S7111 Sigma-AldrichApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

Maxillofacial dysmorphogenesis is found in 5% of the population. To begin to understand the mechanisms required for maxillofacial morphogenesis, we employed the inhibitors of the differentiation 2 (Id2) knock-out mouse model, in which Id proteins, members of the regulator of basic helix-loop-helix (bHLH) transcription factors, modulate cell proliferation, apoptosis, and differentiation. We now report that spatially-restricted growth defects are localized at the skull base of Id2 KO mice. Curiously, at birth, neither the mutant Id2 KO nor wild-type (WT) mice differed, based upon cephalometric and histological analyses of cranial base synchondroses. In postnatal week 2, a narrower hypertrophic zone and an inhibited proliferative zone in presphenoid synchondrosis (PSS) and spheno-occipital synchondrosis (SOS) with maxillary hypoplasia were identified in the Id2 mutant mice. Complementary studies revealed that exogenous bone morphogenetic proteins (BMPs) enhanced cartilage growth, matrix deposition, and chondrocyte proliferation in the WT but not in the mutant model. Id2-deficient chondrocytes expressed more Smad7 transcripts. Based on our results, we assert that Id2 plays an essential role, acting downstream of BMP signaling, to regulate cartilage formation at the postnatal stage by enhancing BMP signals through inhibiting Smad7 expression. As a consequence, abnormal endochondral ossification was observed in cranial base synchondroses during the postnatal growth period, resulting in the clinical phenotype of maxillofacial dysmorphogenesis.

Intraventricular ependymal infection by adenoviruses expressing brain-derived neurotrophic factor (BDNF) and noggin is sufficient to induce the heterotopic recruitment of new medium spiny neurons to the adult neostriatum, from endogenous subependymal neural progenitor cells. This approach was found to slow disease progression and extend survival in an R6/2 mouse model of Huntington's disease (HD). However, the practical therapeutic value of this strategy is limited by the transient expression and immunogenicity of adenoviral vectors. In addition, it has been unclear whether sustained overexpression of BDNF and noggin would yield similarly sustained neuronal production and striatal recruitment, or whether progenitor depletion or tachyphylaxis might supervene to limit the therapeutic potential of this approach. To address these issues, we used adeno-associated virus serotype 4 (AAV4), an ependymotrophic vector that is neither immunogenic nor neurotoxic, to achieve sustained BDNF and noggin expression. Using AAV4, we found that BDNF and noggin achieved levels sufficient to initiate and maintain, for at least 4 months, ongoing neuronal addition to the neostriatum and olfactory bulb. Over this period, we noted no diminution of treatment-associated neuronal recruitment from resident progenitors. AAV4:BDNF and noggin-induced neuronal addition may thus provide a means to provide longlasting and persistent striatal neuronal replacement in conditions of striatal neuronal loss, such as HD.

Development of the adult olfactory system of the moth Manduca sexta depends on reciprocal interactions between olfactory receptor neuron (ORN) axons growing in from the periphery and centrally-derived glial cells. Early-arriving ORN axons induce a subset of glial cells to proliferate and migrate to form an axon-sorting zone, in which later-arriving ORN axons will change their axonal neighbors and change their direction of outgrowth in order to travel with like axons to their target areas in the olfactory (antennal) lobe. These newly fasciculated axon bundles will terminate in protoglomeruli, the formation of which induces other glial cells to migrate to surround them. Glial cells do not migrate unless ORN axons are present, axons fail to fasciculate and target correctly without sufficient glial cells, and protoglomeruli are not maintained without a glial surround. We have shown previously that Epidermal Growth Factor receptors and the IgCAMs Neuroglian and Fasciclin II play a role in the ORN responses to glial cells. In the present work, we present evidence for the importance of glial Fibroblast Growth Factor receptors in glial migration, proliferation, and survival in this developing pathway. We also report changes in growth patterns of ORN axons and of the dendrites of olfactory (antennal lobe) neurons following blockade of glial FGFR activation that suggest that glial FGFR activation is important in reciprocal communication between neurons and glial cells.

Biomaterials that can drive stem cells to an appropriate differentiation level and decrease apoptosis of transplanted cells are needed in regenerative medicine. Nanomaterials are promising novel materials for such applications. Here we reported that carboxylated multiwalled carbon nanotube (MWCNT 1) promotes myogenic differentiation of mouse myoblast cells and inhibits cell apoptosis under the differentiation conditions by regulating basic helix-loop-helix transcription factors. MWCNT 1 attenuates bone morphogenetic protein receptor (BMPR) signaling activity by binding to BMPR2 and attenuating the phosphorylation of BMPR1. This molecular understanding allowed us to tune stem cell differentiation to various levels by chemical modifications, demonstrating human control of biological activities of nanoparticles and opening an avenue for potential applications of nanomaterials in regenerative medicine.

Myocardin belongs to the SAF-A/B, Acinus, PIAS (SAP) domain family of transcription factors and is specifically expressed in cardiac and smooth muscle. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. We have previously found that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of cardiac and smooth muscle genes through recruiting chromatin-modifying enzyme p300, yet no studies have determined whether myocardin itself is similarly modified. In this study, we show that myocardin is a direct target for p300-mediated acetylation. p300 acetylates lysine residues at the N terminus of the myocardin protein. Interestingly, a direct interaction between p300 and myocardin, which is mediated by the C terminus of myocardin, is required for the acetylation event. Acetylation of myocardin by p300 enhances the association of myocardin and SRF as well as the formation of the myocardin-SRF-CArG box ternary complex. Conversely, acetylation of myocardin decreases the binding of histone deacetylase 5 (HDAC5) to myocardin. Acetylation of myocardin is required for myocardin to activate smooth muscle genes. Our study demonstrates that acetylation plays a key role in modulating myocardin function in controlling cardiac and smooth muscle gene expression.

In the degenerative disc, overproduction of reactive oxygen species (ROS) involves in apoptosis and senescence of nucleus pulposus (NP) cells that could accelerate the degenerative process. Ferulic acid (FA) has been reported to have an excellent antioxidant property. In the study, injectable thermosensitive chitosan/gelatin/glycerol phosphate (C/G/GP) hydrogel was applied as a controlled release system for FA delivery. The study was aimed to evaluate possible therapeutic effects of FA-incorporated C/G/GP hydrogel on hydrogen peroxide (H(2)O(2))-induced oxidative stress NP cells. The results showed that the release of FA from C/G/GP hydrogel could decrease the H(2)O(2)-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H(2)O(2)-induced oxidative stress NP cells showed up-regulation of Aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated-glycosaminoglycans (GAGs) to DNA ratio and alcian blue staining revealed that the GAGs production of H(2)O(2)-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H(2)O(2)-induced oxidative stress NP cells. The results suggested that the C/G/GP hydrogel was very suitable for sustained delivery of FA. The FA-incorporated C/G/GP hydrogel would be used to treat the degenerative disc in the early stage before it developed into the latter irreversible stages.

Paclitaxel, a potent chemotherapeutic utilized in a variety of cancers, can be limited in its effectiveness due to inherent insolubility in aqueous media and acquired chemoresistance within certain cells. An approach has been developed for increasing Paclitaxel solubility and effectiveness by covalent attachment to gold nanoparticles via DNA linkers. The resulting conjugates are highly soluble in aqueous buffer, exhibiting greater than a 50-fold increase in solubility over the unconjugated drug. DNA linkers are labeled with a fluorophore, which affords a convenient means of visualizing resultant conjugates within cells. Internalized conjugates demonstrate increased activity as compared with free drug across a variety of cell types, including a Paclitaxel-resistant cell line. Attachment to DNA-nanoparticle conjugates may become a general strategy for solubilizing and enhancing a wide variety of therapeutic agents in aqueous media.

Background and aims: Acute liver failure (ALF) is frequently complicated by cerebral oedema, systemic inflammation and multiorgan dysfunction. Vascular endothelial growth factor (VEGF) may stimulate liver regeneration but it can also be pro-inflammatory, activating endothelial cells and increasing permeability, actions mediated through Src kinase signalling. We therefore examined whether a Src inhibitor could have therapeutic potential in ALF. Methods: Murine ALF was induced with azoxymethane. Liver pathology was graded by a blinded examiner and apoptosis quantified by immunohistochemistry. Cerebral VEGF expression was imaged using VEGF-green fluorescent protein transgenic mice. Circulating and macrophage-secreted VEGF levels were measured. Experimental animals received a Src inhibitor or vehicle controls. Results: VEGF was undetectable in normal plasma but reached a mean of 835 pg/ml at grade III encephalopathy (P<0.001). Ammonia, lipopolysaccharide and interferon-gamma acted synergistically to enhance VEGF secretion by macrophages. Production of VEGF by cerebral cortical astrocytes increased with disease progression. Late treatment with inhibitors of Src or VEGF did not improve liver histology, encephalopathy or survival. However, early use of a Src kinase inhibitor significantly reduced hepatic injury, delayed encephalopathy and allowed 25% of mice to survive an otherwise lethal insult. Conclusion: Systemic and cerebral VEGF levels are significantly elevated during experimental ALF and may be exacerbated by hyperammonemia and macrophage activation. Early use of a Src inhibitor reduced hepatocellular injury and enabled survival, indicating such agents may have some promise in the treatment of ALF.© 2011 John Wiley & Sons A/S.

The primary aim of this study was to compare the effects of pentoxifylline (PTX) versus placebo on the histological features of nonalcoholic steatohepatitis (NASH). In all, 55 adults with biopsy-confirmed NASH were randomized to receive PTX at a dose of 400 mg three times a day (n = 26) or placebo (n = 29) over 1 year. The primary efficacy endpoint was defined as improvement in histological features of NASH through reduction in steatosis, lobular inflammation, and/or hepatocellular ballooning as reflected by a decrease of ≥2 points in the nonalcoholic fatty liver disease (NAFLD) activity score (NAS). After 1 year, intention-to-treat analysis showed a decrease of ≥2 points in the NAS in 38.5% of patients on PTX versus 13.8% of those on placebo (P = 0.036). Per protocol analysis, a decrease of ≥2 points in the NAS from baseline was observed in 50% of the patients on PTX versus 15.4% of those on placebo (P = 0.01). The mean change in NAS score from baseline was -1.6 in the PTX group, versus -0.1 in the placebo group (P < 0.001). PTX significantly improved steatosis (mean change in score -0.9 versus -0.04 with placebo, P < 0.001) and lobular inflammation (median change -1 versus 0 with placebo, P = 0.02). No significant effects in hepatocellular ballooning were observed. PTX also improved liver fibrosis (mean change in fibrosis score was -0.2 among those on PTX versus +0.4 among those on placebo, P = 0.038). Although not statistically significant (P = 0.17), improvement in fibrosis was observed in a greater proportion (35%) of patients in the PTX group compared to placebo (15%). Adverse effects were similar in both groups. Conclusion: PTX improved histological features of NASH compared to placebo. PTX was well tolerated in patients with NASH (ClinicalTrials.gov number NCT00590161). (HEPATOLOGY 2011).

We used the erythropoietin enhancer and Simian virus-40 promoter to create a hypoxia-inducible gene expression system to investigate the effect of vascular endothelial growth factor (VEGF) gene therapy on neuroprotection and neurogenesis in organotypic spinal cord slice culture. The organotypic spinal cord slice culture transfected with pEpo-SV-VEGF expressed the highest amount of VEGF under hypoxic conditions and showed decreased apoptosis and increased proliferation, and evidence of neurogenesis. Our results show that the hypoxia-induced VEGF expression in an organotypic spinal cord slice culture may lead to an optimal treatment for spinal cord injury.