Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.     

Responses of aquatic algae and cyanobacteria to solar UV-B

Continuous depletion of the stratospheric ozone layer has resulted in an increase in solar ultraviolet-B (UV-B; 280–315 nm) radiation reaching the Earth's surface. The consequences for aquatic phototrophic organisms of this small change in the solar spectrum are currently uncertain. UV radiation has been shown to adversely affect a number of photochemical and photobiological processes in a wide variety of aquatic organisms, such as cyanobacteria, phytoplankton and macroalgae. However, a number of photosynthetic organisms counteract the damaging effects of UV-B by synthesizing UV protective compounds such as mycosporine-like amino acids (MAAs) and the cyanobacterial sheath pigment, scytonemin. The aim of this contribution is to discuss the responses of algae and cyanobacteria to solar UV-B radiation and the role of photoprotective compounds in mitigating UV-B damage.     

P301L tau expression affects glutamate release and clearance in the hippocampal trisynaptic pathway

Individuals at risk of developing Alzheimer's disease (AD) often exhibit hippocampal hyperexcitability. A growing body of evidence suggests that perturbations in the glutamatergic tripartite synapse may underlie this hyperexcitability. Here, we used a tau mouse model of AD (rTg(TauP301L)4510) to examine the effects of tau pathology on hippocampal glutamate regulation. We found a 40% increase in hippocampal vesicular glutamate transporter, which packages glutamate into vesicles, and has previously been shown to influence glutamate release, and a 40% decrease in hippocampal glutamate transporter 1, the major glutamate transporter responsible for removing glutamate from the extracellular space. To determine whether these alterations affected glutamate regulation in vivo, we measured tonic glutamate levels, potassium‐evoked glutamate release, and glutamate uptake/clearance in the dentate gyrus, cornu ammonis 3(CA3), and cornu ammonis 1(CA1) regions of the hippocampus. P301L tau expression resulted in a 4‐ and 7‐fold increase in potassium‐evoked glutamate release in the dentate gyrus and CA3, respectively, and significantly decreased glutamate clearance in all three regions. Both release and clearance correlated with memory performance in the hippocampal‐dependent Barnes maze task. Alterations in mice expressing P301L were observed at a time when tau pathology was subtle and before readily detectable neuron loss. These data suggest novel mechanisms by which tau may mediate hyperexcitability.

     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores.

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microwave‐assisted solid‐phase peptide synthesis at 60 °C: alternative conditions with low enantiomerization

Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS‐ET), but we have elected the following as our first choice: 2.5‐fold molar excess of 0.04–0.08 M Boc or Fmoc‐amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 °C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave‐SPPS, peptides containing L ‐Ser, L ‐His, L ‐Cys and/or L ‐Met were manually synthesized traditionally, at 60 °C using conventional heating and at 60 °C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave‐assisted coupling of L ‐Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 °C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave‐assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS‐ET and suggests alternative conditions for microwave‐assisted SPPS. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Catalase Synthesis and Turnover during Peroxisome Transition in the Cotyledons of Helianthus annuus L

Based on measurements of total catalase hematin and the degradation constants of catalase hematin, zero order rate constants for the synthesis of catalase were determined during the development of sunflower cotyledons (Helianthus annuus L.). Catalase synthesis reached a sharp maximum of about 400 picomoles hematin per day per cotyledon at day 1.5 during the elaboration of glyoxysomes in the dark. During the transition of glyoxysomes to leaf peroxisomes (greening cotyledons, day 2.5 to 5) catalase synthesis was constant at a level of about 30 to 40 picomoles hematin per day per cotyledon. In the cotyledons of seedlings kept in the dark (day 2.5 to 5) catalase synthesis did not exceed 10 picomoles hematin per day per cotyledon. During the peroxisome transition in the light, total catalase hematin was maintained at a high level, whereas total catalase activity rapidly decreased. In continuous darkness, total catalase hematin decreased considerably from a peak at day 2. The results show that both catalase synthesis and catalase degradation are regulated by light. The turnover characteristics of catalase are in accordance with the concept that glyoxysomes are transformed to leaf peroxisomes as described by the one population model and contradict the two population model and the enzyme synthesis changeover model which both postulate de novo formation of the leaf peroxisome population and degradation of the glyoxysome population.     

Phenylacetic acid in an anaerobic swine manure digester

Summary The presence of phenylacetic acid (PAA) in an anaerobic swine manure digester was determined by gas chromatography of the butyl ester and confirmed by mass spectroscopy. PAA concentration increased during start-up of a digester and with low carbon, high nitrogen loading. Unlike acetate, propionate and butyrate, the concentration of PAA varied little through the day in a stable digester loaded once per day. The laboratory scale digester was loaded at 4 g of swine manure solids/liter digester volume per day. The retention time and temperature were 15 days and 37°C. PAA is a microbial intermediate which is produced by one group of anaerobic bacteria and converted to methane by other members of the bacterial community in the digester. As such, it may be a useful indicator of the relative metabolic activity of the bacterial groups and thus of the overall stability of the anaerobic process.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation.

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Protein Kinase B Localization and Activation Differentially Affect S6 Kinase 1 Activity and Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 Phosphorylation

Recent studies indicate that phosphatidylinositide-3OH kinase (PI3K)-induced S6 kinase (S6K1) activation is mediated by protein kinase B (PKB). Support for this hypothesis has largely relied on results obtained with highly active, constitutively membrane-localized alleles of wild-type PKB, whose activity is independent of PI3K. Here we set out to examine the importance of PKB signaling in S6K1 activation. In parallel, glycogen synthase kinase 3beta (GSK-3beta) inactivation and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation were monitored as markers of the rapamycin-insensitive and -sensitive branches of the PI3K signaling pathway, respectively. The results demonstrate that two activated PKBalpha mutants, whose basal activity is equivalent to that of insulin-induced wild-type PKB, inhibit GSK-3beta to the same extent as a highly active, constitutively membrane-targeted wild-type PKB allele. However, of these two mutants, only the constitutively membrane-targeted allele of PKB induces S6K1 activation. Furthermore, an interfering mutant of PKB, which blocks insulin-induced PKB activation and GSK-3beta inactivation, has no effect on S6K1 activation. Surprisingly, all the activated PKB mutants, regardless of constitutive membrane localization, induce 4E-BP1 phosphorylation and the interfering PKB mutant blocks insulin-induced 4E-BP1 phosphorylation. The results demonstrate that PKB mediates S6K1 activation only as a function of constitutive membrane localization, whereas the activation of PKB appears both necessary and sufficient to induce 4E-BP1 phosphorylation independently of its intracellular location.