Kinetic study of the H103A mutant yeast transketolase

Data from site-directed mutagenesis and X-ray crystallography show that His103 of holotransketolase (holoTK) does not come into contact with thiamin diphosphate (ThDP) but stabilizes the transketolase (TK) reaction intermediate, alpha,beta-dihydroxyethyl-thiamin diphosphate, by forming a hydrogen bond with the oxygen of its beta-hydroxyethyl group [Eur. J. Biochem. 233 (1995) 750; Proc. Natl. Acad. Sci. USA 99 (2002) 591]. We studied the influence of His103 mutation on ThDP-binding and enzymatic activity. It was found that mutation does not affect the affinity of the coenzyme to apotransketolase (apoTK) in the presence of Ca(2+) (a cation found in the native holoenzyme) but changes all the kinetic parameters of the ThDP-apoTK interaction in the presence of Mg(2+) (a cation commonly used in ThDP-dependent enzymes studies). It was concluded that the structures of TK active centers formed in the presence of Mg(2+) and Ca(2+) are not identical. Mutation of His103 led to a significant acceleration of the one-substrate reaction but a slow down of the two-substrate reaction so that the rates of both types of catalysis became equal. Our results provide evidence for the intermediate-stabilizing function of His103.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

An Examination of the Association between 5-HTTLPR,Combat Exposure,and PTSD Diagnosis among U.S. Veterans

Objective

To examine the association between the 5-HTTLPR polymorphism of the serotonin transporter (SLC6A4) gene, combat exposure, and posttraumatic stress disorder (PTSD) diagnosis and among two samples of combat-exposed veterans.

Method

The first sample included 550 non-Hispanic Black (NHB) combat-exposed veterans. The second sample included 555 non-Hispanic White (NHW) combat-exposed veterans. Participants were genotyped for the 5-HTTLPR/rs25531 variants of the SLC6A4 gene. A structured clinical interview was used to diagnose PTSD. Combat and civilian trauma exposure were assessed with validated self-report instruments. Logistic regression was used to test for main effects of 5-HTTLPR on PTSD diagnosis as well as gene x environment (GxE) interactions after adjusting for sex, ancestry proportion scores, civilian trauma exposure, and combat exposure.

Results

Within the NHB sample, a significant additive effect was observed for 5-HTTLPR (OR = 1.502, p = .0025), such that the odds of having a current diagnosis of PTSD increased by 1.502 for each additional S’ allele. No evidence for an association between 5-HTTLPR and PTSD was observed in the NHW sample. In addition, no evidence for combat x 5-HTTLPR effects were observed in either sample.

Conclusion

The present study suggests that there may be an association between 5-HTTLPR genotype and PTSD diagnosis among NHB veterans; however, no evidence for the hypothesized 5-HTTLPR x combat interaction was found.     

Synthesis, structure, and biological properties of some 8alpha-analogues of steroid estrogens with fluorine in position 2

A total synthesis of 8alpha analogues of steroid estrogens with fluorine in position 2 was achieved. Structural features of these compounds were studied by the example of 17beta-acetoxy-2-fluoro-3-methoxy-8alpha-estra-1,3,5(10)-triene. It was shown that the 8alpha analogues of 2-fluorosubstituted steroid estrogens have a low uterotropic activity and retain the osteoprotective and hypocholesterolemic activities.     

Reorganization of chloroplast ultrastructure associated with low-temperature hardening of Arabidopsis plants

When following low-temperature acclimation (5 days at 2°C) of cold-resistant plants of Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) in relation to the changes in chloroplast ultrastructure, we registered the high efficiency of hardening and the ability of hardened plants to lower a threshold of frost damage by about 3°C. During hardening, the area of grana in the chloroplasts more than doubled, with considerably increased numbers of thylakoids per granum and thylakoids per chloroplast. The rate of apparent photosynthesis decreased to lesser extent than the rate of dark respiration, as a result the content of soluble sugars increased fourfold, ensuring an adaptive reorganization of metabolism, which enabled the hardened plants to survive even at below-zero temperatures (up to ?7°C). The authors conclude that a considerable increase in the number of thylakoids in the chloroplasts helps maintain photosynthesis at low above-zero temperatures and is a prerequisite for the accumulation of soluble sugars in Arabidopsis leaves.     

Isolation and purification of Mn-peroxidase from Azospirillum brasilense SP245

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2′-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and 1 mM as inductors increased the Mn-peroxidase activity by a factor of 3.     

Sequential colonization by river periphyton analysed by microscopy and molecular fingerprinting

1. An artificial glass substratum was incubated in the River Danube for a period of 28 days in order to detect the sequential colonization of microorganisms.
2. Light and fluorescent microscopy showed that microalgae and the picoalgal fraction on the slides increased rapidly over the first 2 weeks of colonization. Diatoms were numerically the most abundant component of the periphyton and their species richness and diversity increased rapidly in the early phase of colonization whereas diversity subsequently increased moderately.
3. Evenness of the diatom community was initially high, lower in the intermediate phase and again higher later on. Succession involving early, intermediate and late colonizer species was observed. Community composition during the first 5 days of colonization was very different from later stages whereas there were only minor changes subsequently.
4. Molecular community analysis by means of terminal restriction fragment length polymorphism analysis of PCR amplified 16S rRNA and 18S rRNA genes pointed to even larger differences between the composition of samples obtained early and late in the period.
5. The number of 18S rRNA and 16S rRNA terminal restriction fragments (T-RF-s) was variable over the colonization period and the fragment patterns of both the bacterial and eukaryotic portion of the microbial community were variable, with most T-RF-s unique to a single sample, suggesting a wide diversity and dynamic properties of periphytic organisms.     

Structure of plant communities in the early succession stages on anthropogenic sandy outcrops of the forest tundra and northern taiga of Western Siberia

The results of a study of the taxonomic, ecological, and phytocenotic structure of plant communities formed in the early stages of overgrowth of the anthropogenic sandy outcrops within the forest-tundra zone and northern taiga forest subzone of Western Siberia are presented. In the early stages of vegetativecover restoration, there are from 2 to 11 species growing into sparse communities of the classes Loiseleurio-Vaccinietea, Artemisietea vulgaris, and Koelerio-Corynephoretea. The participation of some meso-xerophytic forest-tundra, meadow-margin, and weed herbaceous plant and dwarf shrubs species, mostly with ruderal and stress-tolerant eco-phytocenotic strategies, as well as with secondary strategies—violent-ruderal and ruderal–stress-tolerant—in the secondary succession on the sandy outcrops have been shown. The primary succession begins at the bottom and slopes of the pit ditches, as well on the surface of the sand dunes, and proceeds at an accelerated scenario. Hygro-, meso- and xerophilous species, predominantly oligo-mesotrophic rhizome and densely firm-bunch grasses and shrubs of the native flora, as well as ground lichens, participate in the early stages of succession.     

Mathematical model of oscillations in mammalian erythrocytes. The role of calmodulin-dependent Ca-activated Ca-pump

A kinetic model of ionophore-induced oscillation of ion fluxes in mammalian erythrocytes is proposed. The model is based on the regulation by Ca2(+)-ions and calmodulin of (Ca2(+) + Mg2+)-dependent ATPase of erythrocytes. The results obtained are in a good agreement with the experimental data available.