Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Inactivation of <Emphasis Type="Italic">pycA</Emphasis>, encoding pyruvate carboxylase activity,increases poly-β-hydroxybutyrate accumulation in <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> on solid medium

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly--hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene (pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly--hydroxybutyrate.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Tailoring of bioresorbable polymers for elaboration of sugar-functionalized nanoparticles

Maleic copolymers with different contents of galactose moieties and dodecyl chains were synthesized and used as both a stabilizer and a surface coating for the preparation of poly(epsilon-caprolactone) nanoparticles by the emulsification-diffusion technique. The size of the nanoparticles was controlled by varying the initial concentration of the modified maleic copolymers. As the concentration of the latter increased, the particle size decreased, indicating that the copolymers serve as a stabilizer. Moreover, surface modification of nanoparticles was confirmed by xi-potential measurements. Nanoparticles were also shown to be recognized by a galactose-specific lectin, demonstrating the presence of galactose units on the particle surface. This approach offers opportunities for the production of novel targeted drug delivery systems.     

Preparation and physicochemical characterization of 5 niclosamide solvates and 1 hemisolvate

The purpose of the study was to characterize the physicochemical, structural, and spectral properties of the 1∶1 niclosamide and methanol, diethyl ether, dimethyl sulfoxide, N,N' dimethylformamide, and tetrahydrofuran solvates and the 2∶1 niclosamide and tetraethylene glycol hemisolvate prepared by recrystallization from these organic solvents. Structural, spectral, and thermal analysis results confirmed the presence of the solvents and differences in the structural properties of these solvates. In addition, differences in the activation energy of desolvation, batch solution calorimetry, and the aqueous solubility at 25°C, 24 hours, showed the stability of the solvates to be in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > methanol solvate > dimethyl sulfoxide solvate > N,N' dimethylformamide solvate. The intrinsic and powder dissolution rates of the solvates were in the order: anhydrate > diethyl ether solvate > tetraethylene glycol hemisolvate > N,N' dimethylformamide solvate > methanol solvate > dimethyl sulfoxide solvate. Although these nonaqueous solvates had higher solubility and dissolution rates than the monohydrous forms, they were unstable in aqueous media and rapidly transformed to one of the monohydrous forms.     

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors

Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buried waters was included in the model and its behavior under dynamic conditions was molecular dynamics simulated, revealing a most interesting similarity pattern to kallikrein. The benzamidine-based thrombin inhibitors alpha-NAPAP, 3-TAPAP, and 4-TAPAP were docked into the refined model, allowing for a more insightful functional characterization of the enzyme and a better understanding of the reported comparatively low affinity of KN-BJ2 toward those inhibitors.     

Molar absorption coefficients for the reduced Ellman reagent: reassessment

The Ellman method for assaying thiols is based on the reaction of thiols with the chromogenic DTNB (5,5'-dithiobis-2-nitrobenzoate) whereby formation of the yellow dianion of 5-thio-2-nitrobenzoic acid (TNB) is measured. The TNB molar absorption coefficient, 13.6 x 10(3)M(-1)cm(-1), as published by Ellman in 1959 has been almost universally used until now. Over the years, however, slightly different values have been published, and it has further been shown that TNB reveals thermochromic properties. This should be taken into account when the Ellman method is used for determination of enzyme activities, such as in cholinesterase assays. Our data show that the absorbance spectra of TNB are shifted to longer wavelengths when temperature increases, while absorbance maxima decrease. Our recommended molar absorption coefficients at 412 nm are 14.15 x 10(3)M(-1)cm(-1) at 25 degrees C and 13.8 x 10(3)M(-1)cm(-1) at 37 degrees C (0.1M phosphate buffer, pH 7.4). Molar absorption coefficients for other temperatures and wavelengths are included in the paper.     

Do capuchin monkeys, Cebus apella, know what conspecifics do and do not see?

Capuchin monkeys were tested in five experiments in which two individuals competed over food. When given a choice between retrieving a piece of food that was visible or hidden from the dominant, subordinate animals preferred to retrieve hidden food. This preference is consistent with the hypotheses that either (1) the subordinate knew what the dominant could and could not see or (2) the subordinate was monitoring the behaviour of the dominant and avoiding the piece of food that it approached. To test between these alternatives, we released subordinates with a slight head start forcing them to make their choice (between a piece of food hidden or visible to the dominant) before the dominant entered the area. Unlike chimpanzees, Pan troglodytes, subordinates that were given a head start did not preferentially approach hidden pieces of food first. Therefore, our experiments provide little support for the hypothesis that capuchin monkeys are sensitive to what another individual does or does not see. We compare our results with those obtained with chimpanzees in the same paradigm and discuss the evolution of primate social cognition. Copyright 2003 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved.      

Origin and diffusion of mtDNA haplogroup X

A maximum parsimony tree of 21 complete mitochondrial DNA (mtDNA) sequences belonging to haplogroup X and the survey of the haplogroup-associated polymorphisms in 13,589 mtDNAs from Eurasia and Africa revealed that haplogroup X is subdivided into two major branches, here defined as “X1” and “X2.” The first is restricted to the populations of North and East Africa and the Near East, whereas X2 encompasses all X mtDNAs from Europe, western and Central Asia, Siberia, and the great majority of the Near East, as well as some North African samples. Subhaplogroup X1 diversity indicates an early coalescence time, whereas X2 has apparently undergone a more recent population expansion in Eurasia, most likely around or after the last glacial maximum. It is notable that X2 includes the two complete Native American X sequences that constitute the distinctive X2a clade, a clade that lacks close relatives in the entire Old World, including Siberia. The position of X2a in the phylogenetic tree suggests an early split from the other X2 clades, likely at the very beginning of their expansion and spread from the Near East.