On membrane transport and internal entropy production

A plausible thermodynamic model for lysis of living cells has been proposed by correlating membrane fluidity and transport properties across biological membranes. The analysis presented here suggests that when the rate of internal entropy production exceeds a certain value, the regulatory mechanism involved in the transport of solutes across the membrane breaks down. Conditions describing the stability of the membrane structure and the role of leakage factors have been analysed.     

Bidens pilosa L.: Exclusive report of vivipary in a non-mangrove taxa from the eastern Himalayas

Vivipary is an extremely evolved type of epigeal germination in seed plants and very common in tropical estuarine plant communities (mangroves), ensuring a greater number of viable settings of offspring. In addition to this, there are some other non-mangrove angiosperm taxa that have a successful existence, and because of their invasive nature, vivipary has been reported as an attribute of their uncongenial abiotic stress. The present observation confirms the incidence of true vivipary in Bidens pilosa L. (a member of the family Asteraceae) from the eastern Himalayan region.     

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation

The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo‐, chondro‐, and adipo‐lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy. J. Cell. Biochem. 113: 3153–3164, 2012. © 2012 Wiley Periodicals, Inc.     

A simple and economical route to generate functional hepatocyte-like cells from hESCs and their application in evaluating alcohol induced liver damage

The in vitro derived hepatocytes from human embryonic stem cells (hESC) is a promising tool to acquire improved knowledge of the cellular and molecular events underlying early human liver development under physiological and pathological conditions. Here we report a simple two-step protocol employing conditioned medium (CM) from human hepatocellular carcinoma cell line, HepG2 to generate functional hepatocyte-like cells from hESC. Immunocytochemistry, flow cytometry, quantitative RT-PCR, and biochemical analyses revealed that the endodermal progenitors appeared as pockets in culture, and the cascade of genes associated with the formation of definitive endoderm (HNF-3β, SOX-17, DLX-5, CXCR4) was consistent and in concurrence with the up-regulation of the markers for hepatic progenitors [alpha-feto protein (AFP), HNF-4α, CK-19, albumin, alpha-1-antitrypsin (AAT)], followed by maturation into functional hepatocytes [tyrosine transferase (TAT), tryptophan-2, 3-dioxygenase (TDO), glucose 6-phosphate (G6P), CYP3A4, CYP7A1]. We witnessed that the gene expression profile during this differentiation process recapitulated in vivo liver development demonstrating a gradual down-regulation of extra embryonic endodermal markers (SOX-7, HNF-1β, SNAIL-1, LAMININ-1, CDX2), and the generated hepatic cells performed multiple liver functions. Since prenatal alcohol exposure is known to provoke irreversible abnormalities in the fetal cells and developing tissues, we exposed in vitro generated hepatocytes to ethanol (EtOH) and found that EtOH treatment not only impairs the survival and proliferation, but also induces apoptosis and perturbs differentiation of progenitor cells into hepatocytes. This disruption was accompanied by alterations in the expression of genes and proteins involved in hepatogenesis. Our results provide new insights into the wider range of destruction caused by alcohol on the dynamic process of liver organogenesis.     

A size dependent folding contour for cytochrome C

The paper describes an experimental construct of the folding route of the heme protein cytochrome-C. The construct highlights a slowing down near the nose of the folding funnel caused by the multiplicity of the energy traps near the native conformation created as a result of complex heme-peptide interaction. Interestingly the hydrodynamic size, the size heterogeneity and peroxidase activity serve as a triple measure of the distance of this near equilibrium departure from native conformation. Accordingly, the folding process is marked with a gradual and reversible reduction of mean hydrodynamic size, size heterogeneity and peroxidase activity (higher in unfolded state). The Dynamic Light Scattering based straightforward illustration of hydrodynamic size variation may serve as a model to slow folding observed in case of heme proteins, the heme itself serving as a natural facilitator for the native peptide conformation.     

A biosensor based on co-immobilized L-glutamate oxidase and L-glutamate dehydrogenase for analysis of monosodium glutamate in food

A monosodium glutamate (MSG) biosensor made by co-immobilized L-glutamate oxidase (L-GLOD) and L-glutamate dehydrogenase (L-GLDH) as the bio-component based on substrate recycling for highly sensitive MSG or L-glutamate determination, has been developed. Regeneration of MSG by substrate recycling provided an amplification of the sensor response. Higher signal amplification was found in the presence of ammonium ion. The sensor was standardized to determine MSG in the range of 0.02-3.0 mg/L. Linearity was obtained from 0.02 to 1.2 mg/L in presence of ammonium ion (10 mM) and NADPH (reduced nicotinamide adenine dinucleotide phosphate) (2 mM), but in absence of L-GLDH, the detection limit of MSG is confined to 0.1 mg/L. The apparent Km for MSG with L-GLOD-L-GLDH coupled reaction was 0.4451 mM but 1.9222 mM when only L-GLOD was immobilized. Cross linking with glutaraldehyde in the presence of bovine serum albumin (BSA) as a spacer molecule has been used for the method of immobilization. The response time of the sensor was 2 min. The optimum pH and temperature of the biosensor has been determined as 7+/-2 and 25+/-2 degrees C, respectively. The enzyme immobilized on the membrane was used for over 50 measurements. The standard error of the sample measurement was 4-5%. The activity of the enzyme-immobilized membrane was tested over a period of 60 days.     

Role of mitochondrial oxidative stress in the apoptosis induced by diospyrin diethylether in human breast carcinoma (MCF-7) cells

Mitochondria and associated oxidative stress have been shown to play critical roles in apoptotic death induced by various stress agents. Previously, we reported the antitumor property of diospyrin (D1), a plant-derived bisnaphthoquinonoid, and its diethylether derivative (D7), which was found to cause apoptotic death in human cancer cell lines. The present study aims to explore the relevant mechanism of apoptosis involving generation of cellular reactive oxygen species (ROS) by D7 in human breast carcinoma (MCF-7) cells. It was found that while D7 inhibited the proliferation of tumor cells, the associated apoptosis induced by D7 was prevented by treating the cells with N-acetyl-L: -cysteine (NAC), an antioxidant, and cyclosporine A (CsA), an inhibitor of mitochondrial permeability transition (MPT). Experiments using suitable inhibitors also demonstrated that D7 could alter the electron flow in mitochondrial electron transport chain by affecting target(s) between complex I and complex III, and indicated the probable site of D7-induced generation of ROS. These results were further supported by confocal microscopic observation on changes in mitochondrial organization and shape in cells treated with D7. Taken together, the results of our study clearly suggested that the apoptosis induced by D7 would involve alteration of MPT, cardiolipin peroxidation, migration of Bax from cytosol to mitochondria, decreased expression of Bcl-2, and release of cytochrome c, indicating oxidative mechanism at the mitochondrial level in the tumor cells.