Proliferation of Myoblast Skeletal Cells on Three-Dimensional Supermacroporous Cryogels

Cardiac and skeletal muscle tissue engineering provides a smart approach to overcome problems associated with organ transplantation and cardiac tissue and also lays a platform for superior alternative approaches in muscle regeneration. The aim of the study was to demonstrate cryogel scaffold potential in the field of skeletal muscle and cardiac tissue engineering. Poly-hydroxyethyl methacrylate (pHEMA)-gelatin cryogel scaffold was synthesized using cryogelation technique and such a designed material is being reported first time. Rheology study of the pHEMA-gelatin (HG) suggested that the cryogel scaffolds were stable at different temperatures and phase angle remained constant in both dry and wet state. HG cryogel was able to bear increased stress without leading to deformation. Monitoring the hydration of HG scaffold showed shift from a stiff to a more pliable material and upon continuing hydration, shear modulus remained constant with no further change observed. However, the change in phase angle <0.24º indicates a gradual increase in stiffness of the material over time. Scaffold synthesised using such polymer combinations gave cells a native environment for proliferation and surface stiffness have shown to help in differentiation of the cells. Myoskeletal cell lines were cultured on these scaffolds to check the biocompatibility and cell proliferation. Alamar blue assay performed over a period of 3 weeks analysed the metabolic activity of cells which showed more than 60% increase in the total cellular activity. DNA content of cells was found to be directly related to number of cells present at a given time point and this was found to have increased by more than 50% in 3 weeks. Since in 3-D scaffold the surface area is more in comparison to 2-D, hence better cell proliferation is observed. Hoechst and DAPI staining showed tubular structure and alignment of the cells during formation of the tubules shows promising cellular response to the cryogel matrix. The mechanical strength, stiffness and elastic measurements of the scaffold indicated potential application of these materials for skeletal and cardiac tissue engineering.     

Chloroquine treatment increases detection of 5-fluorouracil-induced apoptosis index in vivo

Although apoptosis can be readily assessed in vitro with a variety of techniques, the detection of apoptosis in the in vivo setting poses a much more difficult proposition. Apoptosis in an organism is followed almost inevitably by rapid clearance of dying cells via phagocytosis, thus limiting the ability to analyze apoptosis in vivo using classical techniques. To address this issue, we developed a method to enhance in vivo apoptosis detection using pretreatment with chloroquine, an inhibitor of macrophage activity, in Swiss albino mice. This technique resulted in a significant increase in the accumulation of apoptotic cells induced by 5-fluorouracil, as detected by propidium iodide staining in solid and ascitic forms of Ehrlich ascitic tumors and in bone marrow cells. We further validated our technique using DNA fragmentation and endonuclease assays. Our results demonstrated that chloroquine pretreatment can significantly enhance accumulation of apoptotic cells in organisms, and we envision combining this method with modern imaging techniques to optimize in vivo detection of apoptosis.     

Single additional methylene group in the head-group region imparts high gene transfer efficacy to a transfection-incompetent cationic lipid

The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45^Ntf4) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45^Ntf4, is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45^Ntf4. In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss-of-function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45^Ntf4 and/or SIPK, are involved in pollen germination.     

Pyramid environment reduces the wound healing suppressant properties of dexamethasone in albino rats

With a view to investigate the contribution and role of environment within a wooden pyramid model on the wound healing suppressant effect of dexamethasone in rats, wound breaking strength, dry weight, hydroxyproline content and histology of granulation tissue of the dead space wound were studied in rats. The results indicate that the environment within the wooden pyramid not only promotes significant wound healing but also reduces the wound healing suppressant effect of dexamethasone. Histological studies also confirmed the results.     

Enhancement of gp120-specific immune responses by genetic vaccination with the human immunodeficiency virus type 1 envelope gene fused to the gene coding for soluble CTLA4

DNA vaccines exploit the inherent abilities of professional antigen-presenting cells to prime the immune system and to elicit immunity against diverse pathogens. In this study, we explored the possibility of augmenting human immunodeficiency virus type 1 gp120-specific immune responses by a DNA vaccine coding for a fusion protein, CTLA4:gp120, in mice. In vitro binding studies revealed that secreted CTLA4:gp120 protein induced a mean florescence intensity shift, when incubated with Raji B cells, indicating its binding to B7 proteins on Raji B cells. Importantly, we instituted three different vaccination regimens to test the efficacy of DNA vaccines encoding gp120 and CTLA4:gp120 in the induction of both cellular (CD8(+)) and antibody responses. Each of the vaccination regimens incorporated a single intramuscular (i.m.) injection of the DNA vaccines to prime the immune system, followed by two booster injections. The i.m.-i.m.-i.m. regimen induced only modest levels of gp120-specific CD8(+) T cells, but the antibody response by CTLA4:gp120 DNA was nearly 16-fold higher than that induced by gp120 DNA. In contrast, using the i.m.-subcutaneous (s.c.)-i.m. regimen, it was found that gp120 and CTLA4:gp120 DNAs were capable of inducing significant levels of gp120-specific CD8(+) T cells (3.5 and 11%), with antibody titers showing a modest twofold increase for CTLA4:gp120 DNA. In the i.m.-gene gun (g.g.)-g.g. regimen, the mice immunized with gp120 and CTLA4:gp120 harbored gp120-specific CD8(+) T cells at frequencies of 0.9 and 2.9%, with the latter showing an eightfold increase in antibody titers. Thus, covalent antigen modification and the routes of genetic vaccination have the potential to modulate antigen-specific immune responses in mice.     

Down-Regulation of AMPA Glutamate Receptors Reduces Cerebrocortical Metabolic Response to Stimulation

We tested the hypothesis that chronic stimulation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) glutamate receptors with an agonist causes down-regulation of the receptor protein and a decrement in basal and/or stimulated cerebral O2 consumption. Male Wistar rats were intradurally infused with 10 microM AMPA by an osmotic pump at a rate of 1 microl/h for 6 days. As a result, the specific binding of (S)-[3H]-5-fluorowillardiine to AMPA receptors in the cerebral cortex decreased 46% from 2.7 +/- 0.3 to 1.5 +/- 0.6 (density units). Under isoflurane anesthesia and after topical stimulation to the right cerebral cortex with 10(-3) M AMPA, cerebral blood flow (14C-iodoantipyrine method) and O2 consumption (cryomicrospectrophotometrically determined) were determined in control and down-regulated rats. Down-regulation of AMPA receptors did not alter basal O2 consumption. In control, after agonist stimulation, the O2 consumption in the ipsilateral cortex increased by 34%, (4.7 +/- 0.5 ml O2 x min(-1) x 100 g(-1) compared to 3.5 +/- 0.4 in the contralateral cortex). In the down-regulated rats, the O2 consumption did not significantly increase (4.0 +/- 1.5 ml O2 x min(-1) x 100 g(-1) compared to 3.3 +/- 1.7 in the contralateral cortex) after AMPA. In conclusion, following chronic simulation, AMPA receptors underwent down-regulation, but such down-regulation did not alter basal cerebrocortical blood flow or O2 consumption. AMPA down-regulation reduced the agonist stimulated increase in cortical O2 consumption.     

Phosphorylation-regulated inhibition of the Gz GTPase-activating protein activity of RGS proteins by synapsin I

Synapsins are neuronal proteins that bind and cluster synaptic vesicles in the presynaptic space, presumably by anchoring to actin filaments, but specific regulatory functions of the synapsins are unknown. We found that a sub-population of brain synapsin Ia, a splice variant of one of three synapsin isoforms, inhibits the GTPase-activating protein (GAP) activity of several RGS proteins. Inhibition is highly selective for Galphaz, a member of the Gi family that is found in neurons, platelets, adrenal chromaffin cells, and a few other neurosecretory cells. Gz has been indirectly implicated in the regulation of secretion. Synapsin Ia constitutes a major fraction of the total GAP-inhibitory activity in brain, and its inhibitory activity is absent from the brains of synapsin I(-/-)/II(-/-) mice. Inhibition depends on the cationic D/E domain of synapsin. Phosphorylation of synapsin Ia at serine 9 by either cyclic AMP-dependent protein kinase or p21-activated protein kinase (PAK1) attenuates its potency as a GAP inhibitor more than 7-fold. Synapsin can thus act as a phosphorylation-modulated mediator of feedback regulation of Gz signaling by the synaptic machinery.     

Homer 2 tunes G protein-coupled receptors stimulus intensity by regulating RGS proteins and PLCbeta GAP activities

Homers are scaffolding proteins that bind G protein-coupled receptors (GPCRs), inositol 1,4,5-triphosphate (IP3) receptors (IP3Rs), ryanodine receptors, and TRP channels. However, their role in Ca2+ signaling in vivo is not known. Characterization of Ca2+ signaling in pancreatic acinar cells from Homer2-/- and Homer3-/- mice showed that Homer 3 has no discernible role in Ca2+ signaling in these cells. In contrast, we found that Homer 2 tunes intensity of Ca2+ signaling by GPCRs to regulate the frequency of [Ca2+]i oscillations. Thus, deletion of Homer 2 increased stimulus intensity by increasing the potency for agonists acting on various GPCRs to activate PLCbeta and evoke Ca2+ release and oscillations. This was not due to aberrant localization of IP3Rs in cellular microdomains or IP3R channel activity. Rather, deletion of Homer 2 reduced the effectiveness of exogenous regulators of G proteins signaling proteins (RGS) to inhibit Ca2+ signaling in vivo. Moreover, Homer 2 preferentially bound to PLCbeta in pancreatic acini and brain extracts and stimulated GAP activity of RGS4 and of PLCbeta in an in vitro reconstitution system, with minimal effect on PLCbeta-mediated PIP2 hydrolysis. These findings describe a novel, unexpected function of Homer proteins, demonstrate that RGS proteins and PLCbeta GAP activities are regulated functions, and provide a molecular mechanism for tuning signal intensity generated by GPCRs and, thus, the characteristics of [Ca2+]i oscillations.