Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.     

Molecular cloning and expression profile of snow trout GPDH gene in response to abiotic stress

Glycerol-3-phosphate dehydrogenase (GPDH) gene possibly plays a key role for cold acclimation process in snow trout during winter months when water temperature goes down to 4–5?°C. In this study, 1,012?bp nucleotide fragment of GPDH gene was obtained from two snow trout species (Schizothorax richardsonii and S. niger; family: Cyprinidae), distributed in several Himalayan rivers. The gene encoded a protein of 334 amino acids. The encoded protein sequence was very similar to GPDH of Danio rerio (94.36?%) using BLASTx searches. In S. richardsonii the qRT-PCR showed highest expression in muscle tissue followed by liver and also revealed 19 fold gene expression in liver tissue under cold (5?°C) in comparison with warm (15?°C) condition. The elevated expression levels of GPDH cDNA on cold treatment furthermore suggest that GPDH plays a role in stress related responses in S. richardsonii. The phylogenetic analysis showed that the two snow trout species GPDH share the same clade with characterized GPDHs from other teleost fishes suggesting a common evolutionary origin and a similar catalytic function. In addition, the Ka/Ks ratios of these sequences suggested that they are under purifying selection. Moreover, the expression profile of GPDH gene among co generic species of genus Schizothorax showed that GPDH cDNA expression was highest in S. richardsonii and lowest in S. esocinus which gives an indication of species specific adaptation in relation to different geographical areas.     

Berger C. Mayne (1920–2011): a friend and his contributions to photosynthesis research

We provide here insights on the life and work of Berger C. Mayne (1920-2011). We remember and honor Berger, whose study of photosynthesis began with the most basic processes of intersystem electron transport and oxygen evolution, continued with application of fluorescence techniques to the study of photophosphorylation and the unique features of photosystems in specialized cells, and concluded with collaborative study of photosynthesis in certain nitrogen fixing symbioses. Berger loved the outdoors and was dedicated to preserving the environment and to social justice, and was a wonderful friend.     

Hyperhomocysteinemia decreases intestinal motility leading to constipation

Elevated levels of plasma homocysteine (Hcy) called hyperhomocysteinemia (HHcy) have been implicated in inflammation and remodeling in intestinal vasculature, and HHcy is also known to aggravate the pathogenesis of inflammatory bowel disease (IBD). Interestingly, colon is the pivotal site that regulates Hcy levels in the plasma. We hypothesize that HHcy decreases intestinal motility through matrix metalloproteinase-9 (MMP-9)-induced intestinal remodeling leading to constipation. To verify this hypothesis, we used C57BL/6J or wild-type (WT), cystathionine β-synthase (CBS(+/-)), MMP-9(-/-), and MMP-9(-/-) + Hcy mice. Intestinal motility was assessed by barium meal studies and daily feces output. Plasma Hcy levels were measured by HPLC. Expression of ICAM-1, inducible nitric oxide synthase, MMP-9, and tissue inhibitors of MMPs was studied by Western blot and immunohistochemistry. Reactive oxygen species (ROS) including super oxide were measured by the Invitrogen molecular probe method. Tissue nitric oxide levels were assessed by a commercially available kit. Plasma Hcy levels in the treated MMP-9 group mice were comparable to CBS(+/-) mice. Barium meal studies suggest that intestinal motility is significantly decreased in CBS(+/-) mice compared with other groups. Fecal output-to-body weight ratio was significantly reduced in CBS(+/-) mice compared with other groups. There was significant upregulation of MMP-9, iNOS, and ICAM-1 expression in the colon from CBS(+/-) mice compared with WT mice. Levels of ROS, superoxide, and inducible nitric oxide were elevated in the CBS(+/-) mice compared with other groups. Results suggest that HHcy decreases intestinal motility due to MMP-9-induced intestinal remodeling leading to constipation.     

Mitochondrial division/mitophagy inhibitor (Mdivi) ameliorates pressure overload induced heart failure

Background

We have previously reported the role of anti-angiogenic factors in inducing the transition from compensatory cardiac hypertrophy to heart failure and the significance of MMP-9 and TIMP-3 in promoting this process during pressure overload hemodynamic stress. Several studies reported the evidence of cardiac autophagy, involving removal of cellular organelles like mitochondria (mitophagy), peroxisomes etc., in the pathogenesis of heart failure. However, little is known regarding the therapeutic role of mitochondrial division inhibitor (Mdivi) in the pressure overload induced heart failure. We hypothesize that treatment with mitochondrial division inhibitor (Mdivi) inhibits abnormal mitophagy in a pressure overload heart and thus ameliorates heart failure condition.

Materials and Methods

To verify this, ascending aortic banding was done in wild type mice to create pressure overload induced heart failure and then treated with Mdivi and compared with vehicle treated controls.

Results

Expression of MMP-2, vascular endothelial growth factor, CD31, was increased, while expression of anti angiogenic factors like endostatin and angiostatin along with MMP-9, TIMP-3 was reduced in Mdivi treated AB 8 weeks mice compared to vehicle treated controls. Expression of mitophagy markers like LC3 and p62 was decreased in Mdivi treated mice compared to controls. Cardiac functional status assessed by echocardiography showed improvement and there is also a decrease in the deposition of fibrosis in Mdivi treated mice compared to controls.

Conclusion

Above results suggest that Mdivi inhibits the abnormal cardiac mitophagy response during sustained pressure overload stress and propose the novel therapeutic role of Mdivi in ameliorating heart failure.     

Homology inference of protein-protein interactions via conserved binding sites

The coverage and reliability of protein-protein interactions determined by high-throughput experiments still needs to be improved, especially for higher organisms, therefore the question persists, how interactions can be verified and predicted by computational approaches using available data on protein structural complexes. Recently we developed an approach called IBIS (Inferred Biomolecular Interaction Server) to predict and annotate protein-protein binding sites and interaction partners, which is based on the assumption that the structural location and sequence patterns of protein-protein binding sites are conserved between close homologs. In this study first we confirmed high accuracy of our method and found that its accuracy depends critically on the usage of all available data on structures of homologous complexes, compared to the approaches where only a non-redundant set of complexes is employed. Second we showed that there exists a trade-off between specificity and sensitivity if we employ in the prediction only evolutionarily conserved binding site clusters or clusters supported by only one observation (singletons). Finally we addressed the question of identifying the biologically relevant interactions using the homology inference approach and demonstrated that a large majority of crystal packing interactions can be correctly identified and filtered by our algorithm. At the same time, about half of biological interfaces that are not present in the protein crystallographic asymmetric unit can be reconstructed by IBIS from homologous complexes without the prior knowledge of crystal parameters of the query protein.     

Mitochondrial mitophagic mechanisms of myocardial matrix metabolism and remodelling

High levels of homocysteine (Hcy), known as hyperhomocysteinmia (HHcy), are correlated with an increase in extracellular matrix remodelling (ECM) via the matrix metalloproteinases (MMPs) and plasminogen/plasmin system. This results in an increase deposition of collagen that leads to endothelial-myocyte (EM) and myocyte-myocyte (MM) uncoupling; the physiological consequences are a plethora of cardiovascular pathologies. Homocysteine-induced increase in intracellular and mitochondrial Ca(2+) plays an important role in increasing reactive oxygen species (ROS) within mitochondria and instigating mitophagy within the cell. This occurs via several Hcy-mitigated processes: agonizing N-methyl-d-aspartate receptor-1 (NMDA-R1), decreasing expression of peroxisome proliferator activator receptor (PPAR) [thereby increasing oxidation], impairing Ca(2+) handling via Na(+)/Ca(2+) exchanger (NCX1) and Sarco endoplasmic reticulum Ca(2+) ATPase (SERCA-2a). The end result is an increase in ROS that directly or indirectly lead to MMP activation within mitochondria or the cytoplasm. Hcy induces a mitochondrial permeability transition that allows MMPs to be released from mitochondria thereby metabolizing matrix and impairing cardiac function. Further work remains to be elucidated concerning the specific mitochondrial mitophagic mechanisms under which matrix metabolism and remodelling occurs. Moreover, the therapeutic implications of NMDA and PPAR ligands are some promise to patient.