Effect of ultraviolet B (302 nm) irradiation on viability, metabolic and detoxification functions of goat hepatocytes – in vitro study

The object of the present study was to investigate the effect(s) of UV-B irradiation on the functional integrity, metabolic and detoxifying capacity of the isolated goat hepatocytes. Isolated goat hepatocytes were subjected to UV-B irradiation invitro for 0, 250, 500, 1250, 2500 and 7500 Joules/m² which correspond to the irradiation time of 0, 1, 2, 5, 10 and 30 min. Cells were then analysed for Viability (Trypan blue exclusion test [TBE], 3-[4,5-dimethylthiozol-2yl]-2,5-diphenyltetrazolium bromide [MTT] assay, Membrane integrity (Lactate dehydrogenase [LDH] leakage, Lipid peroxidation) Detoxification (Ureagenesis, Cytochrome P450 activity [CYP450, Diazepam metabolism] and Glutathione-S-Transferase [GST] activity. The results show that there was no difference in functional, metabolic as well as detoxifying parameters of the hepatocytes when irradiated from 0–1250 Joules/m², whereas a significant alteration was appreciable in the parameters such as LDH leakage, lipid peroxidation, and CYP450 activity when irradiated beyond 1250 Joules/m². Our present findings suggest that the biologically compatible and feasible dose of UV-B irradiation for xenotransplantation appears to be 1250 Joules/m².     

Development of a simple and rapid fluorogenic procedure for identification of vibrionaceae family members

We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.     

Oxidation of gum arabic by soluble colloidal MnO2

In the present work, the oxidative degradation of gum arabic by colloidal manganese dioxide (MnO2) was carried out. Monitoring the disappearance of the MnO2 spectrophotometrically at 375 nm was used to follow the kinetics. The oxidation obeyed fractional-order kinetics with respect to the [gum arabic]. Effect of various experimental parameters such as the initial colloidal [MnO2], [HClO4], temperature, and complexing agents (P2O7(4-), F-, and Mn2+) for the oxidation of gum arabic was studied. The reaction was acid catalyzed. Addition of P2O(7)4-, F-, and Mn2+ ions enhances the rate of oxidation significantly. Gum arabic adsorbs onto the surface of the colloidal MnO2 through the equatorial -OH groups of the rhamnose moiety, and the complex breaks down into products. The Arrhenius equation was valid for the oxidation kinetics between 40 and 60 degrees C. To explain the observed kinetic results, a suitable mechanism and rate law for the reaction taking place at the surface of the colloidal particle has been proposed. The reducing nature of gum arabic is found be due to the presence of -OH group in the skeleton.     

EXPERIMENTAL STUDIES IN PONDEROSA PINE. I. RELATIONSHIP BETWEEN VARIATION IN PROTEINS AND MORPHOLOGY

Ponderosa pine from California (Pinus ponderosa var. ponderosa) were crossed to one another and also to individuals from the Rocky Mountains (P. p. var. scopulorum). All crosses involved a single mother and a single pollen donor. Patterns of inheritance of electrophoretically-detectable loci followed Mendelian expectations with one exception. Shikimate dehydrogenase showed unpredictable banding patterns in intervarietal crosses. Variability at these biochemical loci was compared to variability at 14 morphometric characters. The relationship between these two levels of variation is complex and permits only one generalization: variability at one level is not a good predictor of variability at the other level.     

Saturated fat-induced changes in Sf 60-400 particle composition reduces uptake of LDL by HepG2 cells

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.     

Genome-wide expression analysis of yeast response during exposure to 4°C

Adaptation to temperature fluctuation is essential for the survival of all living organisms. Although extensive research has been done on heat and cold shock responses, there have been no reports on global responses to cold shock below 10°C or near-freezing. We examined the genome-wide expression in Saccharomyces cerevisiae, following exposure to 4°C. Hierarchical cluster analysis showed that the gene expression profile following 4°C exposure from 6 to 48 h was different from that at continuous 4°C culture. Under 4°C exposure, the genes involved in trehalose and glycogen synthesis were induced, suggesting that biosynthesis and accumulation of those reserve carbohydrates might be necessary for cold tolerance and energy preservation. The observed increased expression of phospholipids, mannoproteins, and cold shock proteins (e.g., TIP1) is consistent with membrane maintenance and increased permeability of the cell wall at 4°C. The induction of heat shock proteins and glutathione at 4°C may be required for revitalization of enzyme activity, and for detoxification of active oxygen species, respectively. The genes with these functions may provide the ability of cold tolerance and adaptation to yeast cells.     

Biocompatible surface preparation using amino-functionalized amylose

Aminopropyl amyloses with various degrees of substitution (DS) were prepared and investigated with respect to their surface modification properties. Poly(acrylic acid) was grafted to plasma-activated PVDF films, and the functional amylose was bound via amide linkage formation. Layer formation was confirmed by X-ray photoelectron spectroscopy. Contact angle measurements and surface MALDI-TOF mass spectrometry indicated a hydrophilic surface and minimization of protein adsorption.     

Effect of albumin conformation on the binding of ciprofloxacin to human serum albumin: a novel approach directly assigning binding site

Human serum albumin (HSA) is known to exist as N (pH approximately 7), B (pH approximately 9), and F (pH approximately 3.5) isomeric forms and an equilibrium intermediate state (I) accumulate in the urea induced unfolding pathway of HSA around 4.8-5.2 M urea concentrations. These states displayed characteristic structure and functions. To elucidate the ciprofloxacin (CFX) binding behavior of HSA, the binding of ciprofloxacin with these conformational states of human serum albumin (HSA) has been investigated by fluorescence spectroscopy. The binding constant (K) for N, B, F, and I conformation of HSA were 6.92 x 10(5), 3.87 x 10(5), 4.06 x 10(5), and 2.7 x 10(5) M(-1) and the number of binding sites (n) were 1.26,1.21, 1.16, and 1.19, respectively. The standard free energy changes (DeltaGbinding(0)) of interaction were found to be -33.3 (N isomer), -31.8 (B isomer), -32 (F isomer), and -30.0 kJ mol(-1) respectively. By using unfolding pathway of HSA, domain II of HSA has been assigned to possess binding site of ciprofloxacin. Plausible correlation between stability of CFX-N and CFX-B complexes and drug distribution have been discussed. At plasma concentration of HSA fraction of free CFX, which contributes potential to its rate of transport across cell membrane, was found to be approximately 80% more for B isomers compared to N isomers of HSA. The conformational changes in two physiologically important isomers of HSA (N and B isomers) upon ciprofloxacin binding were evaluated by measuring far, near-UV CD, and fluorescence properties of the CFX-HSA complex.     

Is proline the quintessential sentinel of plants? A case study of postharvest flower senescence in Dianthus chinensis L.

The present investigation primarily focussed on evaluating the efficacy of exogenous proline on the flower longevity of Dianthus chinensis L. Floral buds were harvested at the paint brush stage (i.e., a day prior to anthesis) and divided into 6 sets, with one set of buds (i.e., control) held in distilled water and rest of the 5 sets were supplemented with various concentrations of proline, viz., 10 mM, 20 mM, 30 mM, 40 mM and 50 mM. The application of proline at 40 mM concentration proved out to be most effective in improving the longevity of the flowers by about 4 days as compared to the control. The ameliorated longevity coincided with enhanced floral diameter, fresh mass, dry mass and water content. The flowers with delayed senescence also maintained higher soluble proteins, sugars and phenols. The results suggest that exogenous proline effectively alleviates oxidative stress in the petal tissue, as evident by a relatively lower maloendialdehyde content, which is manifested in the form of reduced lipid peroxidation (LPO). Reduced LPO was commensurate with increased membrane stability, quantified by membrane stability index. Moreover, the flowers with improved longevity exhibited a decline in lipoxygenase activity and significant augmentation of antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase.     

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.