In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

2,6-Dithienyl-4-furyl pyridines: Synthesis,topoisomerase I and II inhibition,cytotoxicity, structure–activity relationship,and docking study

For the development of novel antitumor agents, 2,6-dithienyl-4-furyl pyridine derivatives were prepared and evaluated for their topoisomerase I and II inhibitory activity as well as cytotoxicity against several human cancer cell lines. Among the 21 prepared compounds, compound 24 exhibited strong topoisomerase I inhibitory activity. In addition, a docking study with topoisomerase I and compound 24 was performed.     

Insights into the Initiation of JC Virus DNA Replication Derived from the Crystal Structure of the T-Antigen Origin Binding Domain

JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel “pocket”; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.     

Green tea constituent epigallocatechin-3-gallate inhibits angiogenic differentiation of human endothelial cells

Several independent research studies have shown that consumption of green tea reduces the development of cancer in many animal models. Epidemiological observations, though inconclusive, are suggesting that green tea consumption may also reduce the risk of some cancers in humans. These anti-carcinogenic effects of green tea have been attributed to its constituent polyphenols. Angiogenesis is a crucial step in the growth and metastasis of cancers. We have investigated the effect of the major polyphenolic constituent of green tea, epigallocatechin-3-gallate (EGCG), on the tube formation of human umbilical vein endothelial cells (HUVEC) on matrigel. Tube formation was inhibited by treatment both prior to plating and after plating endothelial cells on matrigel. EGCG treatment also was found to reduce the migration of endothelial cells in matrigel plug model. The role of matrix metalloproteinases (MMP) has been shown to play an important role during angiogenesis. Zymography was performed to determine if EGCG had any effect on MMPs. Zymographs of EGCG-treated culture supernatants modulated the gelatinolytic activities of secreted proteinases indicating that EGCG may be exerting its inhibitory effect by regulating proteinases. These findings suggest that EGCG acts as an angiogenesis inhibitor by modulating protease activity during endothelial morphogenesis.     

Identification and characterization of endoglucanases for fungicidal activity in <Emphasis Type="Italic">Anabaena laxa</Emphasis> (Cyanobacteria)

A cyanobacterial strain (Anabaena laxa RPAN8) exhibiting fungicidal activity and β-1,3 and 1,4 endoglucanase activities was selected for identifying the gene(s) involved. Functional analyses of the genomic library revealed that four clones (8, 64, 116, and 248) of RPAN8 exhibited fungicidal activity and induced structural deformities in the cell wall of the growing mycelia of Pythium aphanidermatum. Higher expression of fungicidal and β-1,4 endoglucanase activities, along with low expression of β-1,3 endoglucanase activity, was recorded in two E. coli clones (8 and 64). Clones 8 and 64 exhibited identical sequences while clones 116 and 248 were also similar. Bioinformatic analyses were undertaken only for the two non-identical clones 8 and 116 which showed open reading frames (ORFs) of 348 (end 1) and 656 amino acid residues (end 2), respectively. The amino acid sequence analyses revealed that the end 1 encoding endoglucanases belonged to peptidase M20 family while end 2 showed significant similarities with several known genes. The putative promoters and ribosomal binding sites were identified and amino acid exchanges were observed in both end 1 and 2. The presence of signal peptides of 24 and 20 amino acid residues respectively revealed the secretory nature of these proteins.     

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.     

Screening of chemopreventive effect of naringenin-loaded nanoparticles in DMBA-induced hamster buccal pouch carcinogenesis by FT-IR spectroscopy

The aim of the present study is to investigate the chemopreventive effects of the prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) in modifying the functional, structural, and compositional changes at the molecular level during 7, 12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by Fourier transform infrared (FT-IR) spectroscopy. The results revealed that a significant increase in the amount of proteins and nucleic acid contents and a decrease in the amount of lipids and glycogen contents are observed in DMBA-induced tumor tissues. In addition, in tumor tissues a decrease in lipid order and a significant increase in membrane dynamics were noticed. Further, the composition and secondary structure of proteins were found to be altered, which indicates some important structural alterations in the existing proteins and/or the expression of new types of proteins occurring under the tumor transformation. Furthermore, oral administration of free NAR and NARNPs significantly increased lipids and their order as well as increased the glycogen contents and decreased the levels of proteins and nucleic acid contents. On a comparative basis, NARNPs were found to have a more potent antitumor effect than free NAR in completely preventing the formation of squamous cell carcinoma and in improving the biochemical constituents to a normal range in DMBA-induced HBP carcinogenesis. The present study further shows a great potential of FT-IR spectroscopy as a complimentary tool for the screening of various anticancer drugs and follow-up, which may allow faster response to critical problems arising during treatment.     

Major vault protein (MVP) gene polymorphisms and drug resistance in mesial temporal lobe epilepsy with hippocampal sclerosis

The human major vault protein (MVP) has been implicated in the development of drug resistance in cancer cells. Over expression of MVP has also been reported in brain tissue samples from antiepileptic drug (AED)-resistant human focal epilepsies. To investigate the relationship between single nucleotide polymorphisms (SNPs) involving the MVP gene and AED-resistance, we compared the distribution of three SNPs in the MVP gene, rs4788187, rs3815824 and rs3815823, among 220 patients with mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS) (prototype of AED-resistant epilepsy syndrome), 201 patients with juvenile myoclonic epilepsy (JME) (prototype of AED-responsive epilepsy syndrome) and 213 ethnically matched non-epilepsy controls. All the patients and controls were residents of the South Indian state of Kerala for more than three generations. We did not find any significant difference in allele and genotypic frequencies of the studied SNPs between AED-resistant and AED-responsive cohorts, and between AED-resistant and AED-responsive cohorts independently and pooled together when compared with the controls. We conclude that rs4788187, rs3815824, rs3815823 variants of the MVP gene are associated neither with predisposition for epilepsy nor with AED-resistance in the population that we have studied. Our results suggest the need for further research into the link between MVP and AED-resistance.     

Late‐onset retinal degeneration pathology due to mutations in CTRP5 is mediated through HTRA1

Late‐onset retinal degeneration (L‐ORD) is an autosomal dominant macular degeneration characterized by the formation of sub‐retinal pigment epithelium (RPE) deposits and neuroretinal atrophy. L‐ORD results from mutations in the C1q‐tumor necrosis factor‐5 protein (CTRP5), encoded by the CTRP5/C1QTNF5 gene. To understand the mechanism underlying L‐ORD pathology, we used a human cDNA library yeast two‐hybrid screen to identify interacting partners of CTRP5. Additionally, we analyzed the Bruch's membrane/choroid (BM‐Ch) from wild‐type (Wt), heterozygous S163R Ctrp5 mutation knock‐in (Ctrp5^S163R/wt), and homozygous knock‐in (Ctrp5^S163R/S163R) mice using mass spectrometry. Both approaches showed an association between CTRP5 and HTRA1 via its C‐terminal PDZ‐binding motif, stimulation of the HTRA1 protease activity by CTRP5, and CTRP5 serving as an HTRA1 substrate. The S163R‐CTRP5 protein also binds to HTRA1 but is resistant to HTRA1‐mediated cleavage. Immunohistochemistry and proteomic analysis showed significant accumulation of CTRP5 and HTRA1 in BM‐Ch of Ctrp5^S163R/S163R and Ctrp5^S163R/wt mice compared with Wt. Additional extracellular matrix (ECM) components that are HTRA1 substrates also accumulated in these mice. These results implicate HTRA1 and its interaction with CTRP5 in L‐ORD pathology.     

Evolutionary divergence of the nuclear pore complex from fungi to metazoans

Nuclear pore complex (NPC) is the largest multimeric protein assembly of the eukaryotic cell, which mediates the nucleocytoplasmic transport. The constituent proteins of this assembly (nucleoporins) are present in varying copy numbers to give a size from ~ 60 MDa (yeast) to 112 MDa (human) and share common ancestry with other membrane‐associated complexes such as COPI/COPII and thus share the same structural folds. However, the nucleoporins across species exhibit very low percentage sequence similarity and this reflects in their distinct secondary structure and domain organization. We employed thorough sequence and phylogenetic analysis guided from structure‐based alignments of all the nucleoporins from fungi to metazoans to understand the evolution of NPC. Through evolutionary pressure analysis on various nucleoporins, we deduced that these proteins are under differential selection pressure and hence the homologous interacting partners do not complement each other in the in vitro pull‐down assay. The super tree analysis of all nucleoporins taken together illustrates divergent evolution of nucleoporins and notably, the degree of divergence is more apparent in higher order organisms as compared to lower species. Overall, our results support the hypothesis that the protein–protein interactions in such large multimeric assemblies are species specific in nature and hence their structure and function should also be studied in an organism‐specific manner.