Preparations utilizing monoclonal antibodies against S100A4 provide useful tools for functional studies to investigate the clinical applications of the human S100A4 protein. In the present study, human S100A4 protein was expressed in Escherichia coli (E. coli) BL21 (DE3), successfully purified by diethylaminoethyl cellulose anion-exchange chromatography and identified by western blot analysis. Soluble S100A4 bioactivity was confirmed by Transwell migration and invasion assays in the human HeLa cell line. Monoclonal antibodies (mAbs) were generated utilizing the standard hybridoma method and were validated by enzyme-linked immunosorbent assay and western blot analysis. The antibody was then used to examine human gastric carcinoma specimens by immunohistochemistry. Recombinant S100A4 was functionally expressed in E. coli and promoted the migration and invasion of HeLa cells. Four hybridoma cell lines, which secreted mAbs specifically against human S100A4 protein, were obtained. One of the four mAbs, namely 2A12D10B2, recognized human S100A4 as indicated by immunohistochemical staining of human gastric carcinoma specimens and recombinant S100A4 was functionally expressed in E. coli. The mAbs of recombinant S100A4 were suitable for detecting S100A4 expression in human tissues and for investigating the subsequent clinical applications of the protein.
Quantum dots (QDs) have attracted great attention in recent years due to their promising applications in bioimaging. Compared with traditional ultraviolet excitation of QDs, near-infrared laser (NIR) excitation has many advantages, such as being less harmful, little blinking effects, zero autofluorescence and deep penetration in tissue. Composing QDs with upconverting properties is promising to enable NIR excitation. This article provides a review of QDs with upconverting luminescence and their applications in bioimaging. Based on the mechanisms of luminescence, discussion will be divided into four groups: nanoheterostructures/mixtures of QDs and upconverting nanoparticles, graphene quantum dots, lanthanide-doped QDs, and double QDs. The content includes synthetic routes, upconverting luminescence properties, and their applications in bioimaging.
There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) (13) C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously.
S100A4 protein is associated with Ca(2+)-dependent regulation of intracellular activities and is significant in the invasion, growth and metastasis of cancer. In order to express rat S100A4 functionally and identify its biological activity following purification, an S100A4 gene fragment was optimized and fully synthesized via overlapping polymerase chain reaction. The gene was inserted into the prokaryotic expression vector, pBV220, with phage ? PRPL promoters following confirmation by DNA sequencing. The pBV220-S100A4 plasmid was constructed and transformed into Escherichia coli DH5?. Following temperature induction, rat S100A4 was overexpressed and the protein was observed to be located in the supernatant of the lysates, which was ~30-40% of the total protein within the host. The protein was isolated and purified by metal-chelate affinity chromatography. High purity protein (>98% purity) was obtained and in vitro western blot analysis identified that the recombinant S100A4 was able to bind to the antibody against wild-type S100A4. The bioactivity of the recombinant protein was detected via Transwell migration and invasion assays. The polyclonal antibody of rat S100A4 protein was prepared for rabbit immunization and exhibited similar efficacies when compared with commercial S100A4. Therefore, rat S100A4 was functionally expressed in E. coli; thus, the production of active recombinant S100A4 protein in E. coli may further aid with the investigation and application of S100A4.
Caffeic acid phenethyl ester (CAPE), the major bioactive component of honeybee propolis, is a potent selective inhibitor of aldo-keto reductase family member 1B10 (AKR1B10), and a number of derivatives hold promise as potential anticancer agents. However, sequence homology between AKR1B10 and other members of the superfamily, including critical phase I metabolizing enzymes, has resulted in a concern over the selectivity of any potential therapeutic agent. To elucidate the binding mode of CAPE with AKR1B10 and to provide a tool for future in silico efforts towards identifying selective inhibitors, the crystal structure of AKR1B10 in complex with CAPE was determined. The observed interactions provide an explanation for the selectivity exhibited by CAPE for AKR1B10, and could be used to guide further derivative design.
Natural hybridization can lead to various evolutionary outcomes in plants, including hybrid speciation and interspecific gene transfer. It can also cause taxonomic problems, especially in plant genera containing multiple species. In this study, the hybrid status of Melastoma affine, the most widespread taxon in this genus, and introgression between its putative parental species, M. candidum and M. sanguineum, were assessed on two sites, Hainan and Guangdong, using 13 SSR markers and sequences of a chloroplast intergenic spacer. Bayesian-based STRUCTURE analysis detected two most likely distinct clusters for the three taxa, and 76.0% and 73.9% of the morphologically identified individuals of M. candidum and M. sanguineum were correctly assigned, respectively. 74.5% of the M. affine individuals had a membership coefficient to either parental species between 0.1 and 0.9, suggesting admixture between M. candidum and M. sanguineum. Furthermore, NewHybrids analysis suggested that most individuals of M. affine were F2 hybrids or backcross hybrids to M. candidum, and that there was extensive introgression between M. candidum and M. sanguineum. These SSR data thus provides convincing evidence for hybrid origin of M. affine and extensive introgression between M. candidum and M. sanguineum. Chloroplast DNA results were consistent with this conclusion. Much higher hybrid frequency on the more disturbed Guangdong site suggests that human disturbance might offer suitable habitats for the survival of hybrids, a hypothesis that is in need of further testing.
Ionizing radiation (IR) cytotoxicity is primarily mediated through reactive oxygen species (ROS). Since tumor cells neutralize ROS by utilizing reducing equivalents, we hypothesized that measurements of reducing potential using real-time hyperpolarized (HP) magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) can serve as a surrogate marker of IR induced ROS. This hypothesis was tested in a pre-clinical model of anaplastic thyroid carcinoma (ATC), an aggressive head and neck malignancy. Human ATC cell lines were utilized to test IR effects on ROS and reducing potential in vitro and [1-¹³C] pyruvate HP-MRS/MRSI imaging of ATC orthotopic xenografts was used to study in vivo effects of IR. IR increased ATC intra-cellular ROS levels resulting in a corresponding decrease in reducing equivalent levels. Exogenous manipulation of cellular ROS and reducing equivalent levels altered ATC radiosensitivity in a predictable manner. Irradiation of ATC xenografts resulted in an acute drop in reducing potential measured using HP-MRS, reflecting the shunting of reducing equivalents towards ROS neutralization. Residual tumor tissue post irradiation demonstrated heterogeneous viability. We have adapted HP-MRS/MRSI to non-invasively measure IR mediated changes in tumor reducing potential in real time. Continued development of this technology could facilitate the development of an adaptive clinical algorithm based on real-time adjustments in IR dose and dose mapping.
Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.
The antineoplastic target aldo-keto reductase family member 1B10 (AKR1B10) and the critical polyol pathway enzyme aldose reductase (AKR1B1) share high structural similarity. Crystal structures reported here reveal a surprising Trp112 native conformation stabilized by a specific Gln114-centered hydrogen bond network in the AKR1B10 holoenzyme, and suggest that AKR1B1 inhibitors could retain their binding affinities toward AKR1B10 by inducing Trp112 flip to result in an "AKR1B1-like" active site in AKR1B10, while selective AKR1B10 inhibitors can take advantage of the broader active site of AKR1B10 provided by the native Trp112 side-chain orientation.
The complete mitogenome was first determined for a minnow Pseudohemiculter dispar in this study. It is 16,621 bp in length, including 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and 1 control region. Overall nucleotide base composition is 30.30% A, 27.10% C, 17.10% G, and 25.50% T, with a A+T bias. Except for the tRNA-Ser2 that lost the dihydrouridine arm and replaced with one 11-bp loop, the other 22 tRNA genes can fold into the typical cloverleaf secondary structures. The origin of light-strand replication (OL) was found between the tRNA-Asn and tRNA-Cys genes, while the termination-associated sequence (TAS) was found in the control region. Both OL and TAS can form the hairpin structure that acts as the signals participated in the mitochondrial DNA replication.
The aim of this study was to determine the correlation of S100A4 expression with the progression, prognosis and clinical pathology of gastric cancer (GC) in young pateints. A total of 85 tumor tissues with corresponding adjacent normal tissues and 62 non-metastatic lymph nodes (LNs) with corresponding metastatic LNs were obtained from young GC patients (<40 years old) who underwent surgery between January 2001 and December 2006. The expression of S100A4 was detected by RT-PCR and immunohistochemistry. Differences in the expression of S100A4 mRNA or protein were observed among the GC tissues, matched normal gastric mucosa, non-metastatic LNs and metastatic LNs. The expression of S100A4 mRNA and protein in GC tissues and metastatic LNs was significantly higher compared with that in the matched normal gastric mucosa and non-metastatic LNs, respectively (P<0.05). The overexpression of S100A4 was significantly associated with parameters involved in tumor progression and poor prognosis, including tumor size (P=0.017), Lauren classification (P=0.002), histological classification (P= 0.010), histological differentiation (P= 0.000), Borrmann classification (P=0.020), tumor-node-metastasis (TNM) stage (P=0.000), LN metastasis (P=0.000) and distant metastasis (P=0.024). Multivariate analysis suggested that patient age (P=0.035), tumor size (P=0.002), TNM stage (P=0.001) and S100A4 upregulation (P=0.000) were independent prognostic indicators for the disease. The overexpression of S100A4 in young GC patients is significantly associated with the clinicopathological characteristics. S100A4 may be used as a biomarker to predict the progression and poor prognosis of GC in young patients.
A series of arene ruthenium(II) complexes coordinated by phenanthroimidazole derivates, [(C6H6)Ru(L)Cl]Cl·2H2O (1b L = IP, 2b L = p-NMe2PIP, 3b L = p-MeOPIP, 4b L = p-HOPIP, 5b L = p-COOHPIP, 6b L = p-CF3PIP, 7b L = p-BrPIP) have been synthesized in yields of 89-92% under microwave irradiation in 30 min, and the crystal structure of 1b by XRD gives a typical "piano stool" conformation. The antitumor activity of these complexes against various tumor cells have been evaluated by MTT assay, and the results show that this type of arene Ru(II) complexes exhibit acceptable inhibitory effect against all of these tumor cells, especially osteosarcoma MG-63 cells, but with low toxicity toward HK-2 human normal cells. Studies on the mechanism revealed that cell cycle arrest at S-phase in MG-63 cells induced by the arene Ru(II) complex 2b, which was confirmed by the increase in the percentage of cells at S-phase and down-regulator of cyclin A. The further studies by Comet assay at single cell level indicated that DNA damage in MG-63 cells was triggered by 2b, following with the up-regulation of phosphorylated p53 and histone. The studies by spectroscopy in vitro also indicate that 2b bind to DNA molecule by intercalative mode to disturb the bio-function of tumor cells. In conclusion, the synthetic arene Ru(II) complexes could serve as novel p53 activator with potential application in cancer chemotherapy.
The complete mitochondrial genome of Sinibrama macrops was first determined in this study. The genome is 16,626 bp in length, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and 1 control region. Its gene arrangement and translation direction were identical to those of other typical vertebrates. The overall base composition is 30.7% A, 27.9% C, 16.7% G and 24.7% T, with the negative GC skew value ( - 0.251). In a long term, S. macrops was confused with Sinibrama wui, which was confirmed as an invalid species by recent morphological study. Therefore, the mitochondrial genome of S. macrops can contribute to the molecular identification of the species and further phylogenetic study of Cyprinidae.
We determined the complete mitochondrial genome (mitogenome) of Acrossocheilus wenchowensis. It is the first report of mitogenome in genus Acrossocheilus. The total length of A. wenchowensis is 16,591 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 transfer RNA genes and a A+T rich control region. The gene order and transcriptional orientation are similar to most vertebrates. The overall base composition is 31.00% A, 27.70% C, 16.30% G and 25.00% T. The termination-associated sequence and conserved sequence blocks were identified in the control region.
Sulindac (SLD) exhibits both the highest inhibitory activity towards human aldose reductase (AR) among popular non-steroidal anti-inflammatory drugs and clear beneficial clinical effects on Type 2 diabetes. However, the molecular basis for these properties is unclear. Here, we report that SLD and its pharmacologically active/inactive metabolites, SLD sulfide and SLD sulfone, are equally effective as un-competitive inhibitors of AR in vitro. Crystallographic analysis reveals that ?-? stacking favored by the distinct scaffold of SLDs is pivotal to their high AR inhibitory activities. These results also suggest that SLD sulfone could be a potent lead compound for AR inhibition in vivo.
Four new dammarane-type saponins, operculinosides A-D (1-4), were isolated from the aerial parts of Operculina turpethum, of which 1 and 2 are the first two dammarane-type triterpenoids having an oxymethyl group at C-24. Their structures were determined by spectroscopic analysis and acid hydrolysis. The absolute configuration of operculinoside A (1) was confirmed by X-ray crystallography. Compounds 1 and 3 showed significant protective activities against d-galactosamine-induced toxicity in L-02 human hepatic cells.
RPB5 is an essential subunit of eukaryotic RNA polymerase II. It has been proposed to interact with DNA and several key transcription factors during transcription. These interactions are crucial for transcription and its regulation. Here, prior to obtaining complex structures of human RPB5 and its binding partners, recombinant human RPB5 was crystallized alone by vapour diffusion in hanging drops. A complete data set was collected from a single frozen crystal employing an in-house X-ray source. The crystal diffracted to 2.8 Å resolution and belonged to space group P4(3)2(1)2. The likely Matthews coefficient and solvent content of 2.67 Å(3) Da(-1) and 53.92%, respectively, suggested the presence of two protein subunits in the asymmetric unit. The structure was solved using molecular replacement.
Anaplastic thyroid carcinoma (ATC) is one of the most lethal human cancers with a median survival of 6 months. The inhibition of epidermal growth factor receptor (EGFR) alone, or with VEGF receptor 2 (VEGFR2), represents an attractive approach for treatment of ATC. Several reports have examined agents that target these receptors. However, with the misidentification of as many as 60% of all commonly used ATC cell lines, the significance of these past findings is unclear.
Papillary thyroid carcinomas (PTC) are the most common type of thyroid malignancy. Most PTC carry one of the two mutations, RET/PTC rearrangement or BRAF mutation. Both mutations are able to activate the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling transduction pathway leading to cellular proliferation, differentiation, and apoptosis. PD0325901 is a specific MEK1/2 inhibitor and therefore is a promising drug to treat thyroid cancers with either RET/PTC or BRAF mutation. In this study we tested the effects of PD0325901 on PTC cells harboring either mutation in vitro by growth curves and Western blots and in vivo using a murine orthotopic xenograft model. We found that 50% growth inhibition (GI(50)) by PD0325901 was 11 nmol/L for the PTC cells with the RET/PTC1 rearrangement and 6.3 nmol/L for PTC cells with a BRAF mutation, with both concentrations readily achievable in serum. After 1 week of oral administration of PD0325901 (20-25 mg/kg/day) in mice, no tumor growth was detected in mice inoculated with PTC cells bearing a BRAF mutation. For PTC with the RET/PTC1 rearrangement, the average tumor volume of the orthotopic tumor was reduced by 58% as compared with controls. In conclusion, our data suggested that PTC cells carrying a BRAF mutation were more sensitive to PD0325901 than were PTC cells carrying the RET/PTC1 rearrangement. Our findings support the clinical evaluation of PD0325901 for patients with PTC and potentially other carcinomas with BRAF mutations.
Double grating interferometer is usually used to achieve phase information from distorted wave front by its temporal phase-shifting characteristic. In this paper, the spatial phase-shifting characteristic of double grating interferometer is presented. The explicit intensity distributions of interferograms produced by double gratings are derived with the scalar diffraction theory, and the stable phase shift is found between plus-first, zero and minus-first order interferograms. Results indicate that the phase shift only depends on the grating period and the distance between two gratings if no phase object exists. If phase object exists, it varies on the interferograms. But the phase shifts are equal at any special point of interferograms. In particular, the triple grating interferometer is presented to generate at least four phase shift interferograms simultaneously with the similar method.
The complete mitogenome of the pale-edged stingray (Dasyatis zugei) was first confirmed in this study. The mitogenome is 18,264 bp in length and contains 13 protein-coding genes, 2 rRNAs, 22 tRNAs and 1 control region, with the typical gene order in vertebrates. Similar to nucleotide composition of most vertebrate mitogenomes, the nucleotide composition of the pale-edged stingray demonstrates low G and high A+T components. The control region of the pale-edged stingray is 2523 bp in length, the longest known in Chondrichthyes. The termination-associated sequence and two short conserved blocks (CSB II and CSB III) are found in the control region.
A simple and reliable HPLC method was developed and validated for the simultaneous quantification of four major constituents in Semen Vaccariae. The chromatographic separation was performed on an Agilent Zorbax SB-C18 column with gradient elution using methanol and water. The calibration curves showed good linearity of R2 > 0.9999 with LOQs (S/N = 10) of 0.20-1.16 microg/mL. The precision was evaluated by intra- and inter-day assays and R.S.D. values were less than 2.09%. The recovery rates were between 97.0% and 105.0%. The developed method was applied to the quantitative analysis of Semen Vaccariae and its stir-fried products. During the stir-frying process, vaccarin degraded and yielded isovitexin-2"-O-arabinoside. The preferable stir-frying temperature is around 120 degrees C. The developed HPLC method can be applied to the quality control of crude and stir-fried Semen Vaccariae.
A little is more than enough: Aldose reductase (AR) is a potential target in a wide range of diseases but its utility may be limited by the side effects caused by complete inhibition. Furthermore, known inhibitors of AR have suffered in clinical evaluation due to poor bioavailability. Here, the clinically used antiprotozoal drug nitazoxanide with proven bioavailability has been shown to partially inhibit AR, potentially circumventing the negatives effects of complete enzyme inhibition.
A spatial phase-shifting shearing interferometry is presented in this paper. The whole optical configuration is simple and consists of three Ronchi gratings. Four phase-shifted shearing interferograms can be obtained simultaneously. The explicit intensity distributions of shearing interferograms are given and a corresponding four-step spatial phase-shifting algorithm is proposed to extract phase information from the new interferometry. This spatial phase-shifting configuration is applied to extract phase projection of a propane flame and a mathematical error analysis is presented.
IN THE TITLE COMPOUND [SYSTEMATIC NAME: 2-hy-droxy-N-(5-nitro-1,3-thia-zol-2-yl)benzamide pyridine monosolvate], C(10)H(7)N(3)O(4)S·C(5)H(5)N, the dihedral angle between the pyridine and benzamide rings is 80.55?(7)°. An intamolecular O-H?N hydrogen bond occurs in the tizoxanide. In the crystal, the components are linked by an O-H?N hydrogen bond, forming a zigzag chain along the c axis. Aromatic ?-? inter-actions between inversion-related pyridine rings [centroid-centroid distance = 3.803?(6)?Å] are also observed.
Anaplastic thyroid carcinoma (ATC) accounts for more than 50% of thyroid cancer mortality and is generally refractory to conventional treatment. On the basis of recent studies, we hypothesized that ATC metabolism can be targeted to improve response to chemoradiotherapy. Eight established and authenticated ATC cell lines were sequenced at 140 sites contained within 26 commonly mutated genes to identify novel potential therapeutic targets. Cellular proliferation, energy, and reducing potential stores were measured under conditions of specific nutrient deprivation. Tumor metabolism was evaluated using hyperpolarized (13)C MRI in a murine orthotopic xenograft model of ATC. Sensitivity to chemotherapeutic agents and radiation (XRT) was assayed using cytotoxicity assays. We identified mutations in BRAF, NRAS, and KIT but failed to identify generalized novel targets for therapeutic intervention. ATC cell lines exhibited a mesenchymal phenotype and generalized dependence on glucose for energy, reducing potential and survival. Glycolytic inhibition using 2-deoxyglucose (2-DG) sensitized ATC cells to conventional chemotherapy and external beam radiation. In vivo, 2-DG induced a transient, but significant reduction in ATC metabolic activity. Generalized dependence of ATC cells on glucose catabolism makes them susceptible to the sensitizing effects of 2-DG for radiation therapy and chemotherapy. Under in vivo conditions, 2-DG can inhibit ATC metabolism. However, the modest magnitude and transient nature of this effect suggest the need for antimetabolic agents with more favorable pharmacodynamics to achieve therapeutic effects.
Talaromyces flavus containing a constitutive epoxide hydrolase (EH) resolved racemic benzyl glycidyl ether and nine derivatives into their (R)-enantiomers. After optimization of the fermentation conditions, the specific EH activity and biomass concentration were improved from 13.5 U/g DCW and 14.8 g DCW/l to 26.2 U/g DCW and 31.3 g DCW/l, respectively, with final values for e.e. ( s ) of 96 % and E of 13 with (R)-benzyl glycidyl ether. Substituents on the phenyl ring, however, gave low enantioselectivities.
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