Isoamyl alcohol-induced morphological change in Saccharomyces cerevisiae involves increases in mitochondria and cell wall chitin content

Isoamyl alcohol reduced growth and induced filament formation in Saccharomyces cerevisiae. Isoamyl alcohol-induced filamentation was accompanied by an almost threefold greater increase in the specific activity of succinate dehydrogenase than in untreated cells, which suggested that isoamyl alcohol treatment caused the cells to produce more mitochondria than in normal yeast form proliferation. This was supported by measuring the dry weight of purified, isolated mitochondria. Filaments have an increased chitin content which is distributed over the majority of their surface, and is not confined to bud scars and the chitin ring between mother and daughter cells as in yeast-form cells.     

Suzuki cross-coupling approaches to the synthesis of bioactive 3-substituted and 5-substituted-4-methoxy-6-methyl-2-pyrones

Suzuki cross-coupling has been used to access a wide range of 3- and 5-substituted 2-pyrones, which show remarkable inhibitory activity against bacteria, yeasts and fungi. 3-Octenyl and 5-octenyl 2-pyrones inhibit human ovarium carcinoma (A2780) and human chronic myelogenous leukaemia (K562) cell lines at the micromolar level.     

Nasal and cloacal bacteria in free-ranging desert tortoises from the western United States

Aerobic bacteria were collected from three free-ranging desert tortoise (Gopherus agassizii) populations in the eastern Mojave Desert (Arizona, Utah; USA) from 1989 to 1993, and from two free-ranging populations in the central Sonoran Desert (Arizona, USA) from 1990 to 1994. Six species of nasal bacteria and 18 species of cloacal bacteria were identified. At least one potential pathogen was found in the nasal cavity (Pasteurella testudinis), and at least two potential pathogens in the cloaca (Pseudomonas spp., Salmonella spp.).     

Gene targeting in primary fetal fibroblasts from sheep and pig

Nuclear transfer offers a new cell-based route for introducing precise genetic modifications in a range of animal species. However, significant challenges, such as establishment of somatic gene targeting techniques, must be overcome before the technology can be applied routinely. In this report, we describe targeted deletion at the GGTA1 (alpha 1,3-galactosyl transferase) and PrP (prion protein) loci in primary fibroblasts from livestock. We place particular emphasis on the growth characteristics of the primary cell cultures, since these are key to determining success.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.     

Using electroporation and lipid-mediated transfection of GFP-expressing plasmids to label embryonic avian cells for vital confocal and two-photon microscopy

Fluorescent proteins have emerged as an ideal fluorescent marker for studying cell morphologies in vital systems. These proteins were first applied in whole organisms with established germ-line transformation protocols, but now it is possible to label cells with fluorescent proteins in other organisms. Here we present two ways to introduce GFP expressing plasmids into avian embryos for vital confocal and two-photon imaging. First, electroporation is a powerful approach to introduce GFP into the developing neural tube, offering several advantages over dye labeling. Second, we introduce a new lipid-based transfection system for introducing plasmid DNA directly to a small group of injected cells within live, whole embryos. These complementary approaches make it possible to transfect a wide-range of cell types in the avian embryo and the bright, stable, uniform expression of GFP offers great advantages for vital fluorescence imaging.     

4-hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells

Glutathione is the most abundant non-protein thiol in the cell, with roles in cell cycle regulation, detoxification of xenobiotics, and maintaining the redox tone of the cell. The glutathione content is controlled at several levels, the most important being the rate of de novo synthesis, which is mediated by two enzymes, glutamate cysteine ligase (GCL), and glutathione synthetase (GS), with GCL being rate-limiting generally. The GCL holoenzyme consists of a catalytic (GCLC) and a modulatory (GCLM) subunit, which are encoded by separate genes. In the present study, the signaling mechanisms leading to de novo synthesis of GSH in response to physiologically relevant concentrations of 4-hydroxy-2-nonenal (4HNE), an endproduct of lipid peroxidation, were investigated. We demonstrated that exposure to 4HNE resulted in increased content of both Gcl mRNAs, both GCL subunits, phosphorylated JNK1 and c-Jun proteins, as well as Gcl TRE sequence-specific AP-1 binding activity. These increases were attenuated by pretreating the cells with a novel membrane-permeable JNK pathway inhibitor, while chemical inhibitors of the p38 or ERK pathways were ineffective. These data reveal that de novo GSH biosynthesis in response to 4HNE signals through the JNK pathway and suggests a major role for AP-1 driven expression of both Gcl genes in HBE1 cells.     

Internalization of bioluminescent Escherichia coli and Salmonella Montevideo in growing bean sprouts

AIMS: Investigate the interaction of bioluminescent Escherichia coli and Salmonella Montevideo with germinating mung bean sprouts. METHODS AND RESULTS: E. coli or Salm. Montevideo introduced on mung beans became established both internally and externally on sprouts after the initial 24 h germinating period. In both cases the inoculated bacterium formed the predominant microflora on the sprouted beans throughout. From the bioluminescent profile of inoculated sprouting beans, bacterial growth was found to be in close proximity to the roots but not on the hypocotyls. Clumps (biofilms) of cells with low viability were observed within the grooves between epidermal cells on hypocotyls. Treatment with 20,000 ppm sodium hypochlorite removed the majority of bacteria from the surface of hypocotyls although nonviable single cells were occasionally observed. However, viable bacteria were recovered from the apoplastic fluid, and extracts of surface-sterilized sprouts indicating that the internal bacterial populations had been protected. This was confirmed using in situ beta-glucuronidase staining of surface-sterilized sprouts where cleaved enzyme substrate (by the action of internalized E. coli) was visualized within the plant vascular system. CONCLUSIONS: E. coli or Salmonella present on seeds become internalized within the subsequent sprouts and cannot be removed by postharvest biocidal washing. SIGNIFICANCE AND IMPACT OF THE STUDY: Mung bean production should be carefully controlled to prevent contamination occurring in order to minimize the health risk associated with raw bean sprouts.