Elevated lipid peroxides induced angiogenesis in proliferative diabetic retinopathy

Oxidative stress is associated with causation of diabetic vascular complications. A case–control study was undertaken to evaluate the association of platelet thiobarbituric acid reacting substances (TBARS) with the severity of diabetic retinopathy for the first time. Platelet TBARS levels were estimated using standard protocol. Platelet TBARS levels in the cases with non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, and healthy controls were 0.56 ± 0.09, 0.69 ± 0.11 and 0.41 ± 0.1 nmol/h/10⁸ platelets, respectively. A significant increase in platelet TBARS levels was observed in the cases as compared to controls (p < 0.001). Elevated TBARS levels were observed to significantly increase further during the proliferative stage of the disease (p < 0.01). The increase in platelet TBARS levels, and thereby at retinal level, is associated with angiogenesis in diabetic retinopathy. Supplemental anti-oxidant therapy in diabetic retinopathy may prevent ocular angiogenesis resulting as a consequence of oxidative stress.     

An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells

Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in?vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low, suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as?a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication, nor did the DDR marks colocalize with latent episomes. Rather, a transient period of EBV-induced hyperproliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoprotein EBNA3C was required to attenuate the EBV-induced DDR. We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR that is attenuated by viral latency products to induce cell immortalization.     

Hepatic stem cells: A viable approach for the treatment of liver cirrhosis

Liver cirrhosis is characterized by distortion of liver architecture, necrosis of hepatocytes and regenerative nodules formation leading to cirrhosis. Various types of cell sources have been used for the management and treatment of decompensated liver cirrhosis. Knowledge of stem cells has offered a new dimension for regenerative therapy and has been considered as one of the potential adjuvant treatment modality in patients with end stage liver diseases (ESLD). Human fetal hepatic progenitor cells are less immunogenic than adult ones. They are highly propagative and challenging to cryopreservation. In our earlier studies we have demonstrated that fetuses at 10-18 wk of gestation age contain a large number of actively dividing hepatic stem and progenitor cells which possess bi-potent nature having potential to differentiate into bile duct cells and mature hepatocytes. Hepatic stem cell therapy for the treatment of ESLD is in their early stage of the translation. The emerging technology of decellularization and recellularization might offer a significant platform for developing bioengineered personalized livers to come over the scarcity of desired number of donor organs for the treatment of ESLD. Despite these significant advancements long-term tracking of stem cells in human is the most important subject nowadays in order to answer several unsettles issues regarding the route of delivery, the choice of stem cell type(s), the cell number and the time-point of cell delivery for the treatment in a chronic setting. Answering to these questions will further contribute to the development of safer, noninvasive, and repeatable imaging modalities that could discover better cell therapeutic approaches from bench to bed-side. Combinatorial approach of decellularization and nanotechnology could pave a way towards the better understanding in determination of cell fate post-transplantation.     

Sulfo-N-hydroxysuccinimide interferes with bicinchoninic acid protein assay

A high-performance liquid chromatography (HPLC)-based fluorometric method for measuring serine hydroxymethyltransferase (SHMT) activity toward formation of serine and (6S)-H₄PteGlu_n has been developed. In this method, serine formed by SHMT activity is reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to form the fluorescent adduct NBD–serine. The fluorescent assay components are then separated by reversed-phase chromatography, and NBD–serine is quantified by comparison with standards. This method was used to determine the K_m and k_cat values for 5,10-CH₂–H₄PteGlu₅ of an SHMT from Arabidopsis thaliana. These data represent the first determination of kinetic parameters for (6S)-5,10-CH₂–H₄PteGlu₅ for an SHMT from any organism.     

Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential

To better define the still unclear role of proline (Pro) metabolism in drought resistance, we analyzed Arabidopsis (Arabidopsis thaliana) Δ(1)-pyrroline-5-carboxylate synthetase1 (p5cs1) mutants deficient in stress-induced Pro synthesis as well as proline dehydrogenase (pdh1) mutants blocked in Pro catabolism and found that both Pro synthesis and catabolism were required for optimal growth at low water potential (ψ(w)). The abscisic acid (ABA)-deficient mutant aba2-1 had similar reduction in root elongation as p5cs1 and p5cs1/aba2-1 double mutants. However, the reduced growth of aba2-1 but not p5cs1/aba2-1 could be complemented by exogenous ABA, indicating that Pro metabolism was required for ABA-mediated growth protection at low ψ(w). PDH1 maintained high expression in the root apex and shoot meristem at low ψ(w) rather than being repressed, as in the bulk of the shoot tissue. This, plus a reduced oxygen consumption and buildup of Pro in the root apex of pdh1-2, indicated that active Pro catabolism was needed to sustain growth at low ψ(w). Conversely, P5CS1 expression was most highly induced in shoot tissue. Both p5cs1-4 and pdh1-2 had a more reduced NADP/NADPH ratio than the wild type at low ψ(w). These results indicate a new model of Pro metabolism at low ψ(w) whereby Pro synthesis in the photosynthetic tissue regenerates NADP while Pro catabolism in meristematic and expanding cells is needed to sustain growth. Tissue-specific differences in Pro metabolism and function in maintaining a favorable NADP/NADPH ratio are relevant to understanding metabolic adaptations to drought and efforts to enhance drought resistance.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

PKCγ is required for ethanol-induced increases in GABA(A) receptor α4 subunit expression in cultured cerebral cortical neurons

Ethanol exposure produces alterations in GABA(A) receptor function and expression associated with CNS hyperexcitability, but the mechanisms of these effects are unknown. Ethanol is known to increase both GABA(A) receptor α4 subunits and protein kinase C (PKC) isozymes in vivo and in vitro. Here, we investigated ethanol regulation of GABA(A) receptor α4 subunit expression in cultured cortical neurons to delineate the role of PKC. Cultured neurons were prepared from rat pups on postnatal day 0-1 and tested after 18?days. GABA(A) receptor α4 subunit surface expression was assessed using P2 fractionation and surface biotinylation following ethanol exposure for 4?h. Miniature inhibitory post-synaptic currents were measured using whole cell patch clamp recordings. Ethanol increased GABA(A) receptor α4 subunit expression in both the P2 and biotinylated fractions, while reducing the decay time constant in miniature inhibitory post-synaptic currents, with no effect on γ2 or δ subunits. PKC activation mimicked ethanol effects, while the PKC inhibitor calphostin C prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression. PKCγ siRNA knockdown prevented ethanol-induced increases in GABA(A) receptor α4 subunit expression, but inhibition of the PKCβ isoform with PKCβ pseudosubstrate had no effect. We conclude that PKCγ regulates ethanol-induced alterations in α4-containing GABA(A) receptors.     

Rab13-dependent trafficking of RhoA is required for directional migration and angiogenesis

Angiogenesis requires concomitant remodeling of cell junctions and migration, as exemplified by recent observations of extensive endothelial cell movement along growing blood vessels. We report that a protein complex that regulates cell junctions is required for VEGF-driven directional migration and for angiogenesis in vivo. The complex consists of RhoA and Syx, a RhoA guanine exchange factor cross-linked by the Crumbs polarity protein Mupp1 to angiomotin, a phosphatidylinositol-binding protein. The Syx-associated complex translocates to the leading edge of migrating cells by membrane trafficking that requires the tight junction recycling GTPase Rab13. In turn, Rab13 associates with Grb2, targeting Syx and RhoA to Tyr(1175)-phosphorylated VEGFR2 at the leading edge. Rab13 knockdown in zebrafish impeded sprouting of intersegmental vessels and diminished the directionality of their tip cells. These results indicate that endothelial cell mobility in sprouting vessels is facilitated by shuttling the same protein complex from disassembling junctions to the leading edges of cells.     

Advances in carbon nanotube based electrochemical sensors for bioanalytical applications

Electrochemical (EC) sensing approaches have exploited the use of carbon nanotubes (CNTs) as electrode materials owing to their unique structures and properties to provide strong electrocatalytic activity with minimal surface fouling. Nanofabrication and device integration technologies have emerged along with significant advances in the synthesis, purification, conjugation and biofunctionalization of CNTs. Such combined efforts have contributed towards the rapid development of CNT-based sensors for a plethora of important analytes with improved detection sensitivity and selectivity. The use of CNTs opens an opportunity for the direct electron transfer between the enzyme and the active electrode area. Of particular interest are also excellent electrocatalytic activities of CNTs on the redox reaction of hydrogen peroxide and nicotinamide adenine dinucleotide, two major by-products of enzymatic reactions. This excellent electrocatalysis holds a promising future for the simple design and implementation of on-site biosensors for oxidases and dehydrogenases with enhanced selectivity. To date, the use of an anti-interference layer or an artificial electron mediator is critically needed to circumvent unwanted endogenous electroactive species. Such interfering species are effectively suppressed by using CNT based electrodes since the oxidation of NADH, thiols, hydrogen peroxide, etc. by CNTs can be performed at low potentials. Nevertheless, the major future challenges for the development of CNT-EC sensors include miniaturization, optimization and simplification of the procedure for fabricating CNT based electrodes with minimal non-specific binding, high sensitivity and rapid response followed by their extensive validation using “real world” samples. A high resistance to electrode fouling and selectivity are the two key pending issues for the application of CNT-based biosensors in clinical chemistry, food quality and control, waste water treatment and bioprocessing.     

Plasmid relaxation induced by copper metalated diglycine conjugates under heterogeneous reaction conditions

This paper reports synthesis and plasmid modification activities of a new class of insoluble copper-metalated diglycine conjugates, containing aliphatic linkers of varying length. Besides providing significant rate enhancement for model phosphate ester cleavage, these constructs also displayed efficient supercoiled plasmid scission, in the absence of co-oxidants, under heterogeneous catalytic conditions.