Hypoxia-ischemia (HI) is a model of cerebral ischemia used to model neonatal hypoxia and to study brain damage. Interpreting the results of experiments that use HI rests partly on the assumption that only the brain suffers major damage. In this study, we demonstrate that HI also has adverse consequences on the heart. Both infarction scoring and measurements of troponin I indicate cardiac damage subsequent to HI. These results indicate that the effects of HI on the heart may confound the interpretation of experiments that have used HI to study neuroprotection or other aspects of brain damage.
Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.
Public awareness work regarding palliative and end-of-life care is increasingly promoted within national strategies for palliative care. Different approaches to undertaking this work are being used, often based upon broader educational principles, but little is known about how to undertake such initiatives in a way that equally engages both the health and social care sector and the local communities. An asset-based community engagement approach has been developed that facilitates community-led awareness initiatives concerning end-of-life conversations and care by identifying and connecting existing skills and expertise.
The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.
Cerebral ischaemia triggers various physiological processes, some of which have been considered deleterious and others beneficial. These processes have been characterized in one influential model as being part of a transition from injury to repair processes. We argue that another important distinction is between dysregulated and regulated processes. Although intervening in the course of dysregulated processes may be neuroprotective, this is unlikely to be true for regulated processes. This is because from an evolutionary perspective, regulated complex processes that are conserved across many species are likely to be adaptive and provide a survival advantage. We argue that the neuroinflammatory cascade is an adaptive process in this sense, and contrast this with a currently popular theory which we term the maladaptive immune response theory. We review the evidence from clinical and preclinical pharmacology with respect to this theory, and deduced that the evidence is inconclusive at best, and probably falsifies the theory. We argue that this is why there are no anti-inflammatory treatments for cerebral ischaemia, despite 30years of seemingly promising preclinical results. We therefore propose an opposing theory, which we call the adaptive immune response hypothesis.
Statins reduce the risk of death from cardiovascular disease in millions of people worldwide. Recent pharmacovigilance data has suggested that people taking statins have an increased risk of psychiatric adverse events such as amnesia and anxiety. This study aimed to investigate the possibility of statin-induced amnesia through animal models of memory and learning. We conducted extracellular field recordings of synaptic transmission in area CA1 of hippocampal slices to examine the effects of acute cholesterol lowering with lipid lowering drugs. We also assessed the effect of six weeks of simvastatin (2mg/kg/d) and atorvastatin (1mg/kg/d) treatment using the Morris water maze. Long Term Potentiation (LTP) was significantly diminished in the presence of 3µM atorvastatin or simvastatin and by the cholesterol sequestering agent methyl-?-cyclodextrin (MBCD). The effects were reversed in the MBCD but not the statin treated slices by the addition of cholesterol. In the water maze, statin treatment did not cause any deficits in the first five days of reference memory testing, but statin treated guinea pigs preformed significantly worse than control animals in a working memory test. The deficits observed in our experiments in water maze performance and hippocampal LTP are suggestive of statin induced changes in hippocampal plasticity. The effects on LTP are independent of cholesterol regulation, and occur at concentrations that may be relevant to clinical use. Our results may help to explain some of the behavioural changes reported in some people after beginning statin treatment.
The delivery of some classes of drugs is challenging. Solubility, absorption, distribution, and duration of action may all be altered by combination with vehicle molecules. It has already been discovered that polyethylene glycol - which is used as a stabiliser in peptide drug formulations - has biological activity in its own right, including potential neuroprotective properties. In this article we review the evidence for confounding activity for four distinct compounds that have been used as solvents and/or carrier molecules for the delivery of lipophilic drugs under investigation for potential neuroprotective properties. We discuss the evidence that cyclodextrins, ethanol, dimethyl sulphoxide, and a castor oil derivative - Cremophor™ EL - have all been found to have mild to moderate neuroprotective effects. We argue that this has probably reduced the statistical power and increased the Type II error rates of neuroprotection experiments that have employed these vehicles, and suggest experimental design considerations to help correct the problem. However, we also note that the properties of these compounds may represent an opportunity for drug development, particularly for the newer compounds that have been subject to only limited experimental investigation.
In recent decades, growth of computing power has facilitated powerful techniques for reconstructing evolutionary relationships from online genetic and proteomic databases. These methods are useful tools for pharmacologists for analyzing relationships between receptors and associated enzymes. Phylogenetic analysis can help generate hypotheses and leads for experimentation. Reconstruction of molecular phylogenies for the nonspecialist is described in this article using the example of the orphaned g protein coupled receptor GPR18.
The question of whether cannabinoid CB? receptors are expressed on neurons in the brain and under what circumstances they are expressed is controversial in cannabinoid neuropharmacology. While some studies have reported that CB? receptors are not detectable on neurons under normal circumstances, other studies have reported abundant neuronal expression. One reason for these apparent discrepancies is the reliance on incompletely validated CB? receptor antibodies and immunohistochemical procedures. In this study, we demonstrate some of the methodological problems encountered using three different commercial CB? receptor antibodies. We show that (1) the commonly used antibodies that were confirmed by many of the tests used for antibody validation still failed when examined using the knockout control test; (2) the coherence between the labeling patterns provided by two antibodies for the same protein at different epitopes may be misleading and must be validated using both low- and high-magnification microscopy; and (3) although CB? receptor antibodies may label neurons in the brain, the protein that the antibodies are labeling is not necessarily CB?. These results showed that great caution needs to be exercised when interpreting the results of brain immunohistochemistry using CB? receptor antibodies and that, in general, none of the tests for antibody validity that have been proposed, apart from the knockout control test, are reliable.
We used an in vivo small hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase Syk. In contrast, loss of Itgb3 in normal hematopoietic stem and progenitor cells did not affect engraftment, reconstitution, or differentiation. Finally, using an Itgb3 knockout mouse model, we confirmed that Itgb3 is dispensable for normal hematopoiesis but is required for leukemogenesis. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
Biomedical animal models predict clinical efficacy with varying degrees of success. An important feature of in vivo modeling is matching the age of the animals used in preclinical research to the age of peak incidence for a disease state in humans. However, growth and development are highly variable between mammalian species, and age matching is always based on assumptions about the nature of development. We propose that researchers commonly make the assumption that developmental sequences are highly conserved between mammalian species--an assumption that we argue is often incorrect. We instead argue that development is often a modular process. Consideration of the modular nature of development highlights the difficulty in matching animal ages to human ages in a one-to-one scalar manner. We illustrate this with a discussion of the problem of age matching rodents to humans for neuroprotection experiments, and argue that researchers should pay deliberate attention to the modularity of developmental processes in order to optimally match ages between species in biomedical research.
Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed "ROS-low"). Second, ROS-low LSCs aberrantly overexpress BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.
Neuroblastoma is a common, frequently fatal, neural crest tumor of childhood. Chemotherapy-resistant neuroblastoma cells typically have Schwann cell-like ("S-type") morphology and express the p75 neurotrophin receptor (p75NTR). p75NTR has been previously shown to modulate the redox state of neural crest tumor cells. We, therefore, hypothesized that p75NTR expression level would influence the effects of the redox-active chemotherapeutic drug fenretinide on neuroblastoma cells.
O-1602 is an atypical cannabinoid that acts as an agonist at GPR55, a g protein-coupled receptor that previous studies have indicated may have a pronociceptive role in neuropathic pain. We administered O-1602 to both naive rats and rats that had undergone chronic constriction injury surgery. O-1602 did not cause any changes in hind paw responses to Von Frey hair testing in naive rats. However, O-1602 reversed the desensitising effects of ETOH, which was used as an active and opposing vehicle. Our results are consistent with the hypothesis that GPR55 has a pronociceptive role in neuropathic pain.
Pancytopenia is a major cause of morbidity in acute myeloid leukemia (AML), yet its cause is unclear. Normal osteoblastic cells have been shown to support hematopoiesis. To define the effects of leukemia on osteoblastic cells, we used an immunocompetent murine model of AML. Leukemic mice had inhibition of osteoblastic cells, with decreased serum levels of the bone formation marker osteocalcin. Osteoprogenitor cells and endosteal-lining osteopontin(+) cells were reduced, and osteocalcin mRNA in CD45(-) marrow cells was diminished. This resulted in severe loss of mineralized bone. Osteoclasts were only transiently increased without significant increases in bone resorption, and their inhibition only partially rescued leukemia-induced bone loss. In vitro data suggested that a leukemia-derived secreted factor inhibited osteoblastic cells. Because the chemokine CCL-3 was recently reported to inhibit osteoblastic function in myeloma, we tested its expression in our model and in AML patients. Consistent with its potential novel role in leukemic-dependent bone loss, CCL-3 mRNA was significantly increased in malignant marrow cells from leukemic mice and from samples from AML patients. Based on these results, we propose that therapeutic mitigation of leukemia-induced uncoupling of osteoblastic and osteoclastic cells may represent a novel approach to promote normal hematopoiesis in patients with myeloid neoplasms.
Statins play a crucial role in reducing the risk of death from cardiovascular disease in millions of people worldwide. Recently, pharmacovigilance data has suggested that statin drugs may have rare but significant adverse psychiatric effects, such as amnesia, anxiety and even aggression. In order to investigate the effects of statins on cognitive function in an animal model, we studied the effect of 6 weeks of daily administration of oral simvastatin (1 mg/kg) or atorvastatin (0.5mg/kg) in guinea pig on performance in the Morris water maze (MWM). Animals were also re-tested in the MWM, 2 weeks after drug cessation, to test for any changes in performance as a result of drug de-challenge. Guinea pigs treated with either statin showed a significant (P<0.001) decrease in total cholesterol and low density lipoprotein-cholesterol (LDL-C), which remained partially reduced after the 2 week drug washout period. Guinea pigs receiving either statin did not show any difference in latency to reach the platform, nor any difference in total distance travelled during testing. Also, analysis of probe trials revealed no significant differences between drug and vehicle groups. However, both groups spent a significantly (P<0.01) greater proportion of time in the outer zone of the maze (indication of increased anxiety) and showed an increase in swimming speed (P<0.05) compared with the vehicle group. Differences between groups for swimming speed, and time spent in the outer zone, were not retained in the drug de-challenge phase. Our results show that low dose treatment with statins can induce mild but significant anxiety in guinea pigs.
Polymeric endoaortic paving (PEAP) may improve endovascular repair of abdominal aortic aneurysms (AAA) since it has the potential to treat patients with complex AAA geometries while reducing the incidence of migration and endoleak. Polycaprolactone (PCL)/polyurethane (PU) blends are proposed as PEAP materials due to their range of mechanical properties, thermoformability, and resistance to biodegradation. In this study, the reduction in AAA wall stress that can be achieved using PEAP was estimated and compared to that resulting from stent-grafts. This was accomplished by mechanically modeling the anisotropic response of PCL/PU blends and implementing these results into finite element model (FEM) simulations. We found that at the maximum diameter of the AAA, the 50/50 and 10/90 PCL/PU blends reduced wall stress by 99 and 98%, respectively, while a stent-graft reduced wall stress by 99%. Our results also show that wall stress reduction increases with increasing PEAP thickness and PCL content in the blend ratio. These results indicate that PEAP can reduce AAA wall stress as effectively as a stent-graft. As such, we propose that PEAP may provide an improved treatment alternative for AAA, since many of the limitations of stent-grafts have the potential to be solved using this approach.
Perinatal and early childhood asphyxia is common, debilitating and has few efficacious treatments. A hypoxia ischemia (HI) rat model that involves a unilateral ligation of the common carotid artery followed by a 60 min period of 8% oxygen hypoxia is often used to test proposed treatments. However, this HI protocol produces inconsistent infarction volumes due to the variability of individual rats to compensate for the ligated artery and hypoxia. Therefore, this HI model is problematic for experiments that prevent measurement of infarction volume, such as those that require analysis of homogenised brain tissue. We therefore aimed to find a simple and non-invasive predictor of infarction volume. Observations made prior, during and following HI in p26 rats showed that weight change 24 h following surgery was a strong predictor of infarction volume. The occurrence of a tonic clonic seizure during hypoxia was highly correlated with success of inducing an infarction, and for this reason we assessed whether ceasing the hypoxia for each rat following a tonic clonic seizure would produce a more consistent infarction volume. Using this procedure, infarction volumes measured at 3 and 15 days after surgery were significantly less variable, resulting in considerable improvements in statistical power compared with the original model.
Ageing women may choose to drink soya milk to reduce menopausal symptoms. As fermentation enriches soya milk with isoflavone aglycones, its beneficial qualities may improve. To reduce osteoporotic risk, however, soya milk must be Ca enriched, and it is not known how fermentation affects Ca bioavailability. A randomised crossover pilot study was undertaken to compare the Ca absorption of fortified soya milk with that of fermented and fortified soya milk in twelve Australian osteopenic post-menopausal women. The fortified soya milk was inoculated with Lactobacillus acidophilus American Type Culture Collection (ATCC) 4962 and fermented for 24 h at 37°C. Ca absorption from soya milk samples was measured using a single isotope radiocalcium method. Participants had a mean age of 54·8 (sd 12·3) years, with mean BMI of 26·5 (sd 5·5) kg/m2 and subnormal to normal serum 25-hydroxyvitamin D (mean 62·5 (sd 19·1) nmol/l). Participants consumed 185 kBq of 45Ca in 44 mg of Ca carrier. The mean fractional Ca absorption (?) from soya milk and fermented soya milk was 0·64 (sd 0·23) and 0·71 (sd 0·29), respectively, a difference not of statistical significance (P = 0·122). Although fermentation of soya milk may provide other health benefits, fermentation had little effect on acute Ca absorption.
The presence of phytate in calcium-fortified soymilk may interfere with mineral absorption. Certain lactic acid bacteria (LAB) produce the enzyme phytase that degrades phytates and therefore may potentially improve mineral bioavailability and absorption. This study investigates the phytase activity and phytate degradation potential of 7 strains of LAB including: Lactobacillus acidophilus ATCC4962, ATCC33200, ATCC4356, ATCC4161, L. casei ASCC290, L. plantarum ASCC276, and L. fermentum VRI-003. Activity of these bacteria was examined both in screening media and in calcium-fortified soymilk supplemented with potassium phytate. Most strains produced phytase under both conditions with L. acidophilus ATCC4161 showing the highest activity. Phytase activity in fortified soymilk fermented with L. acidophilus ATCC4962 and L. acidophilus ATCC4161 increased by 85% and 91%, respectively, between 12 h and 24 h of fermentation. All strains expressed peak phytase activity at approximately pH 5. However, no phytate degradation could be observed.
Calcium loss after menopause increases the risk of osteoporosis in aging women. Soymilk is often consumed to reduce menopausal symptoms, although in its native form, it contains significantly less calcium than cows milk. Moreover, when calcium is added as a fortificant, it may not be absorbed efficiently. This study compares calcium absorption from soymilk fortified with a proprietary phosphate of calcium versus absorption from cows milk. Preliminary studies compared methods for labelling the calcium fortificant either before or after its addition to soymilk. It was established that fortificant labelled after it was added to soymilk had a tracer distribution pattern very similar to that shown by fortificant labelled before adding to soymilk, provided a heat treatment (90?C for 30 min) was applied. This method was therefore used for further bioavailability studies. Calcium absorption from fortified soy milk compared to cows milk was examined using a randomised single-blind acute cross-over design study in 12 osteopenic post-menopausal women aged (mean +/- SD) 56.7+/-5.3 years, with a body mass index of 26.5+/-5.6 kg/m2. Participants consumed 20 mL of test milk labelled after addition of fortificant with 185 kBq of 45Ca in 44 mg of calcium carrier, allowing the determination of the hourly fractional calcium absorption rate (alpha) using a single isotope radiocalcium test. The mean hourly fractional calcium absorption from fortified soymilk was found to be comparable to that of cows milk: alpha = 0.65+/-0.19 and alpha =0.66+/-0.22, p>0.05, respectively.
Previous studies have suggested panic induced by cannabinoids may be potentiated by concurrently using selective serotonin reuptake inhibitors. We therefore tested for panic and anxiety-related behaviour in rats coadministered with the selective serotonin reuptake inhibitor fluoxetine hydrochloride and the CB1 agonist delta9-tetrahydrocannabinol. Although each drug alone elicited behaviours consistent with previous studies, we did not find that coadministration of these drugs potentiated or reduced the effects of either drug alone. Specifically, fluoxetine treatment did not cause any increase in freezing behaviour, decrease in social interaction or exploration in an adverse environment, change preference for outer or inner zones, or modulate rearing behaviour in rats receiving delta9-tetrahydrocannabinol.
Two cannabinoids receptors have been characterised in mammals; cannabinoid receptor type 1 (CBI) which is ubiquitous in the central nervous system (CNS), and cannabinoid receptor type 2 (CBII) that is expressed mainly in immune cells. Cannabinoids have been used in the treatment of nausea and emesis, anorexia and cachexia, tremor and pain associated with multiple sclerosis. These treatments are limited by the psychoactive side-effects of CBI activation. Recently CBII has been described within the CNS, both in microglia and neuronal progenitor cells (NPCs), but with few exceptions, not by neurons within the CNS. This has suggested that CBII agonists could have potential to treat various conditions without psycho-activity. This article reviews the potential for CBII agonists as treatments for neurological conditions, with a focus on microglia and NPCs as drug targets. We first discuss the role of microglia in the healthy brain, and then the role of microglia in chronic neuroinflammatory disorders, including Alzheimers disease and Parkinsons disease, as well as in neuroinflammation following acute brain injury such as stroke and global hypoxia. As activation of CBII receptor on microglia results in suppression of the proliferation and activation of microglia, there is potential for the anti-inflammatory properties of CBII agonist to treat neuropathologies that involve heightened microglia activity. In addition, activating CBII receptors may result in an increase in proliferation and affect migration of NPCs. Therefore, it is possible that CBII agonists may assist in the treatment of neuropathologies by increasing neurogenesis. In the second part of the article, we review the state of development of CBII selective drugs with an emphasis on critical aspects of CBII agonist structural activity relationship (SAR).
APOBEC-1 Complementation Factor (ACF) is an RNA-binding protein that interacts with apoB mRNA to support RNA editing. ACF traffics between the cytoplasm and nucleus. It is retained in the nucleus in response to elevated serum insulin levels where it supports enhanced apoB mRNA editing. In this report we tested whether ACF may have the ability to regulate nuclear export of apoB mRNA to the sites of translation in the cytoplasm. Using mouse models of obesity-induced insulin resistance and primary hepatocyte cultures we demonstrated that both nuclear retention of ACF and apoB mRNA editing were reduced in the livers of hyperinsulinemic obese mice relative to lean controls. Coincident with an increase in the recovery of ACF in the cytoplasm was an increase in the proportion of total cellular apoB mRNA recovered in cytoplasmic extracts. Cytoplasmic ACF from both lean controls and obese mouse livers was enriched in endosomal fractions associated with apoB mRNA translation and ApoB lipoprotein assembly. Inhibition of ACF export to the cytoplasm resulted in nuclear retention of apoB mRNA and reduced both intracellular and secreted ApoB protein in primary hepatocytes. The importance of ACF for modulating ApoB was supported by the finding that RNAi knockdown of ACF reduced ApoB secretion. An additional discovery from this study was the finding that leptin is a suppressor ACF expression. Dyslipidemia is a common pathology associated with insulin resistance that is in part due to the loss of insulin controlled secretion of lipid in ApoB-containing very low density lipoproteins. The data from animal models suggested that loss of insulin regulated ACF trafficking and leptin regulated ACF expression may make an early contribution to the overall pathology associated with very low density lipoprotein secretion from the liver in obese individuals.
In the mid-1980s in Liverpool, and the area surrounding it (Merseyside and Cheshire), harm reduction was adopted on a large scale for the first time in the UK. The harm reduction model was based on a population approach to achieve the public health goal of reducing the harm to health associated with drug use. The particular concern at that time was the risk of HIV infection, but there was also the issue of the health of a group of young people who were under-served by health services. To achieve the goal, services were developed that would attract the majority of those at risk within the community, not simply the few who wished to stop using drugs, and which would enable contact with the target group to be maintained so as to bring about the necessary changes in behaviour required to maintain health and reduce risk. This Commentary describes some of the background to the development of the Mersey Model of Harm Reduction from the memories and perspectives of two people who promoted harm reduction within the health service and the region.
An intraluminal thrombus (ILT) forms in the majority of abdominal aortic aneurysms (AAAs). While the ILT has traditionally been perceived as a byproduct of aneurysmal disease, the mechanical environment within the ILT may contribute to the degeneration of the aortic wall by affecting biological events of cells embedded within the ILT. In this study, the drained secant modulus (E(5) approximately modulus at 5% strain) of ILT specimens (luminal, medial, and abluminal) procured from elective open repair was measured and compared using unconfined compression. Five groups of fibrin-based thrombus mimics were also synthesized by mixing various combinations of fibrinogen, thrombin, and calcium. Drained secant moduli were compared to determine the effect of the components concentrations on mimic stiffness. The stiffness of mimics was also compared to the native ILT. Preliminary data on the water content of the ILT layers and mimics was measured. It was found that the abluminal layer (E(5)=19.3kPa) is stiffer than the medial (2.49kPa) and luminal (1.54kPa) layers, both of which are statistically similar. E(5) of the mimics (0.63, 0.22, 0.23, 0.87, and 2.54kPa) is dependent on the concentration of all three components: E(5) decreases with a decrease in fibrinogen (60-20 and 20-15mg/ml) and a decrease in thrombin (3-0.3 units/ml), and E(5) increases with a decrease in calcium (0.1-0.01M). E(5) from two of the mimics were not statistically different than the medial and luminal layers of ILT. A thrombus mimic with similar biochemical components, structure, and mechanical properties as native ILT would provide an appropriate test medium for AAA mechanobiology studies.
Our understanding of the complexity of the endocannabinoid system has evolved considerably since the cloning of the receptors in the early 1990s. Since then several endogenous ligands have been identified and their respective biosynthetic pathways unravelled. This research has revealed the involvement of the cannabinoid system in a number of important physiological processes including the regulation of neurotransmitter release, pain and analgesia, energy homeostasis, and control of immune cell function. All of these events are mediated by two similar receptors, CB1 and CB2, which were initially thought to possess mutually exclusive expression profiles. Recent advances have begun to dissolve such absolutes with the discovery of CB2 in brain tissue and identification of a range of functions for CB1 in peripheral tissues. With improved understanding of the cannabinoid system comes the illumination of various roles in disease pathologies and identification of potential therapeutic targets. This review provides an overview of the current understanding of the endocannabinoid system, and then focuses on recent discoveries that we believe are likely to shape the future directions of the field.
Network formation of whey protein isolate (WPI) with increasing concentrations of native wheat starch (WS) has been examined. Small deformation dynamic oscillation in shear and modulated temperature differential scanning calorimetry enabled analysis of binary mixtures at the macro- and micromolecular level. Following heat induced gelation, textural hardness was measured by undertaking compression tests. Environmental scanning electron microscopy provided tangible information on network morphology of polymeric constituents. Experiments involving in vitro starch digestion also allowed for indirect assessment of phase topology in the binary mixture. The biochemical component of this work constitutes an attempt to utilise whey protein as a retardant to the enzymatic hydrolysis of starch in a model system with ?-amylase enzyme. During heating, rheological profiles of binary mixtures exhibited dramatic increases in G at temperatures more closely related to those observed for single whey protein rather than pure starch. Results from this multidisciplinary approach of analysis, utilising rheology, calorimetry and microscopy, argue for the occurrence of phase separation phenomena in the gelled systems. There is also evidence of whey protein forming the continuous phase with wheat starch being the discontinuous filler, an outcome that is explored in the in vitro study of the enzymatic hydrolysis of starch.
Leukemia stem cells (LSCs) represent a biologically distinct subpopulation of myeloid leukemias, with reduced cell cycle activity and increased resistance to therapeutic challenge. To better characterize key properties of LSCs, we employed a strategy based on identification of genes synergistically dysregulated by cooperating oncogenes. We hypothesized that such genes, termed "cooperation response genes" (CRGs), would represent regulators of LSC growth and survival. Using both a primary mouse model and human leukemia specimens, we show that CRGs comprise genes previously undescribed in leukemia pathogenesis in which multiple pathways modulate the biology of LSCs. In addition, our findings demonstrate that the CRG expression profile can be used as a drug discovery tool for identification of compounds that selectively target the LSC population. We conclude that CRG-based analyses provide a powerful means to characterize the basic biology of LSCs as well as to identify improved methods for therapeutic targeting.
O-1602 is a cannabidiol analogue that does not bind with high affinity to either the cannabinoid CB1 receptor or CB2 receptor. However, there is evidence that O-1602 has significant effects in the central nervous system as well as other parts of the body. Depending upon the model, O-1602 has anti-inflammatory or pronociceptive effects, mediated through a number of distinct receptors. This article reviews the evidence for functional effects of O-1602, particularly in the CNS, and describes its known targets as they relate to these effects. These include the abnormal cannabidiol (Abn- CBD) receptor and GPR55. The GPR18 receptor has been identified with the Abn-CBD receptor, and therefore the evidence that O-1602 also acts at GPR18 is also reviewed. Finally, the evidence that these receptor targets are expressed in the CNS and the phenotypes of cells expressing these targets is discussed, concluding with a discussion of the prospects for O-1602 as a therapeutic agent in the CNS.
Rimonabant is a cannabinoid CB1 receptor antagonist. Other CB1 antagonists have biphasic effects on blood glucose levels following acute administration. We therefore tested the effects of rimonabant on glucose tolerance following acute administration.
In the last decade a number of products have appeared in various countries that contain synthetic cannabinoids. This article reviews the history of the sale of these drugs, and the evidence that they contain synthetic cannabinoids. The biochemistry of the synthetic cannabinoids identified thus far is discussed, including a discussion of chemical structures and biochemical targets. The cannabinoid receptor targets for these drugs are discussed, as well as other possible targets such as serotonin receptors. Evidence for the abuse potential of these drugs is reviewed. The toxicity of synthetic cannabinoids and cannabinoid products is reviewed and compared to that of the phytocannabinoid ?9- tetrahydrocannabinol (THC). As cannabinoids are a structurally diverse class of drugs, it is concluded that synthetic cannabinoids should be classified by biological activity rather than by structure, and that if this isnt done, novel synthetic cannabinoids will continue to emerge that fall outside of current regulatory classification models.
RelB and nuclear factor ?B (NF-?B2) are the main effectors of NF-?B noncanonical signaling and play critical roles in many physiological processes. However, their role in hematopoietic stem/progenitor cell (HSPC) maintenance has not been characterized. To investigate this, we generated RelB/NF-?B2 double-knockout (dKO) mice and found that dKO HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Transplantation of wild-type bone marrow cells into dKO mice to assess the role of the dKO microenvironment showed that wild-type HSPCs cycled more rapidly, were more abundant, and had developmental aberrancies: increased myeloid and decreased lymphoid lineages, similar to dKO HSPCs. Notably, when these wild-type cells were returned to normal hosts, these phenotypic changes were reversed, indicating a potent but transient phenotype conferred by the dKO microenvironment. However, dKO bone marrow stromal cell numbers were reduced, and bone-lining niche cells supported less HSPC expansion than controls. Furthermore, increased dKO HSPC proliferation was associated with impaired expression of niche adhesion molecules by bone-lining cells and increased inflammatory cytokine expression by bone marrow cells. Thus, RelB/NF-?B2 signaling positively and intrinsically regulates HSPC self-renewal and maintains stromal/osteoblastic niches and negatively and extrinsically regulates HSPC expansion and lineage commitment through the marrow microenvironment.
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