Purification and characterization of haloalcohol dehalogenase from Arthrobacter sp. strain AD2.

An enzyme capable of dehalogenating vicinal haloalcohols to their corresponding epoxides was purified from the 3-chloro-1,2-propanediol-utilizing bacterium Arthrobacter sp. strain AD2. The inducible haloalcohol dehalogenase converted 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol, 1-chloro-2-propanol, and their brominated analogs, 2-bromoethanol, as well as chloroacetone and 1,3-dichloroacetone. The enzyme possessed no activity for epichlorohydrin (3-chloro-1,2-epoxypropane) or 2,3-dichloro-1-propanol. The dehalogenase had a broad pH optimum at about 8.5 and a temperature optimum of 50 degrees C. The enzyme followed Michaelis-Menten kinetics, and the Km values for 1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol were 8.5 and 48 mM, respectively. Chloroacetic acid was a competitive inhibitor, with a Ki of 0.50 mM. A subunit molecular mass of 29 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With gel filtration, a molecular mass of 69 kDa was found, indicating that the native protein is a dimer. The amino acid composition and N-terminal amino acid sequence are given.     

Purification and characterization of fatty-acid-binding proteins from rat heart and liver

Fatty-acid-binding proteins were purified from delipidated cytosols of rat heart and liver by gel filtration and anion-exchange chromatography at pH 8.0 and by repeated gel filtration, respectively. Homogeneity of both proteins was demonstrated by a single band on polyacrylamide gels; each had a molecular weight of about 14 000. Liver fatty-acid-binding protein is more basic (pI, 8.1) than that of heart (pI, 7.0) and contains more basic amino acids. Examination of fatty acid binding by the binding proteins from heart and liver revealed the presence of a single class of fatty-acid-binding sites in both cases with an apparent dissociation constant for palmitate of about 1 microM. Liver fatty- acid-binding protein shows similar binding characteristics for palmitate, oleate and arachidonate. Palmitate bound to heart fatty- acid-binding protein was a good substrate for oxidation by rat heart mitochondria. The results show that the fatty-acid-binding proteins from rat heart and liver are closely related, but that they are distinct proteins.     

Molecular phylogeny of Kalyptorhynchia (Rhabdocoela,Platyhelminthes) inferred from ribosomal sequence data

With 556 species described to date, Kalyptorhynchia includes about one‐third of all species of rhabdocoel flatworms. In this study, we present the first molecular phylogenetic analysis of this taxon. The final analysis comprises 110 species. These represent 11 of the 17 known families. The largest family (241 known species), Polycystididae, is represented by nine of 10 subfamilies and 33 of the 58 genera. Sequence data from 18S rDNA and 28S rDNA were analysed using maximum likelihood and Bayesian approaches. Of the two taxa traditionally recognised within Kalyptorhynchia, Eukalyptorhynchia and Schizorhynchia, only Schizorhynchia is monophyletic. All eukalyptorhynch families, except Cicerinidae, are monophyletic. On the other hand, two of the three schizorhynch families included are not monophyletic. Within Polycystididae, the traditional taxonomy was not reflected in our phylogenetic analyses and most subfamilies are polyphyletic. These results suggest that current classification, mostly based on characters of the genital system, suffers from homoplasy. Where possible, a revised classification, taking into account these new findings, is given.     

Propionate formation by Opitutus terrae in pure culture and in mixed culture with a hydrogenotrophic methanogen and implications for carbon fluxes in anoxic rice paddy soil

Propionate-forming bacteria seem to be abundant in anoxic rice paddy soil, but biogeochemical investigations show that propionate is not a correspondingly important intermediate in carbon flux in this system. Mixed cultures of Opitutus terrae strain PB90-1, a representative propionate-producing bacterium from rice paddy soil, and the hydrogenotrophic Methanospirillum hungatei strain SK maintained hydrogen partial pressures similar to those in the soil. The associated shift away from propionate formation observed in these cultures helps to reconcile the disparity between microbiological and biogeochemical studies.     

Glutamine synthetase of Klebsiella aerogenes: genetic and physiological properties of mutants in the adenylylation system.

Mutations resulting in defects in the adenylylation system of glutamine synthetase (GS) affect the expression of glnA, the structural gene for GS. Mutants with lesions in glnB are glutamine auxotrophs and contain repressed levels of highly adenylylated GS. Glutamine-independent revertants of the glnB3 mutant have acquired an additional mutation at the glnE site. The glnE54 mutant is incapable of adenylylating GS and produces high levels of enzyme, even when ammonia is present in the growth medium. The fact that mutations in glnB and glnE simultaneously disturb both the normal adenylylation and repression patterns of GS in Klebsiella aerogenes indicates that the adenylylation system, or adenylylation state, of GS is critical for the regulation of synthesis of GS.     

Karyotype instability with multiple 7/14 and 7/7 rearrangements

Summary Chromosomes were studied in a mentally retarded boy with microcephaly, growth retardation, facial erythema, café-au-lait spots, and IgA deficiency. In the lymphocytes there was a remarkable tendency to exchange parts of the chromosomes Nos. 7 and 14, the translocations almost exclusively taking place in bands 7p13, 7q32 and 14q11. Seven different types of rearrangements between Nos. 7 and 14, and some other chromosomal aberrations were found. No abnormalities could be detected in the bone marrow. The patient somewhat resembles those affected with ataxia-telangiectasia or with Bloom's syndrome, but on clinical and cytogenetic grounds these disorders could be excluded.7/14 Translocations similar to those found in our patient's lymphocytes have been reported to occur very rarely in the lymphocyte cultures of individuals with apparently normal chromosome constitution. A relationship between these phenomena may exist.     

17 years of grassland management leads to parallel local and regional biodiversity shifts among a wide range of taxonomic groups

Conservation management is expected to increase local biodiversity, but uniform management may lead to biotic homogenization and diversity losses at the regional scale. We evaluated the effects of renewed grazing and cutting management carried out across a whole region, on the diversity of plants and seven arthropod groups. Changes in occurrence over 17 years of intensive calcareous grassland management were analysed at the species level, which gave insight into the exact species contributing to regional homogenization or differentiation. Reponses were compared between species differing in habitat affinity, dispersal ability, food specialisation and trophic level. Local species richness increased over the sampling period for true bugs and millipedes, while carabid beetles and weevils declined in local species richness. Species richness remained unchanged for plants, woodlice, ants and spiders. Regional diversity and compositional variation generally followed local patterns. Diversity shifts were only to a limited extent explained by species’ habitat affinity, dispersal ability, trophic level and food specialisation. We conclude that implementation of relatively uniform conservation management across a region did not lead to uniform changes in local species composition. This is an encouraging result for conservation managers, as it shows that there is not necessarily a conflict of interest between local and regional conservation goals. Our study also demonstrates that shifts in diversity patterns differ markedly between taxonomic groups. Single traits provide only limited understanding of these differences. This highlights the need for a wide taxonomic scope when evaluating conservation management and demonstrates the need to understand the mechanisms underlying occurrence shifts.     

Identification of Free Nitric Oxide Radicals in Rat Bone Marrow: Implications for Progenitor Cell Mobilization in Hypertension

Nitric oxide (NO) has been implicated in matrix metallopeptidase 9 (MMP9)-dependent mobilization of hematopoietic stem and progenitor cells from bone marrow (BM). However, direct measurement of NO in the BM remained elusive due to its low in situ concentration and short lifetime. Using NO spin trapping and electron paramagnetic resonance (EPR) spectroscopy we give the first experimental confirmation of free NO radicals in rodent BM. NO production was quantified and attributed to enzymatic activity of NO synthases (NOS). Although endothelial NOS (eNOS) accounts for most (66%) of basal NO, we identified a significant contribution (23%) from inducible NOS (iNOS). Basal NO levels closely correlate with MMP9 bioavailability in BM of both hypertensive and control rats. Our observations support the hypothesis that inadequate mobilization of BM-derived stem and progenitor cells in hypertension results from impaired NOS/NO/MMP9 signalling in BM, a condition that may be corrected with pharmacological intervention.     

Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes.

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.     

Acclimation responses to temperature vary with vertical stratification: implications for vulnerability of soil‐dwelling species to extreme temperature events

The occurrence of summer heat waves is predicted to increase in amplitude and frequency in the near future, but the consequences of such extreme events are largely unknown, especially for belowground organisms. Soil organisms usually exhibit strong vertical stratification, resulting in more frequent exposure to extreme temperatures for surface‐dwelling species than for soil‐dwelling species. Therefore soil‐dwelling species are expected to have poor acclimation responses to cope with temperature changes. We used five species of surface‐dwelling and four species of soil‐dwelling Collembola that habituate different depths in the soil. We tested for differences in tolerance to extreme temperatures after acclimation to warm and cold conditions. We also tested for differences in acclimation of the underlying physiology by looking at changes in membrane lipid composition. Chill coma recovery time, heat knockdown time and fatty acid profiles were determined after 1 week of acclimation to either 5 or 20 °C. Our results showed that surface‐dwelling Collembola better maintained increased heat tolerance across acclimation temperatures, but no such response was found for cold tolerance. Concordantly, four of the five surface‐dwelling Collembola showed up to fourfold changes in relative abundance of fatty acids after 1 week of acclimation, whereas none of the soil‐dwelling species showed a significant adjustment in fatty acid composition. Strong physiological responses to temperature fluctuations may have become redundant in soil‐dwelling species due to the relative thermal stability of their subterranean habitat. Based on the results of the four species studied, we expect that unless soil‐dwelling species can temporarily retreat to avoid extreme temperatures, the predicted increase in heat waves under climatic change renders these soil‐dwelling species more vulnerable to extinction than species with better physiological capabilities. Being able to act under a larger thermal range is probably costly and could reduce maximum performance at the optimal temperature.