083

Graft-versus-host disease (GVHD) is a major complication of hematopoietic cell transplantation, GVHD pathophysiology can be divided into three phases, damage to the gastrointestinal (GI) tract in phase 1, principally by inflammatory cytokines, amplifies LPS release and leads to the "cytokine storm" characteristic of severe acute GVHD. It has been proved that disruption of phase 1 of the GVHD cascade is effective in prophylaxis of acute GVHD. Intestinal trefoil factor (ITF), a member of trefoil factor family (TFF) domain peptides, was proved to be very effective in prevention and healing of acute dextran sodium sulfate-induced colitis, and was also involved in protection against and recovery from intestinal mucositis induced by radiation and chemotherapy. So we hypothesise that ITF protects the intestinal tract mucosa from lesions and that it maybe useful in prophylaxis of acute GVHD. ITF can block GI tract damage in phase 1, preventing the amplification of the cascade. ITF may represent a novel strategy for the separation of GVHD and graft-versus-leukemia (GVL), and may serve as an effective adjunct to clinical regimens of GVHD prophylaxis.

[Identification and Analysis of Genuine and False Flos Rosae Rugosae by FTIR and 2D Correlation IR Spectroscopy]

Guang Pu Xue Yu Guang Pu Fen Xi = Guang Pu. Sep, 2009  |  Pubmed ID: 19950645

The genuine and false Flos Rosae Rugosae (Flos Rosae Chinensis and Flos Rosa multiflora) were examined in terms of their differences by using Fourier transform infrared spectroscopy (FTIR) combined with two-dimensional (2D) correlation IR spectroscopy. The three species were shown very similar in FTIR spectra. The peak of 1318 cm(-1) of genuine Flos Rosae Rugosae is not obvious but this peak could be found sharp in Flos Rosae Chinensis and Flos Rosa multiflora. Generally, the second derivative IR spectrum can clearly enhance the spectral resolution. Flos Rosae Rugosae and Flos rosae Chinensis have aromatic compounds distinct fingerprint characteristics at 1 617 and 1 618 cm(-1), respectively. Nevertheless, FlosRosa multiflora has the peak at 1612 cm(-1). There is a discrepancy of 5 to 6 cm(-1). FlosRosa multiflora has glucide's distinct fingerprint characteristics at 1 044 cm(-1), but Flos Rosae Rugosae and Flos Rosae Chinensis don't. The second derivative infrared spectra indicated different fingerprint characteristics. Three of them showed aromatic compounds with autopeaks at 1620, 1560 and 1460 cm(-1). Flos Rosae Chinensis and Flos Rosa multiflora have the shoulder peak at 1660 cm(-1). In the range of 850-1250 cm(-1), three of them are distinct different, Flos Rosae Rugosae has the strongest autopeak, Flos Rosae Chinensis has the feeble autopeak and Flos Rosa multiflora has no autopeak at 1050 cm(-1). In third-step identification, the different contents of aromatic compounds and glucide in Flos Rosae Rugosae, Flos Rosae Chinensis and Flos Rosa multiflora were revealed. It is proved that the method is fast and effective for distinguishing and analyzing genuine Flos Rosae Rugosae and false Flos Rosae Rugosae (Flos Rosae Chinensis and Flos Rosa multiflora).

Tumors Induced in Mice by Direct Inoculation of Plasmid DNA Expressing Both Activated H-ras and C-myc

International Journal of Biological Sciences. , 2010  |  Pubmed ID: 20376206

Vaccines contain residual DNA derived from the cells used to produce them. As part of our investigation to assess the risk of this cellular DNA, we are developing a quantitative in vivo assay to assess the oncogenicity of DNA. In an earlier study, we had generated expression plasmids for two oncogenes--human activated T24-H-ras and murine c-myc--and had shown that these two plasmids, pMSV-T24-H-ras and pMSV-c-myc, could act in concert to induce tumors in mice, although the efficiency was low. In this study, we took two approaches to increase the oncogenic efficiency: 1) both oncogene-expression cassettes were placed on the same plasmid; 2) transfection facilitators, which increase DNA uptake and expression in vitro, were tested. The dual-expression plasmid, pMSV-T24-H-ras/MSV-c-myc, is about 20-fold more efficient at tumor induction in newborn NIH Swiss mice than the separate expression plasmids, with tumors being induced with 1 microg of the dual-expression plasmid DNA. However, none of the transfection facilitators tested increased the efficiency of tumor induction. Based on these data, the dual-expression plasmid pMSV-T24-H-ras/MSV-c-myc will be used as the positive control to develop a sensitive and quantitative animal assay that can be used to assess the oncogenic activity of DNA.

BACE1 Gene Promoter Single-nucleotide Polymorphisms in Alzheimer's Disease

Journal of Molecular Neuroscience : MN. Sep, 2010  |  Pubmed ID: 20455082

Alzheimer's disease (AD) is the most neurodegenerative disorder leading to dementia. Neuritic plaque formation in brains is a hallmark of AD pathogenesis. Amyloid beta protein (Abeta) is the central component of neuritic plaques. Processing beta-amyloid precursor protein (APP) at the beta-secretase site by the beta-site APP cleaving enzyme 1 (BACE1) is essential for generation of Abeta. Elevation of BACE1 activity and expression has been reported in AD brains. However, no mutation in the BACE1 coding sequence has been identified in AD cases. Human BACE1 expression is tightly regulated at the transcription and translation level. To determine whether there is any single-nucleotide polymorphisms in the BACE1 gene promoter region affecting BACE1 expression in AD pathogenesis, in this study, we screened 2.6 kb of the human BACE1 gene promoter region from late-onset AD patients and found that there was no significant association between single-nucleotide polymorphisms and AD cases.

NF-κB Signaling Inhibits Ubiquitin Carboxyl-terminal Hydrolase L1 Gene Expression

Journal of Neurochemistry. Mar, 2011  |  Pubmed ID: 21210816

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that plays a regulatory role in targeting proteins for proteasomal degradation. UCH-L1 is highly expressed in neurons and has been demonstrated to promote cell viability and maintain neuronal integrity. Reduced UCH-L1 levels have been observed in various neurodegenerative diseases, and expression of UCH-L1 can rescue synaptic dysfunction and memory deficits in Alzheimer's Disease model mice. However, the mechanisms regulating UCH-L1 expression have not been determined. In this study, we cloned a 1782 bp of the 5' flanking region of the human UCH-L1 gene and identified a 43 bp fragment containing the transcription start site as the minimal region necessary for promoter activity. Sequence analysis revealed several putative regulatory elements including NF-κB, NFAT, CREB, NRSF, YY1, AP1, and STAT in the UCH-L1 promoter. A functional NF-κB response element was identified in the UCH-L1 promoter region. Expression of NF-κB suppressed UCH-L1 gene transcription. In the RelA knockout system where NF-κB activity is ablated, UCH-L1 expression was significantly increased. Furthermore, activation of NF-κB signaling by the inflammatory stimulator lipopolysaccharide and TNFα resulted in a decrease of UCH-L1 gene expression by inhibiting its transcription. As NF-κB is an important signaling module in inflammatory response, our study suggests a possibility that inflammation might compromise neuronal functions via the interaction of NF-κB and UCH-L1. A better understanding of the NF-κB-regulated UCH-L1 transcription will provide insights to the role of inflammatory responses in Alzheimer's disease and Parkinson's disease.

Increased NF-κB Signalling Up-regulates BACE1 Expression and Its Therapeutic Potential in Alzheimer's Disease

The International Journal of Neuropsychopharmacology / Official Scientific Journal of the Collegium Internationale Neuropsychopharmacologicum (CINP). Feb, 2011  |  Pubmed ID: 21329555

Elevated levels of β-site APP cleaving enzyme 1 (BACE1) were found in the brain of some sporadic Alzheimer's disease (AD) patients; however, the underlying mechanism is unknown. BACE1 cleaves β-amyloid precursor protein (APP) to generate amyloid β protein (Aβ), a central component of neuritic plaques in AD brains. Nuclear factor-kappa B (NF-κB) signalling plays an important role in gene regulation and is implicated in inflammation, oxidative stress and apoptosis. In this report we found that both BACE1 and NF-κB p65 levels were significantly increased in the brains of AD patients. Two functional NF-κB-binding elements were identified in the human BACE1 promoter region. We found that NF-κB p65 expression resulted in increased BACE1 promoter activity and BACE1 transcription, while disruption of NF-κB p65 decreased BACE1 gene expression in p65 knockout (RelA-knockout) cells. In addition, NF-κB p65 expression leads to up-regulated β-secretase cleavage and Aβ production, while non-steroidal anti-inflammatory drugs (NSAIDs) inhibited BACE1 transcriptional activation induced by strong NF-κB activator tumour necrosis factor-alpha (TNF-α). Taken together, our results clearly demonstrate that NF-κB signalling facilitates BACE1 gene expression and APP processing, and increased BACE1 expression mediated by NF-κB signalling in the brain could be one of the novel molecular mechanisms underlying the development of AD in some sporadic cases. Furthermore, NSAIDs could block the inflammation-induced BACE1 transcription and Aβ production. Our study suggests that inhibition of NF-κB-mediated BACE1 expression may be a valuable drug target for AD therapy.

Transcriptional Regulation of TMP21 by NFAT

Molecular Neurodegeneration. , 2011  |  Pubmed ID: 21375783

TMP21 is a member of the p24 cargo protein family, which is involved in protein transport between the Golgi apparatus and ER. Alzheimer's Disease (AD) is the most common neurodegenerative disorder leading to dementia and deposition of amyloid β protein (Aβ) is the pathological feature of AD pathogenesis. Knockdown of TMP21 expression by siRNA causes a sharp increase in Aβ production; however the underlying mechanism by which TMP21 regulates Aβ generation is unknown, and human TMP21 gene expression regulation has not yet been studied.

Small Molecule Agonists of the Orphan Nuclear Receptors Steroidogenic Factor-1 (SF-1, NR5A1) and Liver Receptor Homologue-1 (LRH-1, NR5A2)

Journal of Medicinal Chemistry. Apr, 2011  |  Pubmed ID: 21391689

The crystal structure of LRH-1 ligand binding domain bound to our previously reported agonist 3-(E-oct-4-en-4-yl)-1-phenylamino-2-phenyl-cis-bicyclo[3.3.0]oct-2-ene 5 is described. Two new classes of agonists in which the bridgehead anilino group from our first series was replaced with an alkoxy or 1-ethenyl group were designed, synthesized, and tested for activity in a peptide recruitment assay. Both new classes gave very active compounds, particularly against SF-1. Structure-activity studies led to excellent dual-LRH-1/SF-1 agonists (e.g., RJW100) as well as compounds selective for LRH-1 (RJW101) and SF-1 (RJW102 and RJW103). The series based on 1-ethenyl substitution was acid stable, overcoming a significant drawback of our original bridgehead anilino-substituted series. Initial studies on the regulation of gene expression in human cell lines showed excellent, reproducible activity at endogenous target genes.

Relationship Between the GTPase, Metal-binding, and Dimerization Activities of E. Coli HypB

Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry. Aug, 2011  |  Pubmed ID: 21544686

Biosynthesis of the metallocenter in the active site of the [NiFe] hydrogenase enzyme requires the accessory protein HypB, which is a metal-binding GTPase. In this study, the interplay between the individual activities of Escherichia coli HypB was examined. The full-length protein undergoes nucleotide-responsive dimerization that is disrupted upon mutation of L242 and L246 to alanine. This mutant HypB is monomeric under all of the conditions investigated but the inability of L242A/L246A HypB to dimerize does not abolish its GTPase activity and the monomeric protein has metal-binding behavior similar to that of wild-type HypB. Furthermore, expression of L242A/L246A HypB in vivo results in hydrogenase activity that is approximately half of the activity produced by the wild-type control, suggesting that dimerization of HypB does not have a critical role in the hydrogenase maturation pathway. In contrast, the GTPase activity of HypB is modulated by metal loading of the protein. These results provide insight into the role of HypB in hydrogenase biosynthesis.

Formalin-inactivated Vaccine Provokes Cross-protective Immunity in a Mouse Model of Human Enterovirus 71 Infection

Vaccine. Jun, 2011  |  Pubmed ID: 21550375

Human enterovirus 71 (HEV71) has emerged as a major cause of epidemics of hand, foot and mouth disease associated with severe neurological sequelae in the Asia-Pacific region. In this study, a passive protection mouse model was used to evaluate the protective efficacy of formalin-inactivated HEV71 vaccines derived from a Chinese C4 genotype strain. Pregnant mice were immunised using a prime/boost strategy and ≥50U of vaccine protected five-day-old pups from lethal challenge with a mouse-adapted (B3 genotype) strain of HEV71. Immunised mice developed a neutralising antibody response to both the immunising C4 strain and to the mouse-adapted strain. Mice born to immunised dams showed significantly less myositis and reduced viral loads in tissues.

Eliminating SF-1 (NR5A1) Sumoylation in Vivo Results in Ectopic Hedgehog Signaling and Disruption of Endocrine Development

Developmental Cell. Aug, 2011  |  Pubmed ID: 21820362

Sumoylation is generally considered a repressive mark for many transcription factors. However, the in vivo importance of sumoylation for any given substrate remains unclear and is questionable because the extent of sumoylation appears exceedingly low for most substrates. Here, we permanently eliminated SF-1/NR5A1 sumoylation in mice (Sf-1(K119R, K194R, or 2KR)) and found that Sf-1(2KR/2KR) mice failed to phenocopy a simple gain of SF-1 function or show elevated levels of well-established SF-1 target genes. Instead, mutant mice exhibited marked endocrine abnormalities and changes in cell fate that reflected an inappropriate activation of hedgehog signaling and other potential SUMO-sensitive targets. Furthermore, unsumoylatable SF-1 mutants activated Shh and exhibited preferential recruitment to Shh genomic elements in cells. We conclude that the sumoylation cycle greatly expands the functional capacity of transcription factors such as SF-1 and is leveraged during development to achieve cell-type-specific gene expression in multicellular organisms.

Control of BACE1 Degradation and APP Processing by Ubiquitin Carboxyl-terminal Hydrolase L1

Journal of Neurochemistry. Dec, 2011  |  Pubmed ID: 22212137

J. Neurochem. (2012) 10.1111/j.1471-4159.2012.07644.x ABSTRACT: Deposition of amyloid β protein (Aβ) in the brain is the hallmark of Alzheimer's disease (AD) pathogenesis. Beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is the β-secretase in vivo essential for generation of Aβ. Previously we demonstrated that BACE1 is ubiquitinated and the degradation of BACE1 is mediated by the ubiquitin-proteasome pathway (UPP). However the mechanism underlying regulation of BACE1 degradation by UPP remains elusive. Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme highly specific to neuron, catalyzing the hydrolysis of ubiquitin conjugates from ubiquitinated substrates. UCHL1 regulates ubiquitin-dependent protein degradation. However, whether UCHL1 is particularly involved in the proteasomal degradation of BACE1 and what is the role of UCHL1 in AD pathogenesis remain elusive. To investigate the effect of UCHL1 on BACE1 degradation, HUCH cells, a UCHL1 stably over-expressed HEK293 cell line, was established. We found that inhibition of UCHL1 significantly increased BACE1 protein level in a time-dependent manner. Half life of BACE1 was reduced in HUCH cells compared with HEK. Over-expression of UCHL1 decreased APP C-terminal fragment C99 and Aβ levels in HUCH cells. Moreover, disruption of Uchl1 gene significantly elevated levels of endogenous BACE1, C99 and Aβ in the Uchl1-null gad mice. These results demonstrated that UCHL1 accelerates BACE1 degradation and affects APP processing and Aβ production. This study suggests that potentiation of UCHL1 might be able to reduce the level of BACE1 and Aβ in brain, which makes it a novel target for AD drug development.

Role of Polyunsaturated Fatty Acids and Lipid Peroxidation on Colorectal Cancer Risk and Treatments

Current Opinion in Clinical Nutrition and Metabolic Care. Mar, 2012  |  Pubmed ID: 22234166

The review aims at elucidating the role of lipid peroxidation of polyunsaturated fatty acids (PUFAs) in colorectal cancer (CRC) risk and treatment.

Hypoxia Regulation of ATP13A2 (PARK9) Gene Transcription

Journal of Neurochemistry. Jan, 2012  |  Pubmed ID: 22288903

Parkinson's disease (PD) is the second most common neurodegenerative disorders with a variable combination of motor and non-motor symptoms. Mutations in several genes including ATP13A2 (PARK9) are reported to be associated with PD. The underlying mechanism of PD is not well defined, however, both genetic and environmental causes contribute to it. ATP13A2 gene locates in chromosome 1 and contains 29 exons encoding for a protein of 1180 amino acids with 10 transmembrane domains. Abnormal gene expression has been implicated in neurodegenerative disorders. The transcriptional regulation of the ATP13A2 gene is unknown. In this report we cloned and functionally characterized the human ATP13A2 gene promoter. We showed that the promoter region of the human ATP13A2 gene contains hypoxia response elements (HREs) which can bind to transcription factor hypoxia-inducible factor 1α (HIF-1α). Hypoxia upregulated ATP13A2 transcription via HIF-1α in HEK293 and dopaminergic MN9D cells. Our study indicates that hypoxia signaling plays a very important role in the regulation of human ATP13A2 gene expression. Further study is needed to determine the role of hypoxia in the pathogenesis of PD and its interaction with other PD causative genes, which will provide insights to the role of hypoxia and dysregulation of gene expression in Parkinson's disease. © 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.

Combination of Fucoxanthin and Conjugated Linoleic Acid Attenuates Body Weight Gain and Improves Lipid Metabolism in High-fat Diet-induced Obese Rats

Archives of Biochemistry and Biophysics. Jan, 2012  |  Pubmed ID: 22289788

The present study investigated the effects of combined fucoxanthin (Fc) and conjugated linoleic acid (CLA) on high-fat diet-induced obese rats. Thirty five rats were divided into four groups, fed a high-fat diet (Control, 15% fat, wt/wt), supplemented with low Fc (FCL, 0.083mg/kg/bw), high Fc (FCH, 0.167mg/kg/bw) and FCL (0.083mg/kg/bw) plus CLA (0.15g/kg/bw) (FCL+CLA) for 52d. Body weight and white adipose tissue (WAT) weight were significantly suppressed in FCL+CLA group than those in control group. WAT weight was also markedly attenuated in FCL and FCH groups. Accumulation of hepatic lipid droplets and the perirenal adipocyte size of FCL, FCH and FCL+CLA groups were diminished compared to control group. Serum total cholesterol level in FCH group, triacylglycerol and leptin levels in FCL, FCH and FCL+CLA groups, and glucose concentration in FCH and FCL+CLA groups were significantly decreased than those in control group. The mRNA expression of adiponectin, adipose triacylglycerol lipase, carnitine palmitoyltransferase 1A was remarkably up-regulated in FCL, FCH and FCL+CLA groups. These results suggest that Fc and FCL+CLA could reduce serum levels of triacylglycerol, glucose and leptin, and FCL+CLA could exert anti-obesity effects by regulating mRNA expression of enzymes related to lipid metabolism in WAT of diet-induced obesity rats.