Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well‐established in vivo. In this work we have performed microarray experiments with a spermidine synthase, spermine oxidase mutant (Δspe3 Δfms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10^?5 M ) or spermine (10^?5 M ) compared to a control culture containing 10^?8 M spermidine. 247 genes were upregulated > two‐fold and 11 genes were upregulated >10‐fold after spermidine addition. Functional categorization of the genes showed induction of transport‐related genes and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were downregulated more than two‐fold, and six genes were downregulated > eight‐fold after spermidine addition. A majority of the downregulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only a few genes (18) were significantly responsive to spermine. Thus, results from global gene expression profiling demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. Copyright © 2009 John Wiley & Sons, Ltd.     

Growth of a tropical marine yeast Yarrowia lipolytica NCIM 3589 on bromoalkanes: relevance of cell size and cell surface properties

Yarrowia lipolytica 3589, a tropical marine yeast, grew aerobically on a broad range of bromoalkanes varying in carbon chain length and differing in degree and position of bromide group. Amongst the bromoalkanes studied, viz. 2‐bromopropane (2‐BP), 1‐bromobutane (1‐BB), 1,5‐dibromopentane (1,5‐DBP) and 1‐bromodecane (1‐BD), the best utilized was 1‐BD, with a maximal growth rate (μ_max) of 0.055 h^?1 and an affinity ratio (μ_max/K_s) of 0.022. Utilization of these bromoalkanes as growth substrates was associated with a concomitant release of bromide (8202.9 µm ) and cell mass (36 × 10⁹ cells/ml), occurring maximally on 1‐BD. Adherence of yeast cells to these hydrophobic bromoalkanes was observed microscopically, with an increase in cell size and surface hydrophobicity. The maximal cell diameter was for 1‐BD (4.66 µm), resulting in an increase in the calculated cell surface area (68.19 µm²) and sedimentation velocity (1.31 µm/s). Cell surface hydrophobicity values by microbial adhesion to solvents (MATS) analysis for yeasts grown on bromoalkanes and glucose were significantly high, i.e. >80%. Similarly, water contact angles also indicate that the cell surface of yeast cells grown in glucose possess a relatively more hydrophilic cell surface (θ = 49.1°), whereas cells grown in 1‐BD possess a more hydrophobic cell surface (θ = 90.7°). No significant change in emulsification activity or surface tension was detected in the cell‐free supernatant. Thus adherence to the bromoalkane droplets by an increase in cell size and surface hydrophobicity leading to debromination of the substrate might be the strategy employed in bromoalkane utilization and growth by Y. lipolytica 3589. Copyright © 2011 John Wiley & Sons, Ltd.     

The influence of nitrate on the physiology of the yeast Dekkera bruxellensis grown under oxygen limitation

A previous study showed that the use of nitrate by Dekkera bruxellensis might be an advantageous trait when ammonium is limited in sugarcane substrate for ethanol fermentation. The aim of the present work was to evaluate the influence of nitrate on the yeast physiology during cell growth in different carbon sources under oxygen limitation. If nitrate was the sole source of nitrogen, D. bruxellensis cells presented slower growth, diminished sugar consumption and growth‐associated ethanol production, when compared to ammonium. These results were corroborated by the increased expression of genes involved in the pentose phosphate (PP) pathway, the tricarboxylic acid (TCA) cycle and ATP synthesis. The presence of ammonium in the mixed medium restored most parameters to the standard conditions. This work may open up a line of investigation to establish the connection between nitrate assimilation and energetic metabolism in D. bruxellensis and their influence on its fermentative capacity in oxygen‐limited or oxygen‐depleted conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

Versican expression by dermal papilla-regenerated hair follicles – a promising tool for hair-regrowth products

Hair follicle stem cells and dermal papilla cells play essential roles in the cyclical hair regeneration process. However, compared with the recent progress of follicular stem cell research, detailed analysis of dermal papilla cells is virtually unknown. We have focused on a large chondroitin sulfate proteoglycan molecule, versican, and prove its pivotal role in hair follicle induction both in vitro and in vivo. First, we examined versican expression with hair cycling by in situ hybridization (mRNA) and immunohistochemistry (protein). Results clearly showed specific versican expression in anagen onset of dermal papilla cells implying a function role of versican in hair induction. We then generated transgenic lines with LacZ or green fluorescent protein reporter genes under a versican regulatory element (promoter) control, and confirmed dermal papilla‐specific activation of the reporter. These transgenic lines were utilized to isolate the dermal papilla cell population by means of a fluorescent activated cell sorter. While freshly isolated dermal papilla cells were able to induce hair growth in a skin reconstitution assay when grafted with undifferentiated epidermal cells, this ability and versican expression were rapidly lost during passages in culture. In order to examine the functional role of versican in hair growth induction, versican full‐length cDNAs were transfected into inactive passaged dermal papilla cells. Impressively, this forced expression of versican molecules partially restored hair inductivity of dermal papilla cells in a skin reconstitution assay. These results demonstrated the pivotal role of versican in hair induction both in vitro and in vivo and will connect the missing link between the signal from dermal papilla cells to follicular stem cells, and initiation of hair regeneration. We have also shown that the hair growth‐promoting compound, cyclosporin A, can upregulate versican expression implying the application of a novel screening approach for hair growth‐promoting compounds by monitoring versican expression as an indicator.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

Production and structural characterisation of dextran from an indigenous strain of Leuconostoc mesenteroides BA08 in Whey

In this work, the production of dextran was carried out in whey‐supplemented media using Leuconostoc mesenteroides BA08. Different growth and nutritional parameters were optimised to maximise the dextran production. Batch production in whey‐based media under optimised nutritional and growth conditions yielded a dextran concentration of 17.25 g/L. Structural analysis of the purified polymer by FT‐IR, 1D ¹H and ¹³C nuclear magnetic resonance methodology revealed that polymer was a linear dextran having 93% α‐(1→6) linkage in the main chain. The morphology of the dried dextran and dextran‐producing cells was also studied by scanning electron microscope which showed a granular and porous or weblike structure, respectively. The results obtained showed that the industrial whey supplemented with nutrients such as sucrose, yeast extract and K₂HPO₄ can serve as an ideal growth medium for dextran production. The optimised fermentation and nutritional parameters can be further scaled up to establish the potential for the commercial production of food‐grade dextran from whey as part of a novel cost‐effective and environment‐friendly approach.     

Solanum nigrum L. polyphenolic extract inhibits hepatocarcinoma cell growth by inducing G2/M phase arrest and apoptosis

BACKGROUND: Hepatocellular carcinoma (HCC) is a rapidly progressive cancer with poor prognosis. However, there have been no significant new developments in treating liver cancer. To search for an effective agent against HCC progression, we prepared a polyphenolic extract of Solanum nigrum L. (SNPE), a herbal plant indigenous to Southeast Asia and commonly used in oriental medicine, to evaluate its inhibitive effect on hepatocarcinoma cell growth. The growth inhibition of HepG2 cells in vitro and in vivo was determined in the presence of SNPE. RESULTS: We found 1 µg mL^?1 SNPE‐fed mice showed decreased tumor weight and tumor volume by 90%. Notably, 2 µg mL^?1 SNPE resulted in almost complete inhibition of tumor weight as well as tumor volume. In line with this notion, SNPE reduced the viability of HepG₂ cells in a dose‐dependent manner. HepG₂ cells were arrested in the G₂/M phase of the cell cycle; meanwhile, the protein levels of cell CDC25A, CDC25B, and CDC25C were clearly reduced. Moreover, sub‐G₁ phase accumulation and caspases‐3, 8, and 9 cleavages were induced by SNPE. CONCLUSION: This study shows that SNPE is a potent agent for HCC treatment through targeting G₂/M arrest and apoptosis induction, achieving cell growth inhibition. Copyright © 2010 Society of Chemical Industry     

Evaluation of the conditions of carotenoids production in a synthetic medium by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor

This work studied the cultivation conditions for the production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) in a bioreactor. A Plackett–Burman design was used for the screening of the most important factors, followed by a complete second order design, to maximise the concentration of total carotenoids. The maximum concentration of 3425.9 μg L^?1 of total carotenoids was obtained in a medium containing 80 g L^?1 glucose, 15 g L^?1 peptone and 5 g L^?1 malt extract, with an aeration rate 1.5 vvm, 180 r.p.m., 25 °C and an initial pH of 4.0. Fermentation kinetics showed that the maximum concentration of total carotenoids was reached after 90 h of fermentation. Carotenoid bio‐production was partially associated with cell growth. The specific carotenoid production (Y_P/X) was 238 μg carotenoids/g cells, whereas Y_P/S (substrate to product yield) was 41.3 μg g^?1. The specific growth rate (μ_x) was 0.045 h^?1. The highest cell and total carotenoid productivity were 0.19 g L^?1 h^?1 and 56.9 μg L^?1 h^?1, respectively.     

Active hexose correlated compound exerts therapeutic effects in lymphocyte driven colitis

Active hexose correlated compound (AHCC) is a commercial extract of Basidiomycetes fungi enriched in oligosaccharides that is used as a human nutritional supplement for various purposes in humans. Our aim was to study the anti‐inflammatory effect of AHCC in the CD4+ CD62L⁺ T cell transfer model of colitis, considered one of the closest to the human disease. Colitis was induced by transfer of CD4⁺ CD62L⁺ T cells to recombination activating gene 1^?/? mice. AHCC (75 mg/d) was administered by gavage as a post‐treatment. Three groups were established: noncolitic, colitic (CD4⁺ CD62L⁺ transferred mice treated with vehicle), and AHCC (colitic treated with AHCC). AHCC improved colitis, as evidenced by a 24% lower colonic myeloperoxidase and a 21% lower alkaline phosphatase activity. In addition, a decreased secretion of proinflammatory genes assessed by RT‐qPCR was observed, particularly TNF‐α and IL‐1β. Ex vivo mesenteric lymph node cells obtained from AHCC treated mice exhibited a fully normalized production of IL‐6, IL‐17, and IL‐10 (p < 0.05). Also, AHCC treated mice exhibited decreased STAT4 and IκB‐α phosphorylation in splenic CD4⁺ cells. Our data provide validation of AHCC colonic anti‐inflammatory activity in a chronic, T cell driven model of inflammatory bowel disease.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

Genome‐wide expression profiling of the osmoadaptation response of Debaryomyces hansenii

The euryhaline marine yeast Debaromyces hansenii is a model system for the study of processes related to osmoadaptation. In this study, microarray‐based gene expression analyses of the entire genome of D. hansenii was used to study its response to osmotic stress. Differential gene expression, compared to control, was examined at three time points (0.5, 3 and 6 h) after exposure of D. hansenii cultures to high salt concentration. Among the 1.72% of genes showing statistically significant differences in expression, only 65 genes displayed at least three‐fold increases in mRNA levels after treatment with 2 M NaCl. On the other hand, 44 genes showed three‐fold repression. Upregulated as well as the downregulated genes were grouped into functional categories to identify biochemical processes possibly affected by osmotic stress and involved in osmoadaptation. The observation that only a limited number of genes are upregulated in D. hansenii in response to osmotic stress supports the notion that D. hansenii is pre‐adapted to survive in extreme saline environments. In addition, since more than one‐half of the upregulated genes encode for ribosomal proteins, it is possible that a translational gene regulatory mechanism plays a key role in D. hansenii's osmoregulatory response. Validation studies for ENA1 and for hyphal wall/cell elongation protein genes, using real‐time PCR, confirmed patterns of gene expression observed in our microarray experiments. To our knowledge, this study is the first of its kind in this organism and provides the foundation for future molecular studies assessing the significance of the genes identified here in D. hansenii's osmoadaptation. Copyright © 2009 John Wiley & Sons, Ltd.     

MALDI‐TOF MS characterization of proanthocyanidins from cranberry fruit (Vaccinium macrocarpon) that inhibit tumor cell growth and matrix metalloproteinase expression in vitro

Proanthocyanidin‐rich extracts were prepared by fractionation of the fruit of the North American cranberry (Vaccinium macrocarpon). In vitro growth inhibition assays in eight tumor cell lines showed that selected fractions inhibited the growth of H460 lung tumors, HT‐29 colon and K562 leukemia cells at GI₅₀ values ranging from 20 to 80 µg ml^?1. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) of one of these fractions found it to be composed of polyflavan‐3‐ols, which are primarily tetramers through heptamers of epicatechin containing one or two A‐type linkages. Whole cranberry extract and the proanthocyanidin fractions were screened for effect on the expression of matrix metalloproteinases in DU 145 prostate carcinoma cells. The expression of MMP‐2 and MMP‐9 was inhibited in response to whole cranberry extract and to a lesser degree by the proanthocyanidin fractions. Copyright © 2005 Society of Chemical Industry