Journal of Biomedical Science - Most accessed articles

Pyrazole compound BPR1P0034 with potent and selective anti-influenza virus activity

Shin-Ru Shih — 2010-02-23T00:00:00Z

Background: Influenza viruses are a major cause of morbidity and mortality around the world. More recently, a swine-origin influenza A (H1N1) virus that is spreading via human-to-human transmission has become a serious public concern. Although vaccination is the primary strategy for preventing infections, influenza antiviral drugs play an important role in a comprehensive approach to controlling illness and transmission. In addition, a search for influenza-inhibiting drugs is particularly important in the face of high rate of emergence of influenza strains resistant to several existing influenza antivirals. Methods: We searched for novel anti-influenza inhibitors using a cell-based neutralization (inhibition of virus-induced cytopathic effect) assay. After screening 20,800 randomly selected compounds from a library from ChemDiv, Inc., we found that BPR1P0034 has sub-micromolar antiviral activity. The compound was resynthesized in five steps by conventional chemical techniques. Lead optimization and a structure-activity analysis were used to improve potency. Time-of-addition assay was performed to target an event in the virus life cycle. Results: The 50% effective inhibitory concentration (IC50) of BPR1P0034 was 0.42 ± 0.11 μM, when measured with a plaque reduction assay. Viral protein and RNA synthesis of A/WSN/33 (H1N1) was inhibited by BPR1P0034 and the virus-induced cytopathic effects were thus significantly reduced. BPR1P0034 exhibited broad inhibition spectrum for influenza viruses but showed no antiviral effect for enteroviruses and echovirus 9. In a time-of-addition assay, in which the compound was added at different stages along the viral replication cycle (such as at adsorption or after adsorption), its antiviral activity was more efficient in cells treated with the test compound between 0 and 2 h, right after viral infection, implying that an early step of viral replication might be the target of the compound. These results suggest that BPR1P0034 targets the virus during viral uncoating or viral RNA importation into the nucleus. Conclusions: To the best of our knowledge, BPR1P0034 is the first pyrazole-based anti-influenza compound ever identified and characterized from high throughput screening to show potent (sub-μM) antiviral activity. We conclude that BPR1P0034 has potential antiviral activity, which offers an opportunity for the development of a new anti-influenza virus agent.

Caffeine and a selective adenosine A2A receptor antagonist induce sensitization and cross-sensitization behavior associated with increased striatal dopamine in mice

Chih Hsu — 2010-01-15T00:00:00Z

Background: Caffeine, a nonselective adenosine A1 and A2A receptor antagonist, is the most widely used psychoactive substance in the world. Evidence demonstrates that caffeine and selective adenosine A2A antagonists interact with the neuronal systems involved in drug reinforcement, locomotor sensitization, and therapeutic effect in Parkinson's disease (PD). Evidence also indicates that low doses of caffeine and a selective adenosine A2A antagonist SCH58261 elicit locomotor stimulation whereas high doses of these drugs exert locomotor inhibition. Since these behavioral and therapeutic effects are mediated by the mesolimbic and nigrostriatal dopaminergic pathways which project to the striatum, we hypothesize that low doses of caffeine and SCH58261 may modulate the functions of dopaminergic neurons in the striatum. Methods: In this study, we evaluated the neuroadaptations in the striatum by using reverse-phase high performance liquid chromatography (HPLC) to quantitate the concentrations of striatal dopamine and its metabolites, dihydroxylphenylacetic acid (DOPAC) and homovanilic acid (HVA), and using immunoblotting to measure the level of phosphorylation of tyrosine hydroxylase (TH) at Ser31, following chronic caffeine and SCH58261 sensitization in mice. Moreover, to validate further that the behavior sensitization of caffeine is through antagonism at the adenosine A2A receptor, we also evaluate whether chronic pretreatment with a selective adenosine A2A antagonist SCH58261 or a selective adenosine A1 antagonist DPCPX can sensitize the locomotor stimulating effects of caffeine. Results: Chronic treatments with low dose caffeine (10 mg/kg) or SCH58261 (2 mg/kg) increased the concentrations of dopamine, DOPAC and HVA, concomitant with increased TH phosphorylation at Ser31 and consequently enhanced TH activity in the striatal tissues in both caffeine- and SCH58261-sensitized mice. In addition, chronic caffeine or SCH58261 administration induced locomotor sensitization, and locomotor cross-sensitization to caffeine was observed following chronic treatment of mice with SCH58261 but not with DPCPX. Conclusions: Our study demonstrated that low dosages of caffeine and a selective adenosine A2A antagonist SCH58261 elicited locomotor sensitization and cross-sensitization, which were associated with elevated dopamine concentration and TH phosphorylation at Ser31 in the striatum. Blockade of adenosine A2A receptor may play an important role in the striatal neuroadaptations observed in the caffeine-sensitized and SCH58261-sensitized mice.

Shorter GT repeat polymorphism in the heme oxygenase-1 gene promoter has protective effect on ischemic stroke in dyslipidemia patients

Chyi-Huey Bai — 2010-02-23T00:00:00Z

Background: The microsatellite polymorphism of heme oxygenase (HO)-1 gene promoter has been shown to be associated with the susceptibility to ischemic event, including coronary artery disease (CAD), myocardial infarction, and peripheral vascular disease. We aimed to examine whether the length of (GT)n repeats in HO-1 gene promoter is associated with ischemic stroke in people with CAD risk factors, especially low level of HDL. Methods: A total of 183 consecutive firstever ischemic stroke inpatients and 164 non-stroke patients were screened for the length of (GT)n repeats in HO-1 promoter. The long (L) and short (S) genotype are defined as the averaged repeat number >26 and ≦26, respectively. Results: Stroke patients tended to have more proportions of hypertension, diabetics and genotype L, than those of genotype S. Patients with genotype L of HO-1 gene promoter have higher stroke risk in comparison with genotype S especially in dyslipidemia individuals. The significant differences on stroke risk in multivariate odds ratios were found especially in people with low HDL-C levels. Conclusions: Subjects carrying longer (GT)n repeats in HO-1 gene promoter may have greater susceptibility to develop cerebral ischemic only in the presence of low HDL-C, suggesting the protective effects in HO-1 genotype S in the process of ischemic stroke, particularly in subjects with poor HDL-C status.

Genetic polymorphisms of matrix metalloproteinases and their inhibitors in potentially malignant and malignant lesions of the head and neck

Ajay Chaudhary — 2010-02-15T00:00:00Z

Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are capable of cleaving all extra cellular matrix (ECM) substrates. Degradation of matrix is a key event in progression, invasion and metastasis of potentially malignant and malignant lesions of the head and neck. It might have an important polymorphic association at the promoter regions of several MMPs such as MMP-1 (-1607 1G/2G), MMP-2 (-1306 C/T), MMP-3 (-1171 5A/6A), MMP-9 (-1562 C/T) and TIMP-2 (-418 G/C or C/C). Tissue inhibitors of metalloproteinases (TIMPs) are naturally occurring inhibitors of MMPs, which inhibit the activity of MMPs and control the breakdown of ECM. Currently, many MMP inhibitors (MMPIs) are under development for treating different malignancies. Useful markers associated with molecular aggressiveness might have a role in prognostication of malignancies and to better recognize patient groups that need more antagonistic treatment options. Furthermore, the introduction of novel prognostic markers may also promote exclusively new treatment possibilities, and there is an obvious need to identify markers that could be used as selection criteria for novel therapies. The objective of this review is to discuss the molecular functions and polymorphic association of MMPs and TIMPs and the possible therapeutic aspects of these proteinases in potentially malignant and malignant head and neck lesions. So far, no promising drug target therapy has been developed for MMPs in the lesions of this region. In conclusion, further research is required for the development of their potential diagnostic and therapeutic possibilities.

Development of biomaterial scaffold for nerve tissue engineering: Biomaterial mediated neural regeneration

Anuradha Subramanian — 2009-11-25T00:00:00Z

Neural tissue repair and regeneration strategies have received a great deal of attention because it directly affects the quality of the patient's life. There are many scientific challenges to regenerate nerve while using conventional autologous nerve grafts and from the newly developed therapeutic strategies for the reconstruction of damaged nerves. Recent advancements in nerve regeneration have involved the application of tissue engineering principles and this has evolved a new perspective to neural therapy. The success of neural tissue engineering is mainly based on the regulation of cell behavior and tissue progression through the development of a synthetic scaffold that is analogous to the natural extracellular matrix and can support three-dimensional cell cultures. As the natural extracellular matrix provides an ideal environment for topographical, electrical and chemical cues to the adhesion and proliferation of neural cells, there exists a need to develop a synthetic scaffold that would be biocompatible, immunologically inert, conducting, biodegradable, and infection-resistant biomaterial to support neurite outgrowth. This review outlines the rationale for effective neural tissue engineering through the use of suitable biomaterials and scaffolding techniques for fabrication of a construct that would allow the neurons to adhere, proliferate and eventually form nerves.

Cyclic stretch enhances the expression of Toll-like Receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-kappaB pathway

Kou-Gi Shyu — 2010-03-05T00:00:00Z

Background: Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. Methods: Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected. Results: Cyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-a antibody, and p38 MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-B after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-B siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-a antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-a and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-a antibody, or p38siRNA 30 min before Ang II and TNF-a stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-a. Conclusions: Cyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-B pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

Region-specific effects on brain metabolites of hypoxia and hyperoxia overlaid on cerebral ischemia in young and old rats: a quantitative proton magnetic resonance spectroscopy study

Maria Macri — 2010-02-23T00:00:00Z

Background: Both hypoxia and hyperoxia, deregulating the oxidative balance, may play a role in the pathology of neurodegenerative disorders underlain by cerebral ischemia. In the present study, quantitative proton magnetic resonance spectroscopy was used to evaluate regional metabolic alterations, following a 24-hour hypoxic or hyperoxic exposure on the background of ischemic brain insult, in two contrasting age-groups of rats: young - 3 months old and aged - 24 months old. Methods: Cerebral ischemia was induced by ligation of the right common carotid artery. Concentrations of eight metabolites (alanine, choline-containing compounds, total creatine, γ-aminobutyric acid, glutamate, lactate, myo-inositol and N-acetylaspartate) were quantified from extracts in three different brain regions (fronto-parietal and occipital cortices and the hippocampus) from both hemispheres. Results: In the control normoxic condition, there were significant increases in lactate and myo-inositol concentrations in the hippocampus of the aged rats, compared with the respective values in the young ones. In the ischemia-hypoxia condition, the most prevalent changes in the brain metabolites were found in the hippocampal regions of both young and aged rats; but the effects were more evident in the aged animals. The ischemia-hyperoxia procedure caused less dedicated changes in the brain metabolites, which may reflect more limited tissue damage. Conclusions: We conclude that the hippocampus turns out to be particularly susceptible to hypoxia overlaid on cerebral ischemia and that old age further increases this susceptibility.

In vitro and ex vivo effect of hyaluronic acid on erythrocyte flow properties

A Luquita — 2010-02-12T00:00:00Z

Background: Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology. Methods: Erythrocyte deformability (by filtration through nucleopore membranes) and erythrocyte aggregability (EA) were tested on blood from healthy donors additioned with purified HA. EA was measured by transmitted light and analyzed with a mathematical model yielding two parameters, the aggregation rate and the size of the aggregates. Conformational changes of cytoskeleton proteins were estimated by electron paramagnetic resonance spectroscopy (EPR). Results: In vitro, erythrocytes treated with HA showed increased rigidity index (RI) and reduced aggregability, situation strongly related to the rigidization of the membrane cytoskeleton triggered by HA, as shown by EPR results. Also, a significant correlation (r: 0.77, p < 0.00001) was found between RI and serum HA in RA patients. Conclusions: Our results lead us to postulate the hypothesis that HA interacts with the erythrocyte surface leading to modifications in erythrocyte rheological and flow properties, both ex vivo and in vitro.

Quantitative evaluation of motor function before and after engraftment of dopaminergic neurons in a rat model of Parkinson's disease

Chieh-Sen Chuang — 2010-02-13T00:00:00Z

Although gait change is considered a useful indicator of severity in animal models of Parkinson's disease, systematic and extensive gait analysis in animal models of neurological deficits is not well established. The CatWalk-assisted automated gait analysis system provides a comprehensive way to assess a number of dynamic and static gait parameters simultaneously. In this study, we used the Catwalk system to investigate changes in gait parameters in adult rats with unilateral 6-OHDA-induced lesions and the rescue effect of dopaminergic neuron transplantation on gait function. Four weeks after 6-OHDA injection, the intensity and maximal area of contact were significantly decreased in the affected paws and the swing speed significantly decreased in all four paws. The relative distance between the hind paws also increased, suggesting that animals with unilateral 6-OHDA-induced lesions required all four paws to compensate for loss of balance function. At 8 weeks post-transplantation, engrafted dopaminergic neurons expressed tyrosine hydroxylase. In addition, the intensity, contact area, and swing speed of the four limbs increased and the distance between the hind paws decreased. Partial recovery of methamphetamine-induced rotational response was also noted.

Functional cooperation between FACT and MCM is coordinated with cell cycle and differential complex formation

Bertrand Chin-Ming Tan — 2010-02-16T00:00:00Z

Background: Functional cooperation between FACT and the MCM helicase complex constitutes an integral step during DNA replication initiation. However, mode of regulation that underlies the proper functional interaction of FACT and MCM is poorly understood.Methods & ResultsHere we present evidence indicating that such interaction is coordinated with cell cycle progression and differential complex formation. We first demonstrate the existence of two distinct FACT-MCM subassemblies, FACT-MCM2/4/6/7 and FACT-MCM2/3/4/5. Both complexes possess DNA unwinding activity and are subject to cell cycle-dependent enzymatic regulation. Interestingly, analysis of functional attributes further suggests that they act at distinct, and possibly sequential, steps during origin establishment and replication initiation. Moreover, we show that the phosphorylation profile of the FACT-associated MCM4 undergoes a cell cycle-dependent change, which is directly correlated with the catalytic activity of the FACT-MCM helicase complexes. Finally, at the quaternary structure level, physical interaction between FACT and MCM complexes is generally dependent on persistent cell cycle and further stabilized upon S phase entry. Cessation of mitotic cycle destabilizes the complex formation and likely leads to compromised coordination and activities. Conclusions: Together, our results correlate FACT-MCM functionally and temporally with S phase and DNA replication. They further demonstrate that enzymatic activities intrinsically important for DNA replication are tightly controlled at various levels, thereby ensuring proper progression of, as well as exit from, the cell cycle and ultimately euploid gene balance.