Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration

Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic melanoma cells, we investigated the effects of hypergravity on the regulation of cGMP levels in cultured human melanocytes and in melanoma cell lines with different metastatic potentials. Hypergravity was produced by horizontal centrifugal acceleration. Here we report that long-term application of hypergravity (up to 5 g for 24 h) stimulated cGMP efflux in cultured melanocytes and in non-metastatic melanoma cells in the presence of 0.1 mM 3-isobutyl-1-methylxanthine (IBMX), a non-selective phosphodiesterase (PDE) inhibitor. Under these conditions, cAMP synthesis and melanin production were up-regulated in pigmented melanocytes and non-metastatic melanoma cells. Hypergravity also stimulated cGMP transport in the presence of 1 microM trequinsin, an inhibitor of cGMP-binding PDE (PDE5) and of transport by multidrug resistance proteins MRP4/5, whereas 50 microM trequinsin partially inhibited cGMP transport. Transport was further inhibited by probenecid, an inhibitor of endogenous non-selective transporters as well as of MRP4/5 and by cycloheximide as an inhibitor of de novo protein synthesis. In contrast, hypergravity did not affect cGMP efflux in metastatic melanoma cells, which might be related to an up-regulated cGMP efflux at 1 g. The results of the present study indicate that hypergravity may stimulate cGMP efflux in melanocytes and in non-metastatic melanoma cells most probably by an enhanced expression of endogenous transporters and/or MRP4/5. Thus, an altered acceleration vector may induce signaling events in melanocytic cells.     

Purification of vitellogenin from the plasma of Indian freshwater murrel, Channa punctatus (Bloch) by different methods: a comparative study

Plasma from estrogenized, [32P] NaH2PO4-injected murrel, Channa punctatus was collected in the presence of proteolytic inhibitors and subjected to different separation procedures singly or in combination, viz., gel filtration chromatography on Ultrogel AcA 34, ion-exchange chromatography on DEAE sephacel, or selective precipitation with dimethylformamide or with Mg2+: EDTA in order to isolate vitellogenin from other plasma proteins. The results show that chromatography on Ultrogel or DEAE sephacel yields intact vitellogenin whereas prior precipitation with DMF or with Mg2+: EDTA results in either co-precipitation of other plasma proteins or in the cleavage of phosvitin-like material from the native vitellogenin molecule.     

An internal segment (residues 58-119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

StAR, a protein synthesized in the cytoplasm and subsequently imported into mitochondria, regulates the rate-determining step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The active form of StAR is the 37 kDa pre-protein, which has a short half-life. To determine whether proteasomes participate in the turnover of StAR, we incubated primary cultures of preovulatory rat granulosa cells and immortalized human granulosa cells in the presence of MG132, a specific inhibitor to proteasome catalysis. This treatment caused accumulation of StAR in unstimulated cells. Moreover, incubation of the cells with MG132 in the presence of forskolin (FK), luteinizing hormone/chorionic gonadotropin or follicular stimulating hormone augmented the accumulation of both the 37 kDa cytoplasmic protein and the 30 kDa mature mitochondrial protein, compared to cells incubated with FK or the gonadotropic hormones alone. Concomitantly, progesterone production was enhanced. In contrast no elevation in the 37 kDa StAR intracellular levels or progesterone production was observed following incubation of the cells with the cysteine protease inhibitor E-64. The increase of the 37 kDa StAR protein was evident after 15 min and 30 min of incubation with MG132 (143% and 187% of control values, respectively) with no significant elevation of the 30 kDa protein. Accumulation of the intermediate mitochondrial 32 kDa protein was evident after 1-2 h and the accumulation of the 30 kDa protein was evident only after 4 h of incubation with MG132. In contrast, no elevation in adrenodoxin, a component of the cytochrome P450scc enzyme system, was found. These data suggest that StAR protein is either directly or indirectly degraded by the proteasome which may explain, in part, its short half-life. Moreover, it seems that the cytosolic 37 kDa protein, which is responsible for the steroidogenic activity of StAR, is the primary proteasomal substrate and that the inhibition of its degradation by MG132 causes the up-regulation of progesterone production.     

Length‐weight relationship of ten indigenous freshwater fish species from Gandak River,Bihar, India

Ten indigenous freshwater fish species belonging to nine genera and seven families were collected from Gandak River, Bihar, India during December, 2014 to September, 2016. Fishes were caught using Cast nets, Gill nets, Drag nets with mesh size of 15–20 mm, 25–30 mm and 5–10 mm respectively along with Traps. Sampling was done during dry and wet seasons and a total of 682 fish were collected for analysis of their Length–Weight Relationships (LWRs). Analyses included seven species for which no previous LWRs information were available in FishBase, and new maximum lengths were recorded for three species.     

Identification and comparative analysis of microRNAs from tomato varieties showing contrasting response to ToLCV infections

Increasing incidence of viral infections in crop plants adversely affects their growth and yield. Tomato (Solanum lycopersicum) is considered to be a favorite host for viruses with over 50 species of begomoviruses naturally infecting this crop. Tomato leaf curl virus (ToLCV) is among the most widespread and devastating begomoviruses affecting tomato production. microRNAs (miRs) have been established as key regulators of gene expression and plant development. The miR pathways are disturbed during infection by viruses. Thus, comprehension of regulatory miR networks is crucial in understanding the effect of viral pathogenicity. To identify key miRs involved in ToLCV infection, a high throughput approach involving next generation sequencing was employed. Healthy and infected leaf tissues of two tomato varieties, differing in their susceptibility to ToLCV infection were analyzed. NGS data analysis followed by computational predictions, led to identification of 91 known miRs, 15 novel homologs and 53 novel miRs covering two different varieties of tomato, susceptible (Pusa Ruby) and tolerant (LA1777) to ToLCV infection. The cleaved targets of these miRs were identified using online available degradome libraries from leaf, flower and fruit of tomato and showed their involvement in various biological pathways through KEGG Orthology. With detailed comparative profiling of expression pattern of these miRs, we could associate the specific miRs with the resistant and infected genotypes. This study depicted that in depth analysis of miR expression patterns and their functions will help in identification of molecules that can be used for manipulation of gene expression to increase crop production and developing resistance against diseases.     

Identification of genetic variation that determines human trehalase activity and its association with type 2 diabetes

A prior linkage scan in Pima Indians identified a putative locus for type two diabetes (T2D) and body mass index (BMI) on chromosome 11q23-25. Association mapping across this region identified single nucleotide polymorphisms (SNPs) in the trehalase gene (TREH) that were associated with T2D. To assess the putative connection between trehalase activity and T2D, we performed a linkage study for trehalase activity in 570 Pima Indians who had measures of trehalase activity. Strong evidence of linkage of plasma trehalase activity (LOD = 7.0) was observed in the TREH locus. Four tag SNPs in TREH were genotyped in these subjects and plasma trehalase activity was highly associated with three SNPs: rs2276064, rs117619140 and rs558907 (p = 2.2 × 10^?11–1.4 × 10^?23), and the fourth SNP, rs10790256, was associated conditionally on these three (p = 2.9 × 10^?7). Together, the four tag SNPs explained 51 % of the variance in plasma trehalase activity and 79 % of the variance attributed to the linked locus. These four tag SNPs were further genotyped in 828 subjects used for association mapping of T2D, and rs558907 was associated with T2D (odds ratio (OR) 1.94, p = 0.002). To assess replication of the T2D association, all four tag SNPs were additionally genotyped in two non-overlapping samples of Native Americans. Rs558907 was reproducibly associated with T2D in 2,942 full-heritage Pima Indians (OR 1.27 p = 0.03) and 3,897 “mixed” heritage Native Americans (OR 1.21, p = 0.03), and the strongest evidence for association came from combining all samples (OR 1.27 p = 1.6 × 10^?4, n = 7,667). However, among 320 longitudinally studied subjects, measures of trehalase activity from a non-diabetic exam did not predict those who would eventually develop diabetes versus those who would remain non-diabetic (hazard ratio 0.94 per SD of trehalase activity, p = 0.29). We conclude that variants in TREH control trehalase activity, and although one of these variants is also reproducibly associated with T2D, it is likely that the effect of the SNP on risk of T2D occurs by a mechanism different than affecting trehalase activity. Alternatively, TREH variants may be tagging a nearby T2D locus.     

Isoiation and Characterization of Vicia FABA Lectin Affinity Purified on Chitin Column

Abstract

A lectin from early long pod var. of Vicia faba seed has been purified to homogeneity on chitin. The purified lectin is shown to be homogeneous in nature by Bio Gel P - 150 gel filtration, fast protein liquid chromatography and polyacrylamide gel electrophoresis. The lectin is a glycoprotein with molecular weight of 51,000. The lectin molecule is possibly composed of two types of subunits devoid of any covalent linking through sulfhydryl groups, with molecular weights 9,000 and 15,000 respectively in the ratio 2:2. The purified lectin shows a high affinity for N-acetyl-D-glucosamine (GlcNAc).

Amino acid analyses show that cysteine and methionine are absent, and a high proportion of aspartic acid and glutamic acid are present in the protein molecule. The extinction coefficient of the purified lectin is 7.22. The lectin behaves as a, cold agglutinin displaying stronger agglutination than the naturally occurring ABO agglutinin in the cold.     

Hypoglycemic activity of the antioxidant saponarin,characterized as α-glucosidase inhibitor present in Tinospora cordifolia

Tinospora cordifolia, used in anti-diabetic herbal drug preparations, was reported [12] to contain an α-glucosidase inhibitor, characterized as saponarin (apigenin-6-C-glucosyl-7-O-glucoside). The leaf extract had appreciable antioxidant and hydroxyl radical scavenging activities and contained the flavonoid in the range of 32.1 ± 1.5–45.5 ± 3.5 mg/g of dry solid. Saponarin showed mixed competitive inhibition on activities of α-glucosidase and sucrase of different origins. IC₅₀, Ki and ki′ values determined were 48 μM, 8 μM and 19.5 μM respectively for intestinal maltase and 35 μM, 6 μM and 13 μM respectively for intestinal sucrase. When given orally to maltose-fed rat, saponarin showed hypoglycemic activity in the range of 20–80 mg/kg compared to 100–200 mg/kg for acarbose as reported [27].