Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae

Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.     

Acyl-homoserine lactone-dependent eavesdropping promotes competition in a laboratory co-culture model

Many Proteobacteria use acyl-homoserine lactone (AHL)-mediated quorum sensing to activate the production of antibiotics at high cell density. Extracellular factors like antibiotics can be considered public goods shared by individuals within a group. Quorum-sensing control of antibiotic production may be important for protecting a niche or competing for limited resources in mixed bacterial communities. To begin to investigate the role of quorum sensing in interspecies competition, we developed a dual-species co-culture model using the soil saprophytes Burkholderia thailandensis (Bt) and Chromobacterium violaceum (Cv). These bacteria require quorum sensing to activate the production of antimicrobial factors that inhibit growth of the other species. We demonstrate that quorum-sensing-dependent antimicrobials can provide a competitive advantage to either Bt or Cv by inhibiting growth of the other species in co-culture. Although the quorum-sensing signals differ for each species, we show that the promiscuous signal receptor encoded by Cv can sense signals produced by Bt, and that this ability to eavesdrop on Bt can provide Cv an advantage in certain situations. We use an in silico approach to investigate the effect of eavesdropping in competition, and show conditions where early activation of antibiotic production resulting from eavesdropping can promote competitiveness. Our work supports the idea that quorum sensing is important for interspecies competition and that promiscuous signal receptors allow eavesdropping on competitors in mixed microbial habitats.     

Attenuation of penicillin resistance in a peptidoglycan O-acetyl transferase mutant of Streptococcus pneumoniae

The level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillin-resistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 microg ml(-1). Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for beta-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes--reduced resistance and lysozyme sensitivity--were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.     

Molecular mechanisms of epithelial-barrier disruption by Helicobacter pylori

Intact intercellular junctions and cell-matrix contacts are important structures in the formation and maintenance of epithelial-barrier functions against microbes. The human gastric pathogen Helicobacter pylori developed a remarkable network of strategies to alter these epithelial cell-cell and cell-matrix adhesions, which are implicated in inflammation, proliferation, cell migration and invasive growth. This review focuses on recent findings on H. pylori-induced host-cell signaling. We propose a stepwise model for how H. pylori interacts with components of focal adhesions and intercellular tight and adherens junctions to disrupt the epithelial layer, providing novel insights into the pathogenesis of H. pylori.     

Enhanced plumbagin accumulation in embryogenic cell suspension cultures of Plumbago rosea L. following elicitation

This study focused on enhancing the production of plumbagin, an anticancer compound, in embryogenic cell suspension cultures of Plumbago rosea. Elicitation techniques have been reported to enhance plumbagin production. Cell suspension cultures raised from embryogenic calli induced from in vitro leaf explants were exposed to different concentrations of jasmonic acid, yeast extract and different auxin combinations. Influence of these on cell growth, biomass and plumbagin production was studied. To our knowledge this is the first report on elicitation of embryogenic cell suspension cultures of P. rosea for enhanced plumbagin production. Elicitor treated suspension cultures exhibited decreased culture viability and increased plumbagin synthesis. A maximum of 5.59-fold enhancement of plumbagin production was observed in cultures added with 1 mg L^?1 naphthalene acetic acid after 6 days of incubation. Viability of cultures decreased with increased concentration of elicitors and prolonged incubation period. Application of elicitors in cell suspension cultures induces defense related responses which lead to increased secondary metabolite production for making the cells adapt to the situation. If the stressed condition persists or is in intolerable level this will eventually lead to programmed cell death and loss of culture viability.     

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.     

Hysteresis light curves: a protocol for characterizing the time dependence of the light response of photosynthesis

Photosynthesis vs. light curves (LCs) have played a central role in photosynthesis research for decades. They are the commonest form of describing how photosynthesis responds to changes in light, being frequently used for characterizing photoacclimation. However, LCs are often interpreted exclusively regarding the response to light intensity, the effects of time of exposure not being explicitly considered. This study proposes the use of ‘hysteresis light curves’ (HLC), an experimental protocol focused on the cumulative effects of light exposure to obtain information on the time dependence of photosynthetic light responses. HLC are generated by exposing samples to a symmetrical sequence of increasing and decreasing light levels. The comparison of the light-increasing and the light-decreasing phases allows the quantification of the hysteresis caused by high-light exposure, the magnitude and direction of which inform on the activation, and subsequent relaxation of high-light-induced photosynthetic processes. HLCs of the chlorophyll fluorescence indices rETR (relative electron transport rate of photosystem II) and Y(NPQ) (index of non-photochemical quenching) were measured on cyanobacteria, algae, and plants, with the aim of identifying main patterns of hysteresis and their diversity. A non-parametric index is proposed to quantify the magnitude and direction of hysteresis in HLCs of rETR and Y(NPQ). The results of this study show that HLCs can provide additional relevant information on the time dependence of the light response of photosynthetic samples, not obtainable from conventional LCs, useful for phenotyping photosynthetic traits, including photoacclimation state and kinetics of light activation and relaxation of electron flow and energy dissipation processes.

     

Zona pellucida characteristics and sperm-binding patterns of in vivo and in vitro produced porcine oocytes inseminated with differently prepared spermatozoa

IVF of porcine oocytes has been carried out in many laboratories. However, polyspermic fertilization is still a major issue to be solved. It is well known that besides the nucleus, oocyte organelles and the cytoplasm have to undergo a final maturation process before they become fully competent for fertilization. Until now, it is still uncertain whether the zona pellucida (ZP) must also undergo a maturation process and what impact the maturation status may have on sperm recognition and monospermic fertilization. Our data show that the ZP undergoes biochemical changes in the final maturation phase of the oocyte prior to fertilization. During zona maturation, the induction of the acrosome reaction in spermatozoa bound to the zona pellucida shows a different time pattern. Additionally, it was shown by 2D gel electrophoresis that after maturation, ZPA moved 0.8 pI units and ZPB/ZPC 1.3 pI units in the direction of the anode, indicating increased acidity. These preliminary studies indicate that the maturation processes of the oocyte involves biochemical and functional alterations in the zona pellucida. In addition, the morphology of the porcine ZP was investigated before and after maturation at the GVI and metaphase II stage as well as 1h after onset of IVF. No significant consistent structural changes were seen between immature oocytes and those matured in vitro for 48 h. However, at 24 h, the zona structures were more similar to those in in vivo matured oocytes. This phenomenon needs to be elucidated. So far, the only way to avoid polyspermic penetration is to reduce the number of spermatozoa per oocyte used for IVF. The amount depends on the treatment of the sperm and has to be set for each individual boar.