Anticancer effect of ursolic acid stearoyl glucoside in chemically induced hepatocellular carcinoma

Hepatocellular carcinoma is one of the leading causes of death in cancer and yet no drug has proven to be a successful candidate for its treatment in advanced stages. Ursolic acid stearoyl glucoside (UASG) is a newly discovered triterpene in Lantana camara and there lies a possibility that it possess anti-hepatocellular carcinoma property. In the present study, we induced hepatocellular carcinoma in Wistar rats by diethylnitrosamine (DENA) and treated it with ursolic acid stearoyl glucoside. The ability to treat hepatocellular carcinoma was measured by comparing biochemical serum markers such as serum alanine aminotransferase, serum aspartate aminotransferase, serum alkaline phosphatase, and the specific marker for hepatocellular carcinoma, alpha fetoprotein. The histological studies of the livers were also performed. The results have shown significant elevated levels of these parameters as compared to normal control and the drug receiving groups have shown significant reduction in these marker levels. Histopathological studies also indicated the reduced liver damage in drug-treated groups. It was noted that a significant and dose-dependent reversal of DENA-diminished activity of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, glutathione transferase, and the reduced DENA-elevated level of lipid peroxidation (LPO) with a marked change. UASG significantly suppressed free radical formation by scavenging the hydroxyl radicals. It also modulates the levels of LPO and markedly increases the endogenous antioxidant enzymes level in DENA-induced hepatocellular carcinogenesis.     

Development of a cost-effective high-throughput process of microsatellite analysis involving miniaturized multiplexed PCR amplification and automated allele identification

Background

Microsatellites are nucleotide sequences of tandem repeats occurring throughout the genome, which have been widely used in genetic linkage analysis, studies of loss of heterozygosity, determination of lineage and clonality, and the measurement of genome instability or the emergence of drug resistance reflective of mismatch repair deficiency. Such analyses may involve the parallel evaluation of many microsatellite loci, which are often limited by sample DNA, are labor intensive, and require large data processing.

Results

To overcome these challenges, we developed a cost-effective high-throughput approach of microsatellite analysis, in which the amplifications of microsatellites are performed in miniaturized, multiplexed polymerase chain reaction (PCR) adaptable to 96 or 384 well plates, and accurate automated allele identification has been optimized with a collective reference dataset of 5,508 alleles using the GeneMapper software.

Conclusions

In this investigation, we have documented our experience with the optimization of multiplex PCR conditions and automated allele identification, and have generated a unique body of data that provide a starting point for a cost-effective, high-throughput process of microsatellite analysis using the studied markers.

     

Molecular Characterization of Whole Genomic RNA2 From Iranian Isolates of Grapevine Fanleaf Virus

Grapevine fanleaf virus (GFLV) is the major causal agent of the grapevine degeneration disease. To characterize the genomic RNA2 segment from Iranian isolates of GFLV, leaf samples were collected from infected vineyards in different locations with a long history of vine cultivation. Four isolates were selected for cloning and sequencing on the basis of the restriction profiles of RT‐PCR products. The sequencing data revealed that the RNA2 of the Iranian GFLV isolates were the shortest compared with that of all previously described GFLV isolates. The sizes were 3730 nucleotides (nt) for Shir‐Amin and Urmia isolates and 3749 nt for Takestan and Bonab isolates (excluding the poly (A) tail), due to deletion events in both 5′ and 3′ non‐coding regions. In the phylogenetic tree based on the full‐length nucleotide sequences of GFLV RNA2, all the GFLV isolates clustered into two groups with the exception of the Hungarian isolate (GHu). The Iranian isolates grouped as a distinct cluster. Recombination analyses showed that GFLV‐NW (Germany), GFLV‐F13 (reference isolate), GFLV isolate Shir‐Amin (Iran) and Arabis mosaic virus isolate Lv were recombinant isolates and one of their parents belonged to the same lineage as the Iranian isolates. These findings suggest that these isolates originated from a common ancestor.     

Fermentation Kinetics of Media Optimization for the Production of Alpha Amylase by a New Isolate of Aspergillus Oryzae

The starch-degrading enzyme‘α-amylase’is widelydistributedin nature .This extracellular enzyme randomlyhydrolyzesα1~4 glucosidic linkage throughout the starchmolecule in an endo-fashion producing oligosaccharidesand monosaccharides including maltose ,…     

Anti-CD3/28 mediated expansion of macaque CD4+ T cells is polyclonal and provides extended survival after adoptive transfer

BACKGROUND: Our lab has previously shown that adoptive transfer of in vitro expanded autologous purified polyclonal CD4(+) T cells using anti-CD3/CD28 coated beads induced antiviral responses capable of controlling simian immunodeficiency virus (SIV) replication in vivo. RESULTS: Expansion on anti-CD3/28 coated beads was found to induce a true polyclonal expansion as CFSE labeled cells uniformly showed dilution of the dye over several days of culture, in contrast to aliquots of the same cells subjected to mitogen stimulation. Of interest was the finding that CD4(+) T cells collected before and during early chronic SIV infection or AIDS stage did not show any or only modest differences in proliferative response or expansion kinetics. The reason for such excellent expansion properties was analyzed by the quantitation of telomerase activity in aliquots of expanding CD4(+) T cells from sample collected at various times post-infection. First, anti-CD3/28 expanded CD4(+) T cells exhibited telomerase levels 2- to 20-fold higher than the starting population of CD4(+) T cells. Moreover, while telomerase activity in ex vivo tested CD4(+) T cells was found to decrease following SIV infection and disease progression, anti-CD3/28 expansion appeared to restore significant levels of telomerase activity as no difference was noted in telomerase expression between CD4(+) T cells expanded from samples collected before or during the chronic SIV infection. When such expanded and CFSE labeled T cells were autologously transferred to monkeys, evidence for extended survival in vivo was provided as CFSE labeled cells were detected to relatively high levels in blood and spleen at 1 week post-infection. CONCLUSION: In summary, the data suggest that anti-CD3/28 mediated expansion of CD4(+) T cells retains its immunotherapeutic potential not only during the early stages of lentiviral infection but also at more advanced stages of disease.     

Roles of Nitric Oxide in Conferring Multiple Abiotic Stress Tolerance in Plants and Crosstalk with Other Plant Growth Regulators

Journal of Plant Growth Regulation - Nitric oxide (NO) is a free-radical gasotransmitter signaling molecule associated with a varied spectrum of signal transduction pathways linked to inducing...     

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae

BackgroundA prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet.MethodsEscherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses.ResultsThe T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition.ConclusionsOur study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae.General significanceWe envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.     

Research Article: Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter

Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.     

Lactate Dehydrogenase Like Crystallin: A Potentially Protective Shield for Indian Spiny-Tailed Lizard (Uromastyx hardwickii) Lens Against Environmental Stress?

Taxon specific lens crystallins in vertebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uromastyx hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in U. hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure–function relationship of these proteins.     

Methylation status and protein expression of RASSF1A in breast cancer patients

Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.