Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.     

Differentiation of interphase domains of the homologous human X-chromosomes

In accordance with molecular biology data reported elsewhere, homologous interphase X-chromosome territories differ greatly in the abundance of inactive condensed chromatin. On the other hand, a three dimensional FISH (3D FISH) method has revealed that domains of both inactive and active X-chromosome have similar volumes and similar maximum section areas (Smax). To solve this contradiction, we examined differences between homologous human interphase X-chromosome territories using two dimensional FISH (2D FISH) preparations of clustered PHA-stimulated lymphocytes. For obtaining such preparations, we developed a new technique to avoid a stage of hypotonic treatment of living cells, since this treatment levels the chromatin compactness degree. According to our 2D FISH data, the mean ratios of Smax for larger and smaller homologous X-chromosomes, calculated for individual flattened nuclei, were 1.83 +/- 0.08 and 2.02 +/- 0.09, respectively, for clumped cells and groups of loosely associated and separated lymphocytes. In comparison, the same ratio calculated for individual 3D nuclei of PHA-stimulated lymphocytes was 1.38 +/- 0.05 (Falk et al., 2002). Our findings give evidence for enrichment of inactive X-chromosomes by low stretchable condensed chromatin. In addition, these findings show that an active X is enriched by a high stretchable form of chromatin, whose content may distinctly vary from cell to cell.     

Characterisation of a thermostable alpha-amylase from Bacillus brevis.

Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The alpha-amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.     

Pancreatic lipase inhibitory activity of monogalactosyldiacylglycerols isolated from the freshwater microalga <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Chemical investigation of the freshwater microalga Chlorella sorokiniana led to the isolation of a monogalactosyldiacylglycerol (MGDG)-rich fraction possessing dose-dependent inhibitory activity against pancreatic lipase activity. The MGDG-rich fraction contains 12 MGDGs identified by LC/HRMS analysis. Among them, three MGDGs were new compounds, namely, (2S)-1-O-(7Z,10Z-hexadecadienoyl)-2-O-(7Z,10Z,13Z-hexadecatrienoyl)-3-O-β-D-galactopyranosylglycerol (1), (2S)-1-O-linoleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (6), and (2S)-1-O-oleoyl-2-O-(7Z,10Z-hexadecadienoyl)-3-O-β-D-galactopyranosylglycerol (8). The major galactolipids were isolated by semipreparative HPLC and tested for their effect toward pancreatic lipase inhibitory activity. All the tested MGDGs showed significant reduction of pancreatic lipase activity indicating possible beneficial use for management of lipase-related disorders such as obesity.     

Evolution of Alzheimer’s disease pathogenesis conception

Alzheimer’s disease (AD) is a neurodegenerative disorder that becomes a cause of dementia during atrophic brain changes. There are two distinguished forms of AD: familial early-onset form (FAD, approximately 5% of all cases, develops before age 65, most commonly 40–50) and sporadic late-onset form (SAD, approximately 95% of all cases, develops after 65). Identification of genetic determinants of FAD development and evidence of amyloid-beta peptide’s (Aβ) neurotoxicity as a central event in the cascade of pathological processes significantly expanded the conception of molecular and genetic mechanisms of the disease. However, the question of whether or not the accumulation of Aβ is the triggering factor of more widespread SAD remains open. There are a growing number of arguments for Aβ overproduction being the secondary, concomitant event of AD pathological processes: synaptic failure, hyperphosphorylation of tau protein, neuroinflammation, neuronal loss, and cognitive decline. As one of triggering risk factors of AD development, mitochondrial dysfunction is considered, with the decrease in ATP synthesis and oxidative stress becoming the consequences. However, the specific molecular and genetic mechanisms of AD remain unclear. This is caused by the lack of relevant animal models for studying mechanisms of the disease and objective estimation of pathogenically justified methods of AD prevention and treatment.     

Physiological significance of isoprenoids and phenylpropanoids in drought response of Arundinoideae species with contrasting habitats and metabolism

Physiological, biochemical and morpho‐anatomical traits that determine the phenotypic plasticity of plants under drought were tested in two Arundinoideae with contrasting habitats, growth traits and metabolism: the fast‐growing Arundo donax, which also is a strong isoprene emitter, and the slow‐growing Hakonechloa macra that does not invest on isoprene biosynthesis. In control conditions, A. donax displayed not only higher photosynthesis but also higher concentration of carotenoids and lower phenylpropanoid content than H. macra. In drought‐stressed plants, photosynthesis was similarly inhibited in both species, but substantially recovered only in A. donax after rewatering. Decline of photochemical and biochemical parameters, increased concentration of CO₂ inside leaves, and impairment of chloroplast ultrastructure were only observed in H. macra indicating damage of photosynthetic machinery under drought. It is suggested that volatile and non‐volatile isoprenoids produced by A. donax efficiently preserve the chloroplasts from transient drought damage, while H. macra invests on phenylpropanoids that are less efficient in preserving photosynthesis but likely offer better antioxidant protection under prolonged stress.     

Thyroid hormonal axis regulates protein C anticoagulation pathway in rats

Effects of the hormones of the hypothalamic-pituitary-thyroid axis on some basic parameters of the activity of protein C anticoagulation pathway in rats are studied. Thyrotropin-releasing hormone (0.06 mg/kg body mass), thyrotropin (1 IU/kg), triiodothyronine (T3) (0.08 mg/kg), thyroxine (T4) (0.08 mg/kg), administered subcutaneously for three consecutive days on four different groups of rats increased significantly activated protein C, free protein S and protein S activity, and reduced the soluble endothelial protein C receptor. Protein C antigen and total protein S were significantly elevated only by thyrotropin-releasing hormone and thyroid-stimulating hormone, but they were not affected by T3 and T4 treatment. The data indicate the hypothalamic-pituitary-thyroid axis is involved in the regulation of the protein C anticoagulation pathway in rats by activation of this system, suggesting a tendency of hypocoagulability.     

Behavior of pericentromere heterochromatin regions under different conditions of chromosome decondensation in isolated nuclei]

In isolated mouse nuclei the chromocenters were shown to be the pericentromeric heterochromatin regions (PCHR). After the decreasing of bivalent ion concentration (0.1 mM Ca2+, 2 mM Mg2+) the main and peripheral parts of the chromatin remained on the contrary as the compact chromatin bodies. The additional ultrasound treatment of isolated nuclei in the presence of 0.1 mM Ca2+ with DNAase II and triton X-100 resulted in the species enriched by the condensed PCHR.     

Cell Fate Analysis of Embryonic Ventral Mesencephalic Grafts in the 6-OHDA Model of Parkinson's Disease

Evidence from carefully conducted open label clinical trials suggested that therapeutic benefit can be achieved by grafting fetal dopaminergic (DAergic) neurons derived from ventral mesencephalon (VM) into the denervated striatum of Parkinson''s disease (PD) patients. However, two double-blind trials generated negative results reporting deleterious side effects such as prominent dyskinesias. Heterogeneous composition of VM grafts is likely to account for suboptimal clinical efficacy.We consider that gene expression patterns of the VM tissue needs to be better understood by comparing the genetic signature of the surviving and functioning grafts with the cell suspensions used for transplantation. In addition, it is crucial to assess whether the grafted cells exhibit the DAergic phenotype of adult substantia nigra pars compacta (SNpc). To investigate this further, we used a GFP reporter mouse as source of VM tissue that enabled the detection and dissection of the grafts 6 weeks post implantation. A comparative gene expression analysis of the VM cell suspension and grafts revealed that VM grafts continue to differentiate post-implantation. In addition, implanted grafts showed a mature SNpc-like molecular DAergic phenotype with similar expression levels of TH, Vmat2 and Dat. However, by comparing gene expression of the adult SNpc with dissected grafts we detected a higher expression of progenitor markers in the grafts. Finally, when compared to the VM cell suspension, post-grafting there was a higher expression of markers inherent to glia and other neuronal populations.In summary, our data highlight the dynamic development of distinctive DAergic and non-DAergic gene expression markers associated with the maturation of VM grafts in vivo. The molecular signature of VM grafts and its functional relevance should be further explored in future studies aimed at the optimization of DAergic cell therapy approaches in PD.