Lentiviral Expression of Rabies Virus Glycoprotein in the Rat Hippocampus Strengthens Synaptic Plasticity

Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two μl (10⁸ T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat’s brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.

     

Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells

Background: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2).Aims: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state).Methods: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay.Results: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells.Conclusions: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.     

The clearance of viruses and transmissible spongiform encephalopathy agents from biologicals

The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small 'naked' viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development.     

Diabetes and hyperglycemia impair activation of mitochondrial K(ATP) channels

Hyperglycemia is an important predictor of cardiovascular mortality in patients with diabetes. We investigated the hypothesis that diabetes or acute hyperglycemia attenuates the reduction of myocardial infarct size produced by activation of mitochondrial ATP-regulated potassium (K(ATP)) channels. Acutely instrumented barbiturate-anesthetized dogs were subjected to a 60-min period of coronary artery occlusion and 3 h of reperfusion. Myocardial infarct size (triphenyltetrazolium chloride staining) was 25 +/- 1, 28 +/- 3, and 25 +/- 1% of the area at risk (AAR) for infarction in control, diabetic (3 wk after streptozotocin-alloxan), and hyperglycemic (15% intravenous dextrose) dogs, respectively. Diazoxide (2.5 mg/kg iv) significantly decreased infarct size (10 +/- 1% of AAR, P < 0.05) but did not produce protection in the presence of diabetes (28 +/- 5%) or moderate hyperglycemia (blood glucose 310 +/- 10 mg/dl; 23 +/- 2%). The dose of diazoxide and the degree of hyperglycemia were interactive. Profound (blood glucose 574 +/- 23 mg/dl) but not moderate hyperglycemia blocked the effects of high-dose (5.0 mg/kg) diazoxide [26 +/- 3, 15 +/- 3 (P < 0.05), and 11 +/- 2% (P < 0.05), respectively]. There were no differences in systemic hemodynamics, AAR, or coronary collateral blood flow (by radioactive microspheres) between groups. The results indicate that diabetes or hyperglycemia impairs activation of mitochondrial K(ATP) channels.     

Effect of intervertebral translational flexibilities on estimations of trunk muscle forces,kinematics, loads,and stability

Due to the complexity of the human spinal motion segments, the intervertebral joints are often simulated in the musculoskeletal trunk models as pivots thus allowing no translational degrees of freedom (DOFs). This work aims to investigate, for the first time, the effect of such widely used assumption on trunk muscle forces, spinal loads, kinematics, and stability during a number of static activities. To address this, the shear deformable beam elements used in our nonlinear finite element (OFE) musculoskeletal model of the trunk were either substantially stiffened in translational directions (SFE model) or replaced by hinge joints interconnected through rotational springs (HFE model). Results indicated that ignoring intervertebral translational DOFs had in general low to moderate impact on model predictions. Compared with the OFE model, the SFE and HFE models predicted generally larger L4–L5 and L5–S1 compression and shear loads, especially for tasks with greater trunk angles; differences reached ～15% for the L4–L5 compression, ～36% for the L4–L5 shear and ～18% for the L5–S1 shear loads. Such differences increased, as location of the hinge joints in the HFE model moved from the mid-disc height to either the lower or upper endplates. Stability analyses of these models for some select activities revealed small changes in predicted margin of stability. Model studies dealing exclusively with the estimation of spinal loads and/or stability may, hence with small loss of accuracy, neglect intervertebral translational DOFs at smaller trunk flexion angles for the sake of computational simplicity.     

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model

Background aimsDelivery of bone marrow–derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing.MethodsA transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro–computed tomography.ResultsAMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84.ConclusionsOur data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.     

Programmed cell death 1 (PDCD1) gene haplotypes and susceptibility of patients to basal cell carcinoma

Molecular Biology Reports - Programmed death-1 (PD-1), as an immunoinhibitory receptor encoded by programmed cell death-1 (PDCD1) gene, has a pivotal role in tolerance to self-antigens. Mutations...     

Three-dimensional finite element modeling of pericellular matrix and cell mechanics in the nucleus pulposus of the intervertebral disk based on in situ morphology

Nucleus pulposus (NP) cells of the intervertebral disk (IVD) have unique morphological characteristics and biologic responses to mechanical stimuli that may regulate maintenance and health of the IVD. NP cells reside as single cell, paired or multiple cells in a contiguous pericellular matrix (PCM), whose structure and properties may significantly influence cell and extracellular matrix mechanics. In this study, a computational model was developed to predict the stress–strain, fluid pressure and flow fields for cells and their surrounding PCM in the NP using three-dimensional (3D) finite element models based on the in situ morphology of cell–PCM regions of the mature rat NP, measured using confocal microscopy. Three-dimensional geometries of the extracellular matrix and representative cell–matrix units were used to construct 3D finite element models of the structures as isotropic and biphasic materials. In response to compressive strain of the extracellular matrix, NP cells and PCM regions were predicted to experience volumetric strains that were 1.9–3.7 and 1.4–2.1 times greater than the extracellular matrix, respectively. Volumetric and deviatoric strain concentrations were generally found at the cell/PCM interface, while von Mises stress concentrations were associated with the PCM/extracellular matrix interface. Cell–matrix units containing greater cell numbers were associated with higher peak cell strains and lower rates of fluid pressurization upon loading. These studies provide new model predictions for micromechanics of NP cells that can contribute to an understanding of mechanotransduction in the IVD and its changes with aging and degeneration.     

Oxygen-sensitive {delta}-opioid receptor-regulated survival and death signals: novel insights into neuronal preconditioning and protection

The detrimental effect of severe hypoxia (SH) on neurons can be mitigated by hypoxic preconditioning (HPC), but the molecular mechanisms involved remain unclear, and an understanding of these may provide novel solutions for hypoxic/ischemic disorders (e.g. stroke). Here, we show that the delta-opioid receptor (DOR), an oxygen-sensitive membrane protein, mediates the HPC protection through specific signaling pathways. Although SH caused a decrease in DOR expression and neuronal injury, HPC induced an increase in DOR mRNA and protein levels and reversed the reduction in levels of the endogenous DOR peptide, leucine enkephalin, normally seen during SH, thus protecting the neurons from SH insult. The HPC-induced protection could be blocked by DOR antagonists. The DOR-mediated HPC protection depended on an increase in ERK and Bcl 2 activity, which counteracted the SH-induced increase in p38 MAPK activities and cytochrome c release. The cross-talk between ERK and p38 MAPKs displays a "yinyang" antagonism under the control of the DOR-G protein-protein kinase C pathway. Our findings demonstrate a novel mechanism of HPC neuroprotection (i.e. the intracellular up-regulation of DOR-regulated survival signals).     

Highly efficient and selective methoxymethylation of alcohols and phenols catalyzed by high-valent tin(IV) porphyrin

An efficient and selective method for methoxymethylation of alcohols and phenols with formaldehyde dimethyl acetal (FDMA) catalyzed by electron deficient tin(IV)tetraphenylporphyrinato trifluoromethanesulfonate, [Sn^IV(TPP)(OTf)₂], is reported. A variety of primary, secondary and tertiary alcohols as well as phenols were converted to their corresponding methoxymethyl ethers with FDMA in the presence of a high-valent tin(IV) porphyrin. This catalyst can be used for selective methoxymethylation of primary, secondary and tertiary alcohols in the presence of phenols or tertiary alcohols. The present method offers several advantages such as short reaction times, high yields, simple procedure, selectivity and applicability for both alcohols and phenols.