[Metabolic engineering of yeast Hansenula polymorpha for construction of efficient ethanol producers].

Until recently, the methylotrophic yeast was not considered as a potential producer of biofuels, particularly of ethanol from lignocellulosic hydrolysates. The first work published 10 years ago reveals the ability of thermotolerant methylotrophic yeast Hansenula polymorpha to ferment xylose--one of the main sugars of lignocellulosic hydrolysates, which has made these yeast promising organism for high temperature alcoholic fermentation. Such feature of the H. polymorpha can be used in the implementation of potentially effective process of simultaneous saccharification and fermentation (SSF) of raw materials. SSF allows combining enzymatic hydrolysis of raw materials with the conversion of produced sugars into ethanol: enzymes hydrolyze polysaccharides to monomers, which are immediately consumed by microorganisms-producers of ethanol. However, the efficiency of alcoholic fermentation of major sugars realized after hydrolysis of lignocellulosic raw materials, and especially xylose, by wild strains of H. polymorpha requires significant improvement. In this review the main results of metabolic engineering of H. polymorpha for the construction of improved producers of ethanol from xylose, starch, xylan, and glycerol, as well as strains with increased tolerance to high temperature and ethanol are represented.

Blockade of nitric oxide formation in the rat brain does not disturb development of endotoxin tolerance.

The involvement of nitric oxide (NO) in tolerance development to endotoxin has been proposed because peripherally administered NG-nitro-L-arginine methyl ester (L-NAME) (NO synthases inhibitor) delays the endotoxin tolerance formation. Since L-NAME is capable of crossing the blood-brain barrier, the question arises of where activity of NO synthases (inside or outside the blood-brain barrier) is crucial for development of endotoxin tolerance. To clarify the role of different NO synthases (NOS) isoforms, acting in the brain, on the tolerance development, effects of highly selective iNOS and nNOS inhibitors on stepwise attenuation of febrile response during tolerance formation were examined in freely moving biotelemetered rats. We monitored changes in febrile response during the development of tolerance to repeated intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) (50 µg/kg) along with intracerebroventricular (i.c.v.) injections of vinyl-L-NIO, a neuronal NOS inhibitor, or aminoguanidine, an inducible NOS inhibitor at a dose of 10 µg/rat. Both inhibitors injected at the selected doses had no effect on normal day-time as well as night-time body temperature. Rats were treated with LPS and NOS inhibitors for three consecutive days. On the fourth day, all rats were injected with LPS alone. Rats repeatedly injected with LPS became tolerant to pyrogenic effect of LPS as early as on the second day of the experiment. The treatment with iNOS or nNOS inhibitors completely suppressed fever due to the first, second and third LPS injection. When rats, which received the three i.c.v. injections of vL-NIO along with i.p. injections of LPS, were then treated the fourth time with LPS alone, they responded with virtually identical changes in body temperature to that of the group of rats that were injected with water i.c.v. and LPS i.p. for three consecutive days. This data indicate that both group of rats became tolerant to pyrogenic effect of LPS. It is, therefore, reasonable to hypothesize that activation of nNOS and iNOS inside the brain is not important for the development of endotoxin tolerance.

Analysis of postural sway in patients with normal pressure hydrocephalus: effects of shunt implantation.

Poor postural balance is one of the major risk factors for falling in normal pressure hydrocephalus (NPH). Postural instability in the clinic is commonly assessed based upon force platform posturography. In this study we focused on the identification of changes in sway characteristics while standing quiet in patients with NPH before and after shunt implantation. Postural sway area and sway radius were analyzed in a group of 9 patients and 46 controls of both genders. Subject's spontaneous sway was recorded while standing quiet on a force platform for 30-60 s, with eyes open and then closed. Both analyzed sway descriptors identified between-group differences and also an effect of shunt implantation in the NPH group. Sway radius and sway area in patients exhibited very high values compared with those in the control group. Importantly, the effect of eyesight in patients was not observed before shunt implantation and reappeared after the surgical treatment. The study documents that static force platform posturography may be a reliable measure of postural control improvement due to shunt surgery.

[Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)].

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.

[Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi].

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.

Improvement in the face/name association performance after three months of physical training in elderly women.

Several lines of evidence suggest that physical exercise not only influences the development of muscles, cardiovascular and respiratory systems, but also exerts a significant influence on the central nervous system. We examined the influence of strength and endurance training on cognitive performance in 33 healthy elderly volunteers (women, mean age 63.5 +/-4.5 yr) over a 3-month period of supervised training program. A control group consisted of 8 age-matched (mean age 66.3 +/-4.6) healthy volunteers who did not participate in any exercise training program. To evaluate the cognitive performance in our subjects we used two tests: face/name association test and Stroop test. The tests were applied shortly before and immediately after the training program. In the experimental group, a significant improvement in the association test performance, on average, from 71.6 +/-7.3% to 79.7 +/-7.2% (P<0.0001) was observed over the 3-month training period. There were no changes in the Stroop test results over the same time. Likewise, there were no changes in the control groups. Our data demonstrate that the training regime that is strictly followed over a relatively short period of time may improve the performance in associative memory tasks in elderly subjects. The study supports the notion that physical exercise influences cognitive performance and extend this notion to be valid for healthy elderly subjects.