Characterization of Ascites-Derived Ovarian Tumor Cells from Spontaneously Occurring Ovarian Tumors of the Chicken: Evidence for E-Cadherin Upregulation

Ovarian cancer, a highly metastatic disease, is the fifth leading cause of cancer-related deaths in women. Chickens are widely used as a model for human ovarian cancer as they spontaneously develop epithelial ovarian tumors similar to humans. The cellular and molecular biology of chicken ovarian cancer (COVCAR) cells, however, have not been studied. Our objectives were to culture COVCAR cells and to characterize their invasiveness and expression of genes and proteins associated with ovarian cancer. COVCAR cell lines (n = 13) were successfully maintained in culture for up to19 passages, cryopreserved and found to be viable upon thawing and replating. E-cadherin, cytokeratin and α-smooth muscle actin were localized in COVCAR cells by immunostaining. COVCAR cells were found to be invasive in extracellular matrix and exhibited anchorage-independent growth forming colonies, acini and tube-like structures in soft agar. Using RT-PCR, COVCAR cells were found to express E-cadherin, N-cadherin, cytokeratin, vimentin, mesothelin, EpCAM, steroidogenic enzymes/proteins, inhibin subunits-α, βA, βB, anti-müllerian hormone, estrogen receptor [ER]-α, ER-β, progesterone receptor, androgen receptor, and activin receptors. Quantitative PCR analysis revealed greater N-cadherin, vimentin, and VEGF mRNA levels and lesser cytokeratin mRNA levels in COVCAR cells as compared with normal ovarian surface epithelial (NOSE) cells, which was suggestive of epithelial-mesenchymal transformation. Western blotting analyses revealed significantly greater E-cadherin levels in COVCAR cell lines compared with NOSE cells. Furthermore, cancerous ovaries and COVCAR cell lines expressed higher levels of an E-cadherin cleavage product when compared to normal ovaries and NOSE cells, respectively. Cancerous ovaries were found to express significantly higher ovalbumin levels whereas COVCAR cell lines did not express ovalbumin thus suggesting that the latter did not originate from oviduct. Taken together, COVCAR cell lines are likely to improve our understanding of the cellular and molecular biology of ovarian tumors and its metastasis.     

Growth and cell survival are unevenly impaired in pixie mutant wing discs

It is largely unknown how growth slows and then stops in vivo. Similar to most organs, Drosophila imaginal discs undergo a fast, near-exponential growth phase followed by a slow growth phase before final target size is reached. We have used a genetic approach to study the role of an ABC-E protein, Pixie, in wing disc growth. pixie mutants, like mutants in ribosomal proteins genes (known as Minutes), show severe developmental delay with relatively mild alterations in final body size. Intriguingly, pixie mutant wing imaginal discs show complex regional and temporal defects in growth and cell survival that are compensated to result in near-normal final size. In S2 cells, Pixie, like its yeast homolog RLI1, is required for translation. However, a comparison of the growth of eukaryotic translation initiation factor eIF4A and pixie mutant clones in wing discs suggests that only a subset of translation regulators, including pixie, mediate regional differences in growth and cell survival in wing discs. Interestingly, some of the regional effects on pixie mutant clone growth are enhanced in a Minute background. Our results suggest that the role of Pixie is not merely to allow growth, as might be expected for a translation regulator. Instead, Pixie also behaves as a target of putative constraining signals that slow disc growth during late larval life. We propose a model in which a balance of growth inhibitors and promoters determines tissue growth rates and cell survival. An alteration in this balance slows growth before final disc size is reached.     

Efficiency of mammalian selenocysteine incorporation

Five components have thus far been identified that are necessary for the incorporation of selenocysteine (Sec) into approximately 25 mammalian proteins. Two of these are cis sequences, a SECIS element in the 3'-untranslated region and a Sec codon (UGA) in the coding region. The three known trans-acting factors are a Sec-specific translation elongation factor (eEFSec), the Sec-tRNA(Sec), and a SECIS-binding protein, SBP2. Here we describe a system in which the efficiency of Sec incorporation was determined quantitatively both in vitro and in transfected cells, and in which the contribution of each of the known factors is examined. The efficiency of Sec incorporation into a luciferase reporter system in vitro is maximally 5-8%, which is 6-10 times higher than that in transfected rat hepatoma cells, McArdle 7777. In contrast, the efficiency of Sec incorporation into selenoprotein P in vitro is approximately 40%, suggesting that as yet unidentified cis-elements may regulate differential selenoprotein expression. In addition, we have found that SBP2 is the only limiting factor in rabbit reticulocyte lysate but not in transfected rat hepatoma cells where SBP2 is found to be mostly if not entirely cytoplasmic despite having a strong putative nuclear localization signal. The significance of these findings with regard to the function of known Sec incorporation factors is discussed.     

Phylogenetically Diverse New Sulfur Chemolithotrophs of α-Proteobacteria Isolated from Indian Soils

Five facultative sulfur chemolithotrophs were isolated from soils to study the diversity of sulfur lithotrophy. Phenotypic characteristics, including sulfur lithotrophic properties and chemotaxonomic features of the isolates, were similar to those of the members of the colorless sulfur bacteria. 16S rDNA sequence analyses rendered placing the isolates to three distinct phylogenetic clusters of -proteobacteria. Three isolates, AS001, AS002, and KCT002, were identified as members of the genus Paracoccus. The strains AS001 and AS002, having identical 16S-rDNA sequence, showed significant 16S rDNA sequence similarity (99.1%) to Paracoccus versutus. The strain KCT002 showed highest (98%) 16S rDNA sequence similarity to P. alcaliphilus and 96% similarity to the pair AS001 and AS002. Isolate KCT001 appeared to be closely related to Pseudaminobacter salicylatoxidans, although sulfur lithotrophy of P. salicylotoxidans is not known. The other isolate, TCK, showed almost identical 16S rDNA (99.9%) sequence with two recently described unclassified chemolithoautotrophic arsenite oxidizing strains. Physiological and chemotaxonomic characteristics and phylogenetic analyses of the five new strains emphasize the need of polyphasic bacterial taxonomy of sulfur lithotrophs.     

Isolation and characterization of genomic microsatellite markers for small cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton) for utility in genetic diversity analysis

Microsatellite markers in small cardamom (Elettaria cardamomum Maton) were developed using the selective hybridization enrichment method. A total of 140 microsatellite repeats were identified from 270 clones. Primers were designed for 58 microsatellites and 44 primer pairs amplified products of expected size in cardamom. These markers were used for studying the diversity of 20 important small cardamom genotypes, and six markers were found to be polymorphic. The number of alleles ranged from 2 to 7 with an average of 3.6 per locus. Polymorphic information content values ranged from 0.14 to 0.38 based on dominant scoring. The two markers ECM 47a and ECMG 28 generated specific banding patterns for the genotypes MCC7 (Pink tiller) and APG434 (MA18) respectively. Dendrogram illustrated the genetic similarity between different genotypes of Kerala and Karnataka regions. It differentiated the closely related genotypes and released varieties into separate groups. Principal coordinate analysis revealed PV1 and ICRI 1 as the most divergent genotypes. The study demonstrated that these markers are informative and can be further utilized for generating reliable molecular data for assisting the crop improvement of small cardamom. Cross generic transferability (71.4 %) of the developed primers proved that they are useful for phylogenetic studies in the family Zingiberaceae. This is the first report of de novo isolation, characterisation and utilization of microsatellite markers for the genetic diversity analysis of small cardamom.     

Iron oxide labeling does not affect differentiation potential of human bone marrow mesenchymal stem cells exhibited by their differentiation into cardiac and neuronal cells

Osteoarthritis (OA) is characterized by articular cartilage degradation and joint inflammation. The purpose of the present study is to elucidate the role of the specific function of PRMT1 in chondrocytes and its association with the pathophysiology of OA. We observed that the expression of PRMT1 was apparently upregulated in OA cartilage, as well as in chondrocytes stimulated with IL-1β. Additionally, knockdown of PRMT1 suppressed interleukin 1 beta (IL-1β)-induced extracellular matrix (ECM) metabolic imbalance by regulating the expression of MMP-13, ADAMTS-5, COL2A1, and ACAN. Furthermore, silencing of PRMT1 dramatically declined the production of prostaglandin E2 (PGE2) and nitric oxide as well as the level of pro-inflammatory cytokine IL-6 and TNF-α. Mechanistic analyses further revealed that IL-1β-induced activation of the Hedgehog/Gli-1 signaling is suppressed upon PRMT1 knockdown. However, the effects of inhibition of PRMT1-mediated IL-1β-induced cartilage matrix degradation and inflammatory response in OA chondrocytes were obviously abolished by Hedgehog agonist Purmorphamine (Pur). Our data collectively suggest that silencing of PRMT1 exerts anti-catabolic and anti-inflammatory effects on IL-1β-induced chondrocytes via suppressing the Gli-1 mediated Hedgehog signaling pathway, indicating that PRMT1 plays a critical role in OA development and serves as a promising therapeutic target for OA.     

On elongation factor eEFSec,its role and mechanism during selenium incorporation into nascent selenoproteins

Background

Selenium, an essential dietary micronutrient, is incorporated into proteins as the amino acid selenocysteine (Sec) in response to in-frame UGA codons. Complex machinery ensures accurate recoding of Sec codons in higher organisms. A specialized elongation factor eEFSec is central to the process.

Identification and Characterization of Highly Divergent Simian Foamy Viruses in a Wide Range of New World Primates from Brazil

Foamy viruses naturally infect a wide range of mammals, including Old World (OWP) and New World primates (NWP), which are collectively called simian foamy viruses (SFV). While NWP species in Central and South America are highly diverse, only SFV from captive marmoset, spider monkey, and squirrel monkey have been genetically characterized and the molecular epidemiology of SFV infection in NWPs remains unknown. We tested a large collection of genomic DNA (n  = 332) comprising 14 genera of NWP species for the presence of SFV polymerase (pol) sequences using generic PCR primers. Further molecular characterization of positive samples was carried out by LTR-gag and larger pol sequence analysis. We identified novel SFVs infecting nine NWP genera. Prevalence rates varied between 14–30% in different species for which at least 10 specimens were tested. High SFV genetic diversity among NWP up to 50% in LTR-gag and 40% in pol was revealed by intragenus and intrafamilial comparisons. Two different SFV strains infecting two captive yellow-breasted capuchins did not group in species-specific lineages but rather clustered with SFVs from marmoset and spider monkeys, indicating independent cross-species transmission events. We describe the first SFV epidemiology study of NWP, and the first evidence of SFV infection in wild NWPs. We also document a wide distribution of distinct SFVs in 14 NWP genera, including two novel co-speciating SFVs in capuchins and howler monkeys, suggestive of an ancient evolutionary history in NWPs for at least 28 million years. A high SFV genetic diversity was seen among NWP, yet these viruses seem able to jump between NWP species and even genera. Our results raise concerns for the risk of zoonotic transmission of NWP SFV to humans as these primates are regularly hunted for food or kept as pets in forest regions of South America.