Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: Modulation by calcium-regulated proteases and protein kinases

Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-6 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium.     

Identification of secreted proteins of Aspergillus oryzae associated with growth on solid cereal substrates

Filamentous growth of Aspergillus oryzae on solid cereal substrates involves secretion of substrate converting enzymes and a solid substrate specific polarised hyphal growth phenotype. To identify proteins produced under these specific conditions, the extracts of A. oryzae grown on wheat-based media were analysed using N-terminal sequence analysis. In a submerged wheat-based growth medium of A. oryzae, besides alpha-amylase, also an arabinosidase and xylanase were abundantly produced. In the extracts of A. oryzae grown on wheat-based solid substrate besides alpha-amylase and chitinase, two new proteins of 16 and 27 kDa were identified. These hypothetical proteins showed only close homologies to filamentous fungal proteins.     

Variable number of tandem repeat markers in the genome sequence of Mycosphaerella fijiensis, the causal agent of black leaf streak disease of banana (Musa spp)

We searched the genome of Mycosphaerella fijiensis for molecular markers that would allow population genetics analysis of this plant pathogen. M. fijiensis, the causal agent of banana leaf streak disease, also known as black Sigatoka, is the most devastating pathogen attacking bananas (Musa spp). Recently, the entire genome sequence of M. fijiensis became available. We screened this database for VNTR markers. Forty-two primer pairs were selected for validation, based on repeat type and length and the number of repeat units. Five VNTR markers showing multiple alleles were validated with a reference set of isolates from different parts of the world and a population from a banana plantation in Costa Rica. Polymorphism information content values varied from 0.6414 to 0.7544 for the reference set and from 0.0400 and 0.7373 for the population set. Eighty percent of the polymorphism information content values were above 0.60, indicating that the markers are highly informative. These markers allowed robust scoring of agarose gels and proved to be useful for variability and population genetics studies. In conclusion, the strategy we developed to identify and validate VNTR markers is an efficient means to incorporate markers that can be used for fungicide resistance management and to develop breeding strategies to control banana black leaf streak disease. This is the first report of VNTR-minisatellites from the M. fijiensis genome sequence.     

A purification method for tag-free human cystatin C recombinant protein expressed in Escherichia coli

To obtain recombinant cystatin C (CysC) protein, which can be used in immunological diagnostic kits, we focused on the preparation of tag-free CysC. The 6?×?His–TF–CysC fusion protein was found to overexpress in soluble form in cells of BL21-Gold (DE3)/pCold TF–CysC, which had been induced with isopropyl-D-1-thiogalactopyranoside. Subsequently, we established a protein purification method for tag-free CysC using immobilized metal-affinity chromatography and size-exclusion chromatography. In this method, glutathione-S-transferase–human rhinovirus 3C proteases were used to remove the protein tags. High homogeneity of the purified CysC was determined by SDS-PAGE, while the purity of the tag-free CysC was ascertained to be above 95%. With a yield of 25?mg/L from bacterial culture, the biological activity of the tag-free CysC was evaluated as inhibitors like natural CysC. The performance of this purification method was successfully evaluated in the preparation of other low molecular weight heterologous proteins in Escherichia coli.     

Proliferation‐ and migration‐enhancing effects of ginseng and ginsenoside Rg1 through IGF‐I‐ and FGF‐2‐signaling pathways on RSC96 Schwann cells

The aim of the present study is to evaluate the proliferation‐ and migration‐enhancing effects of ginseng and its component, ginsenoside (Rg1) on RSC96 Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs‐IGFIR‐Akt‐Bcl2 and proliferative signaling, cell cycle factors and mitogen‐activated protein kinase (MAPK) pathways, (2) migrating and anti‐scar signaling, FGF‐2‐uPA‐MMPs.We treated RSC96 cells with different concentrations (100, 200, 300, 400, 500 µg ml^?1) of ginseng and its constituent, Rg1 (5, 10, 15, 20, 25 µg ml^?1). We observed a proliferative effect in a dose‐dependent manner by PCNA western blotting assay, MTT assay, and wound healing test. Furthermore, we also found in the results of western blotting assay, ginseng and Rg1 enhance protein expression of IGF‐I pathway regulators, cell cycle controlling proteins, and MAPK signaling pathways to promote the cell proliferation. In addition, ginseng and Rg1 also stimulated the FGF‐2‐uPA‐MMP 9 migrating pathway to enhance the migration of RSC96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of ginseng and Rg1 on RSC96 cells were identified to be MAPK signaling‐dependent. On the basis of the results, applying appropriate doses of ginseng and Rg1 with biomedical materials would be a potential approach for enhancing neuron regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

Losartan and its interaction with copper(II): biological effects

Losartan, the potassium salt of 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol, is an efficient antihypertensive drug. The vibrational FTIR and Raman spectra of Losartan (its anionic and protonated forms) are discussed. In addition, the copper(II) complex of Losartan was obtained and characterized as a microcrystalline powder. The metal center is bound to the ligand through the nitrogen atoms of the tetrazolate moiety as determined by vibrational spectroscopy. The compound is a dimer with the metal centers in a tetragonal distorted environment but the presence of a monomeric impurity has been determined by EPR spectroscopy. The antioxidant properties of the complex (superoxide dismutase mimetic activity) and its effect on the proliferation and morphology of two osteoblast-like cells in culture are reported. The new compound exerted more toxic effects on tumoral cells than the copper(II) ion and Losartan.     

Systematic revision of the freshwater snail Margarya Nevill, 1877 (Mollusca: Viviparidae) endemic to the ancient lakes of Yunnan,China, with description of new taxa

The systematics of the viviparid freshwater snail genus Margarya endemic to the ancient lakes of Yunnan, China, is revised based on comparative analyses of morphological features, including shell, operculum, radula, and genital anatomy, and molecular phylogenetic analyses of partial sequences of the mitochondrial 16S rDNA (16S) and cytochrome c oxidase subunit I (COI) genes, as well as the nuclear Internal Transcribed Spacer 2 (ITS2). The taxonomic utility of key anatomical and morphological features in this group is evaluated. The genus Margarya as delimited previously is split into three genera in order to retain monophyletic taxa: (1) Margarya s.s., consisting of four species, i.e. the type species Margarya melanioides plus Margarya francheti, Margarya oxytropoides, and Margarya monodi; (2) the previously introduced subgenus Tchangmargarya is elevated to an independent genus containing two species, Tchangmargarya yangtsunghaiensis and the new species T changmargarya multilabiata sp. nov. ; and (3) a new genus, A nularya gen. nov. , is described, also containing two species, i.e. Anularya mansuyi and Anularya bicostata. Molecular phylogenies based on analyses of three gene fragments have identical topologies, supporting the monophyly of these genera. The sister group of Margarya s.s. is Cipangopaludina, whereas the sister group of Anularya is Sinotaia; Tchangmargarya is sister to a clade containing all the aforementioned groups. Features of the operculum and the right male tentacle (penis) are particularly informative on the generic level, whereas shell and radular characters are especially useful to differentiate species. The phylogenetic relationships recovered here are consistent with orogenic patterns of the Yunnan Mountains. Changes in the river system and water area of ancient lakes caused by tectonic activities probably play an important role in speciation and shaping the current pattern of species distribution in Yunnan. © 2015 The Linnean Society of London     

Fungi colonizing hair-baits from three coastal beaches of Mexico

The abundance of hair-bait colonizing fungi was investigated on the beaches of Caracoles, Mocambo, and lcacos, located on the coasts of the Caribbean Sea, Gulf of Mexico and Pacific Ocean, respectively. On each beach a sample of sandy soil was collected. The samples were analyzed by two selective isolation methods for keratinophilic fungi resulting in a total of 544 occurrences. A total of 17 species was found, of which 4 were ascomycetes and 13 hyphomycetes.Gymnascella dankaliensis andAspergillus terreus were the most abundant.Arthroderma curreyi andChrysosporium tropicum were found in low percentages in this survey. From the three beaches sampled, Icacos beach, on the Pacific Ocean coast had the highest number of isolated species.     

The pygidial gland secretion of the forest caterpillar hunter,Calosoma (Calosoma) sycophanta: the antimicrobial properties against human pathogens

Applied Microbiology and Biotechnology - Recently, various insect secretions have been tested as possible antimicrobial agents. In beetles, these secretions are essentially products of various...     

CDl33＋／ABCG2＋的人肺癌多耐药性肿瘤细胞亚群的分离及鉴定

肿瘤干细胞的多耐药性是导致肿瘤化疗失败的重要因素之一。因此,鉴定和明确耐药性肺癌干细胞亚群（cancer stem-cell subpopulations）的特征和机制是当前的热点。该研究从肺癌病人的肿瘤组织中分离出一个高表达CDl33和ABCG2蛋白的细胞亚群。发现该CDl33＋／ABCG2＋肺癌细胞高表达干细胞的生物标志,如：Nanog、Oct4、Sox2、Nestin、CD44、CDll7、CDl33和ABCG2等。不仅如此,这群细胞在体外具有高增殖性和高侵袈性,对于多种常用的化疗药物（如：顺铂和吉西他滨等）都具有耐受性。而且,少量的CDl33＋／ABCG2＋肺癌细胞即可以在免疫缺陷小鼠体内形成肿瘤。为了研究耐药性机制,我们检测了CDl33＋／ABCG2＋中ABCG2基因启动子区域的cpG岛（cpGislands）DNA甲基化修饰状态。实验结果表日月,该细胞中,ABCG2基因启动子区域的cpG岛处于去甲基化修饰状态。综上所述,作者成功地从肺癌病人肿瘤组织中分离、富集得到了CDl33＋／ABCG2＋细胞亚群,并利用该群细胞在体外建立了肿瘤耐药性研究模型。对于肺癌CDl33＋／ABCG2＋细胞的研究可为开发肺癌个体化治疗和新型化疗药物的设计和研发提供理论依据．