Inhibitory kinetics and mechanism of pentacyclic triterpenoid from endophytic Colletotrichum gigasporum against pancreatic lipase.

The burden of obesity is increasing all over the world. Except for Orlistat, no effective anti-obesity drug is currently available. Therefore, a search for the new anti-obesity compound is need of time. This study demonstrates macromolecular interaction and inhibitory effect of pentacyclic triterpenoids (PTT) on pancreatic lipase (PL). In the present study PTTs from endophytic Colletotrichum gigasporum were found to show significant inhibitory activity against PL with IC50 of 16.62 ± 1.43 µg/mL. The PTT isolated through bioassay-guided isolation showed a dose-dependent (R2 = 0.915) inhibition against porcine PL and the results were comparable with the standard (Orlistat). Based on inhibition kinetic data, the gradual increase in Km (app) with increasing PTT concentration indicated that the mode of interaction of PTT with PL was a competitive type, and it directly competed with the substrate (pNPB) for the active site of PL. In vivo studies in Wistar rats at the oral dose (100 mg/kg body weight) of PTT significantly decreased (p < 0.05) incremental plasma triglyceride levels as compared to group B and TG absorption was down-regulated up to 49.18% vis a vis group D animals. The isolated PTT was identified as lupeol based on chromatographic and spectral data. The endophytic isolate was identified as Colletotrichum gigasporum based on morphology and ITS gene sequencing. The present study indicated that PTT had the potential to be used as a natural PL inhibitor in the treatment of obesity and the isolated endophyte can be a valuable bioresource for it.

Data on the inhibitory effect of endophytic fungi of traditional medicinal plants against pancreatic lipase (PL).

This article describes isolation and pancreatic lipase (PL) inhibitory potential of 18 endophytic fungi isolated from the various parts of six indigenous medicinal plants. PL catalyzes absorption and hydrolysis of triglycerides into di-glycerides into mono-glycerides and free fatty acids. PL inhibitors are well-known for the disruption of pancreatic lipase activity. The quest for novel pancreatic lipase inhibitors is crucially important owing to their therapeutic potential in the treatment of obesity and related chronic diseases. The present dataset provides information about the presence of endophytic fungi in the internal tissues of selected plants and the PL inhibitory potential of their metabolites using bioassay based screening. Absence of the yellow zone surrounding the standard Orlistat and test extract indicated PL inhibition due to the cumulative effect of metabolites present in the extract. The data suggests that TLC bio-autographic method is simple, rapid and reproducible and therefore it could be effectively used for high throughput screening of PL inhibitors from natural sources.

Ethnopharmacology, phytochemistry, and biotechnological advances of family Apocynaceae: A review.

The family Apocynaceae is one of the largest and important families in angiosperm. Several members of this family have medicinal properties and have been in the treatment of various ailments. Most of them are consumed as food by tribal people whereas a few plants are used as source of poison. Members of family Apocynaceae are rich in alkaloids, terpenoids, steroids, flavonoids, glycosides, simple phenols, lactones, and hydrocarbons. Other compounds such as sterols, lignans, sugars, lignans, and lactones have been isolated and systematically studied. Few studies have reported antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities of crude extracts as well as single compound(s) isolated from various members of the family Apocynaceae. Holarrhena antidysenterica, Rauvolfia serpentina, Carissa carandas, and Tabernaemontana divaricata are the extensively studied plants in this family. The present review provides a detailed outlook on ethnopharmacology, phytochemistry, and biological activities of selected members of this family. Moreover, it also covers the biotechnological advances used for large-scale production of bioactive compounds of therapeutic interest along with plant tissue culture-based approaches for conservation of this medicinally valuable family.

Transcription factors of Schizophyllum commune involved in mushroom formation and modulation of vegetative growth.

Mushrooms are the most conspicuous fungal structures. Transcription factors (TFs) Bri1 and Hom1 of the model fungus Schizophyllum commune are involved in late stages of mushroom development, while Wc-2, Hom2, and Fst4 function early in development. Here, it is shown that Bri1 and Hom1 also stimulate vegetative growth, while biomass formation is repressed by Wc-2, Hom2, and Fst4. The Δbri1Δbri1 and the Δhom1Δhom1 strains formed up to 0.6 fold less biomass when compared to wild-type, while Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains formed up to 2.8 fold more biomass. Inactivation of TF gene tea1, which was downregulated in the Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains, resulted in a strain that was severely affected in mushroom development and that produced 1.3 fold more biomass than the wild-type. In contrast, introducing a constitutive active version of hom2 that had 4 predicted phosphorylation motifs eliminated resulted in radial growth inhibition and prompt fructification in both Δhom2 and wild-type strains, even in sterile monokaryons. Together, it is concluded that TFs involved in mushroom formation also modulate vegetative growth. Among these TFs is the homeodomain protein Hom2, being the first time that this class of regulatory proteins is implicated in repression of vegetative growth in a eukaryote.

Genotyping Informatics and Quality Control for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort.

The Kaiser Permanente (KP) Research Program on Genes, Environment and Health (RPGEH), in collaboration with the University of California-San Francisco, undertook genome-wide genotyping of >100,000 subjects that constitute the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The project, which generated >70 billion genotypes, represents the first large-scale use of the Affymetrix Axiom Genotyping Solution. Because genotyping took place over a short 14-month period, creating a near-real-time analysis pipeline for experimental assay quality control and final optimized analyses was critical. Because of the multi-ethnic nature of the cohort, four different ethnic-specific arrays were employed to enhance genome-wide coverage. All assays were performed on DNA extracted from saliva samples. To improve sample call rates and significantly increase genotype concordance, we partitioned the cohort into disjoint packages of plates with similar assay contexts. Using strict QC criteria, the overall genotyping success rate was 103,067 of 109,837 samples assayed (93.8%), with a range of 92.1-95.4% for the four different arrays. Similarly, the SNP genotyping success rate ranged from 98.1 to 99.4% across the four arrays, the variation depending mostly on how many SNPs were included as single copy vs. double copy on a particular array. The high quality and large scale of genotype data created on this cohort, in conjunction with comprehensive longitudinal data from the KP electronic health records of participants, will enable a broad range of highly powered genome-wide association studies on a diversity of traits and conditions.

DNA methylation profiling reveals novel diagnostic biomarkers in renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is the tenth most commonly diagnosed cancer in the United States. While it is usually lethal when metastatic, RCC is successfully treated with surgery when tumors are confined to the kidney and have low tumor volume. Because most early stage renal tumors do not result in symptoms, there is a strong need for biomarkers that can be used to detect the presence of the cancer as well as to monitor patients during and after therapy. METHODS: We examined genome-wide DNA methylation alterations in renal cell carcinomas of diverse histologies and benign adjacent kidney tissues from 96 patients. RESULTS: We observed widespread methylation differences between tumors and benign adjacent tissues, particularly in immune-, G-protein coupled receptor-, and metabolism-related genes. Additionally, we identified a single panel of DNA methylation biomarkers that reliably distinguishes tumor from benign adjacent tissue in all of the most common kidney cancer histologic subtypes, and a second panel does the same specifically for clear cell renal cell carcinoma tumors. This set of biomarkers were validated independently with excellent performance characteristics in more than 1,000 tissues in The Cancer Genome Atlas clear cell, papillary, and chromophobe renal cell carcinoma datasets. CONCLUSIONS: These DNA methylation profiles provide insights into the etiology of renal cell carcinoma and, most importantly, demonstrate clinically applicable biomarkers for use in early detection of kidney cancer.

Role of cyclin D1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis.

Activation of c-Met signaling and beta-catenin mutations are frequent genetic events observed in liver cancer development. Recently, we demonstrated that activated beta-catenin can cooperate with c-Met to induce liver cancer formation in a mouse model. Cyclin D1 (CCND1) is an important cell cycle regulator that is considered to be a downstream target of beta-catenin. To determine the importance of CCND1 as a mediator of c-Met- and beta-catenin-induced hepatocarcinogenesis, we investigated the genetic interactions between CCND1, beta-catenin, and c-Met in liver cancer development using mouse models. We coexpressed CCND1 with c-Met in mice and found CCND1 to cooperate with c-Met to promote liver cancer formation. Tumors induced by CCND1/c-Met had a longer latency period, formed at a lower frequency, and seemed to be more benign compared with those induced by beta-catenin/c-Met. In addition, when activated beta-catenin and c-Met were coinjected into CCND1-null mice, liver tumors developed despite the absence of CCND1. Intriguingly, we observed a moderate accelerated tumor growth and increased tumor malignancy in these CCND1-null mice. Molecular analysis showed an up-regulation of cyclin D2 (CCND2) expression in CCND1-null tumor samples, indicating that CCND2 may replace CCND1 in hepatic tumorigenesis. Together, our results suggest that CCND1 functions as a mediator of beta-catenin during HCC pathogenesis, although other molecules may be required to fully propagate beta-catenin signaling. Moreover, our data suggest that CCND1 expression is not essential for liver tumor development induced by c-Met and beta-catenin.

Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity.

2,7-Dihydroxycadalene and lacinilene C, sesquiterpenoid phytoalexins that accumulate at infection sites during the hypersensitive resistant response of cotton foliage to Xanthomonas campestris pv. malvacearum, have light-dependent toxicity toward host cells, as well as toward the bacterial pathogen. Adaxial epidermal cells surrounding and sometimes covering infection sites turn red. The red cells exhibited 3-4-fold higher absorption at the photoactivating wavelengths of sunlight than nearby colorless epidermal cells. Red epidermal cells protected underlying palisade mesophyll cells from the toxic effects of 2,7-dihydroxycadalene plus sunlight, indicating a role for epidermal pigments in protecting living cells that surround infection sites from toxic effects of the plant's own phytoalexins. A semi-quantitative survey of UV-absorbing substances extracted from epidermal strips from inoculated and mock-inoculated cotyledons indicated that the principal increase in capacity to absorb the photoactivating wavelengths was due to a red anthocyanin and a yellow flavonol, which were identified as cyanidin-3-O-beta-glucoside and quercetin-3-O-beta-glucoside, respectively.

Integration of genomic analysis and in vivo transfection to identify sprouty 2 as a candidate tumor suppressor in liver cancer.

UNLABELLED: Hepatocellular carcinoma (HCC) is 1 of the leading causes of cancer-related deaths worldwide, yet the molecular genetics underlying this malignancy are still poorly understood. In our study, we applied statistical methods to correlate human HCC gene expression data obtained from complementary DNA (cDNA) microarrays and corresponding DNA copy number variation data obtained from array-based comparative genomic hybridization. We have thus identified 76 genes that are up-regulated and show frequent DNA copy number gain, and 37 genes that are down-regulated and show frequent DNA copy loss in human HCC samples. Among these down-regulated genes is Sprouty2 (Spry2), a known inhibitor of receptor tyrosine kinases. We investigated the potential role of Spry2 in HCC by expressing dominant negative Spry2 (Spry2Y55F) and activated beta-catenin (DeltaN90-beta-catenin) in the mouse liver through hydrodynamic injection and sleeping beauty-mediated somatic integration. When stably expressed in mouse hepatocytes, Spry2Y55F cooperates with DeltaN90-beta-catenin to confer a neoplastic phenotype in mice. Tumor cells show high levels of expression of phospho-extracellular signal-regulated kinase (ERK), as well as deregulation of genes involved in cell proliferation, apoptosis, and angiogenesis. CONCLUSION: We identified a set of candidate oncogenes and tumor suppressor genes for human HCC. Our study provides evidence that inhibition of Spry activity cooperates with other oncogenes to promote liver cancer in mouse models, and Spry2 may function as a candidate tumor suppressor for HCC development in vivo. In addition, we demonstrate that the integration of genomic analysis and in vivo transfection is a powerful tool to identify genes that are important during hepatic carcinogenesis.

Inverse expression of Jun activation domain binding protein 1 and cell cycle inhibitor p27Kip1: influence on proliferation in hepatocellular carcinoma.

Recently, the functional role of Jun activation domain binding protein 1 (Jab1) as a putative novel oncogene in hepatocellular carcinoma (HCC) has been postulated. We show that expression of p27(Kip1), a negative cell cycle regulator, correlates inversely with Jab1 expression in HCC (P = .014). We observed nuclear Jab1 expression in 57% (55/97) and p27(Kip1) expression in 32% (31/97) of HCCs. Neither Jab1 nor p27(Kip1) nor inverse Jab1 and p27(Kip1) expression correlated with clinicopathological parameters. However, HCCs lacking p27(Kip1) with increased proliferative activity were frequently found to express Jab1 (P = .048). Normal liver tissue, cirrhosis, and tumor-like lesions (focal nodular hyperplasia, dysplastic nodules in cirrhotic liver) showed no significant Jab1 expression. In transfection studies in the hepatoma cell line Huh 7, Jab1 overexpression resulted in reduced p27(Kip1) protein levels. We conclude that Jab1 expression may lead to down-regulation of the negative cell cycle regulator p27(Kip1), pointing to a possible mechanism that promotes hepatocarcinogenesis.

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.

Increased expression of glycosyl-phosphatidylinositol anchor attachment protein 1 (GPAA1) is associated with gene amplification in hepatocellular carcinoma.

Glycosyl-phosphatidylinositol (GPI) anchor attachment protein 1 (GPAA1) transcript level was frequently up-regulated in our earlier study on gene expression profile. We therefore analyzed the potential involvement of GPAA1 in hepatocellular carcinoma (HCC) as GPAA1 gene locates at chromosome 8q24.3 which chromosome region is frequently amplified in HCCs. In this study, we observed that GPAA1 transcript in the HCCs (n = 93) showed a significantly higher expression level compared with their paralleled adjacent nontumor liver tissues, cirrhosis (n = 15) and normal (n = 16) liver tissues using real-time quantitative RT-PCR (p < 0.005). We also demonstrated that GPAA1 protein up-regulation was common in HCCs (90%, 9/10), and GPAA1 gene was frequently amplified (73%, 11/15) using quantitative microsatellite analysis. Increased GPAA1 expression was significantly associated with HCCs poor cellular differentiation (p = 0.011) and poor prognosis (p = 0.010). We then modulated the GPAA1 expression level in HCC cells (Hep3B) by transfection experiments, which was shown to positively regulate cell adhesion ability (p = 0.004) and proliferation rate (p = 0.037). Our data revealed GPAA1 gene amplification with overexpression of RNA and protein in HCC. GPAA1 is a potential amplification target of chromosome 8q and responsible to regulate tumor cells behavior.

Hedgehog signaling in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth most common malignancy and one of the leading causes of death world wide. Signaling pathways important for tumor initiation and progression in HCC are poorly understood. Hedgehog signaling (Hh) has been implicated in multiple events during development and has also been proposed to play important roles in several tumor types. However, it remains unclear whether this pathway is activated in HCC. Here, we report the detection of transcripts for hedgehog pathway signaling molecules in both HCC cell lines and tumor samples. Quantitative real-time RT-PCR also revealed the decreased expression of Hip1 and increased expression of Gli1 and smo in HCC samples compared with nontumor liver tissues. Blocking the hedgehog pathway with cyclopamine inhibited proliferation, induced apoptosis and repressed c-Myc and cyclin D expression in a subset of HCC cell lines. The study therefore, for the first time, provides evidence that hedgehog signaling may be activated in some HCC tumors. The results also indicate that the hedgehog pathway may be a new candidate for therapeutic targeting in HCC.

Array-based comparative genomic hybridization reveals recurrent chromosomal aberrations and Jab1 as a potential target for 8q gain in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide. We have previously characterized global gene expression patterns in HCC using microarrays. Here, we report the analysis of genomic DNA copy number among 49 HCC samples using BAC array-based comparative genomic hybridization (CGH). We observed recurrent and characteristic chromosomal aberrations, including frequent DNA copy number gains of 1q, 6p, 8q and 20q, and losses of 4q, 8p, 13q, 16q and 17p. We correlated gene expression with array CGH data, and identified a set of genes whose expression levels correlated with common chromosomal aberrations in HCC. Especially, we noticed that high expression of Jab1 in HCC significantly correlated with DNA copy number gain at 8q. Quantitative microsatellite analysis further confirmed DNA copy number gain at the Jab1 locus. Overexpression of Jab1 in HCC was also validated using real-time RT-PCR, and Jab1 protein levels were studied by immunohistochemistry on tissue microarrays. Functional analysis in HCC cell lines demonstrated that Jab1 may regulate HCC cell proliferation, thereby having a potential role in HCC development. In conclusion, this study shows that array-based CGH provides high resolution mapping of chromosomal aberrations in HCC, and demonstrates the feasibility of correlating array CGH data with gene expression data to identify novel oncogenes and tumor suppressor genes.

An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT-PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.