Catabolism of factor VIIa bound to tissue factor in fibroblasts in the presence and absence of tissue factor pathway inhibitor

Vascular injury leads to the exposure of blood to fibroblasts and smooth muscle cells within the vessel wall. These cells constitutively express tissue factor (TF), the cellular receptor for plasma clotting factor VIIa (FVIIa). Formation of TF.FVIIa complexes on cell surfaces triggers the blood coagulation cascade. In the present study, we have investigated the fate of TF.FVIIa complexes formed on the cell surface of fibroblasts in the presence and absence of plasma inhibitor, tissue factor pathway inhibitor (TFPI). FVIIa bound to TF on the cell surface was internalized and degraded without depleting the cell surface TF antigen and activity. TFPI significantly enhanced the TF-specific internalization and degradation of FVIIa. TFPI-enhanced internalization and degradation of FVIIa requires the C-terminal domain of TFPI and factor Xa. TFPI. Xa-mediated internalization of FVIIa was associated with the depletion of TF from the cell surface. A majority of the internalized FVIIa was degraded, but a small portion of the internalized FVIIa recycles back to the cell surface as an intact protein. In addition to TF, other cell surface components, such as low density lipoprotein receptor-related protein (LRP) and heparan sulfates, are essential for TFPI.Xa-induced internalization of FVIIa. Acidification of cytosol, which selectively inhibits the endocytotic pathway via coated pits, inhibited TFPI.Xa-mediated internalization but not the basal internalization of FVIIa. Overall, our data support the concept that FVIIa bound to cell surface TF was endocytosed by two different pathways. FVIIa complexed with TF in the absence of the inhibitor was internalized via a LRP-independent and probably noncoated pit pathway, whereas FVIIa complexed with TF along with the inhibitor was internalized via LRP-dependent coated pit pathway.     

Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis

T lymphocytes expressing the chemokine receptors, CCR2, CCR5, CXCR3, and CXCR6 are increased in inflamed tissues in rheumatoid arthritis. The role of CXCR3 in autoimmune arthritis induced in Lewis rats was investigated. CXCR3+ T cells migrated 2- to 3-fold more than CXCR3- T cells to inflamed joints in arthritic animals. CXCR3-expressing in vivo Ag-activated T lymphoblasts and in vitro-activated lymph node cells from arthritic animals were strongly recruited to the arthritic joints, and treatment with anti-CXCR3 mAb significantly inhibited this T cell recruitment by 40-60%. Immune T cells from the spleen and lymph nodes of actively immunized arthritic donors adoptively transferred arthritis to naive rats. Treatment with anti-CXCR3 mAb delayed the onset of arthritis and significantly reduced the severity of joint inflammation with a >50% decrease in the clinical arthritis score. Blockade of CXCR3 also significantly reduced the weight loss in the arthritic animals and inhibited neutrophil accumulation in the joints by 50-60%. There was a marked reduction in the leukocyte infiltration of the synovium in the presence of CXCR3 blockade and a decrease in the loss of articular cartilage of the joints. In conclusion, CXCR3 on T cells has an essential role in T cell recruitment to inflamed joints and the development of joint inflammation in adjuvant arthritis.     

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.     

A new species of Macrobrachium Bate, 1868 (Decapoda, Palaemonidae) from the dolphin trenches of Kulsi River, N. India, possibly under threat

The Kulsi River, a major tributary of Brahmaputra River, N. India is reported to have resident population of Ganges river dolphin, Platanista gangetica (Roxburgh), which feeds on fishes and prawns. While surveying for the dolphins of the river, a number of fishes and prawns were collected. On identification, one of the prawns was found to be undescribed, and hence is described herein. The ecology of the river consisted of: temperature fluctuating widely from 15 to 28 °C, depth from 0.8 to 10 m, turbidity of 11-19 cm, sand mining @ 12,500 MT annually, and fish catch of 300-800 kg (from 1.5 km area). All these factors pose a great threat to the fish and prawn wealth of the river.Macrobrachium kulsiense sp. nov. is a very small sized prawn (maximum size - 34.5 mm in total length), exhibiting species-specific characters, such as highly elevated and moderately long rostrum with 9-12 dorsal teeth, a single ventral tooth, and percentage ratios of ischium, merus, carpus, palm, dactylus of first and second chelipeds (19.05:28.57:33.33:09.52:09.52 and 21.43:25.00:21.43:14.28:17.86, respectively). The species shows close similarity with Macrobrachium mirabile (Kemp). The females are larger than males, eggs are large in size (1.2×0.9 mm) and fecundity is low (15-20).     

Association of Serum Asymmetric Dimethylarginine with the Severity of Coronary Artery Disease: A Pilot Study

Background:Asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase (NOS), has been implicated in endothelial dysfunction and atherogenesis. Though there is much evidence linking ADMA with atherosclerosis and adverse cardiovascular events, only a few studies have established the independent relationship between elevated ADMA and the angiographic extent of coronary artery disease (CAD). The aim of the study was to analyze serum ADMA levels in patients with varied extent and severity of coronary atherosclerosis and to see whether the levels of ADMA in male and female participants vary significantly.Methods:We analyzed 40 individuals with obstructive CAD, including men and women, between the ages of 30 and 60. According to their coronary angiographic reports, the participants were divided into four groups: minor CAD, single vessel disease (SVD), double vessel disease (DVD) group and triple vessel disease (TVD). Then, serum ADMA levels was measured and compared among these groups.Results:ADMA level was significantly higher in patients with TVD (167.74±16.69) than those in the DVD (159.46±10.40), SVD (149.54±16.39) and minor CAD (144.5± 24.16) group (p-value= 0.0001). There was no significant difference in ADMA levels between male and female participants (p= 0.534). Conclusion:ADMA concentration in the serum may be useful in identifying whether CAD correlates significantly to the extent and severity of coronary atherosclerosis.Key Words: ADMA, CAD, Endothelial dysfunction, NOS, Atherosclerosis     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

RASSF1C modulates the expression of a stem cell renewal gene, PIWIL1

ABSTRACT: BACKGROUND: RASSF1A and RASSF1C are two major isoforms encoded by the Ras association domain family 1 (RASSF1) gene through alternative promoter selection and mRNA splicing. RASSF1A is a well established tumor suppressor gene. Unlike RASSF1A, RASSF1C appears to have growth promoting actions in lung cancer. In this article, we report on the identification of novel RASSF1C target genes in non small cell lung cancer (NSCLC). METHODS: Over-expression and siRNA techniques were used to alter RASSF1C expression in human lung cancer cells, and Affymetrix-microarray study was conducted using NCI-H1299 cells over-expressing RASSF1C to identify RASSF1C target genes. RESULTS: The microarray study intriguingly shows that RASSF1C modulates the expression of a number of genes that are involved in cancer development, cell growth and proliferation, cell death, and cell cycle. We have validated the expression of some target genes using qRT-PCR. We demonstrate that RASSF1C over-expression increases, and silencing of RASSF1C decreases, the expression of PIWIL1 gene in NSCLC cells using qRT-PCR, immunostaining, and Western blot analysis. We also show that RASSF1C over-expression induces phosphorylation of ERK1/2 in lung cancer cells, and inhibition of the MEK-ERK1/2 pathway suppresses the expression of PIWIL1 gene expression, suggesting that RASSF1C may exert its activities on some target genes such as PIWIL1 through the activation of the MEK-ERK1/2 pathway. Also, PIWIL1 expression is elevated in lung cancer cell lines compared to normal lung epithelial cells. CONCLUSIONS: Taken together, our findings provide significant data to propose a model for investigating the role of RASSF1C/PIWIL1 proteins in initiation and progression of lung cancer.     

Genetic association of ADIPOQ gene variants with type 2 diabetes,obesity and serum adiponectin levels in south Indian population

Objective

To investigate the genetic association of eight variants of the adiponectin gene with type 2 diabetes mellitus (T2DM), obesity and serum adiponectin level in the south Indian population.

Methods

The study comprised of 1100 normal glucose tolerant (NGT) and 1100 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. The variants were screened by polymerase chain reaction-restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotype frequencies.

Results

Of the 8 variants, four SNPs namely, + 276 G/T (rs1501299), − 4522 C/T (rs822393), − 11365 C/G (rs266729), and + 712 G/A (rs3774261) were significantly associated with T2DM in our study population. The −3971 A/G (rs822396) and − 11391 G/A (rs17300539) SNPs' association with T2DM diabetes was mediated through obesity (where the association with type 2 diabetes was lost after adjusting for BMI). There was an independent association of + 276 G/T (rs1501299) and − 3971 A/G (rs822396) SNPs with generalized obesity and + 349 A/G (rs2241767) with central obesity. Four SNPs, −3971 A/G (rs822396), + 276 G/T (rs1501299), − 4522 C/T (rs822393) and Y111H T/C (rs17366743) were significantly associated with hypoadiponectinemia. The haplotypes GCCATGAAT and AGCGTGGGT conferred lower risk of T2DM in this south Indian population.

Conclusion

The adiponectin gene variants and haplotype contribute to the genetic risk towards the development of type 2 diabetes, obesity and hypoadiponectinemia in the south Indian population.     

Mapping of a dominant rust resistance gene revealed two R genes around the major Rust_QTL in cultivated peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Key message

A consensus rust QTL was identified within a 1.25 cM map interval of A03 chromosome in cultivated peanut. This map interval contains a TIR–NB–LRR R gene and four pathogenesis-related genes.

Abstract

Disease resistance in plants is manifested due to the specific interaction between the R gene product and its cognate avirulence gene product (AVR) in the pathogen. Puccinia arachidis Speg. causes rust disease and inflicts economic damages to peanut. Till now, no experimental evidence is known for the action of R gene in peanut for rust resistance. A fine mapping approach towards the development of closely linked markers for rust resistance gene was undertaken in this study. Phenotyping of an RIL population at five environments for field rust score and subsequent QTL analysis has identified a 1.25 cM map interval that harbored a consensus major Rust_QTL in A03 chromosome. This Rust_QTL is flanked by two SSR markers: FRS72 and SSR_GO340445. Both the markers clearly identified strong association of the mapped region with rust reaction in both resistant and susceptible genotypes from a collection of 95 cultivated peanut germplasm. This 1.25 cM map interval contained 331.7 kb in the physical map of A. duranensis and had a TIR–NB–LRR category R gene (Aradu.Z87JB) and four glucan endo-1,3 β glucosidase genes (Aradu.RKA6 M, Aradu.T44NR, Aradu.IWV86 and Aradu.VG51Q). Another resistance gene analog was also found in the vicinity of mapped Rust_QTL. The sequence between SSR markers, FRS72 and FRS49, contains an LRR-PK (Aradu.JG217) which is equivalent to RHG4 in soybean. Probably, the protein kinase domain in AhRHG4 acts as an integrated decoy for the cognate AVR from Puccinia arachidis and helps the TIR–NB–LRR R-protein to initiate a controlled program cell death in resistant peanut plants.