Members of a readily enriched beta-proteobacterial clade are common in surface waters of a humic lake

Humic lakes are systems often characterized by irregular high input of dissolved organic carbon (DOC) from the catchment. We hypothesized that specific bacterial groups which rapidly respond to changes in DOC availability might form large populations in such habitats. Seasonal changes of microbial community composition were studied in two compartments of an artificially divided bog lake with contrasting DOC inputs. These changes were compared to community shifts induced during short-term enrichment experiments. Inocula from the two compartments were diluted 1:10 into water from the more DOC-rich compartment, and inorganic nutrients were added to avoid microbial N and P limitation. The dilutions were incubated for a period of 2 weeks. The microbial assemblages were analyzed by cloning and sequencing of 16S rRNA genes and by fluorescence in situ hybridization with specific oligonucleotide probes. beta-Proteobacteria from a cosmopolitan freshwater lineage related to Polynucleobacter necessarius (beta II) were rapidly enriched in all treatments. In contrast, members of the class Actinobacteria did not respond to the enhanced availability of DOC by an immediate increase in growth rate, and their relative abundances declined during the incubations. In lake water members of the beta II clade seasonally constituted up to 50% of all microbes in the water column. Bacteria from this lineage annually formed a significantly higher fraction of the microbial community in the lake compartment with a higher allochthonous influx than in the other compartment. Actinobacteria represented a second numerically important bacterioplankton group, but without clear differences between the compartments. We suggest that the pelagic microbial community of the studied system harbors two major components with fundamentally different growth strategies.     

Structural characterization of LNA and alpha-L-LNA hybridized to RNA

LNA and alpha-L-LNA are promising candidates for the development of efficient oligonucleotide-based therapeutic agents. Here, we present a short overview of the structural results we have obtained for LNA:RNA and alpha-L-LNA:RNA hybrids. Specifically, we have shown that LNA acts as an A-type mimic, while alpha-L-LNA acts as a B-type mimic when built into oligonucleotides.     

Regulation of 5'AMP-activated protein kinase activity and substrate utilization in exercising human skeletal muscle

The metabolic role of 5'AMP-activated protein kinase (AMPK) in regulation of skeletal muscle metabolism in humans is unresolved. We measured isoform-specific AMPK activity and beta-acetyl-CoA carboxylase (ACCbeta) Ser(221) phosphorylation and substrate balance in skeletal muscle of eight athletes at rest, during cycling exercise for 1 h at 70% peak oxygen consumption, and 1 h into recovery. The experiment was performed twice, once in a glycogen-loaded (glycogen concentration approximately 900 mmol/kg dry wt) and once in a glycogen-depleted (glycogen concentration approximately 160 mmol/kg dry wt) state. At rest, plasma long-chain fatty acids (FA) were twofold higher in the glycogen-depleted than in the loaded state, and muscle alpha1 AMPK (160%) and alpha2 AMPK (145%) activities and ACCbeta Ser(221) phosphorylation (137%) were also significantly higher in the glycogen-depleted state. During exercise, alpha2 AMPK activity, ACCbeta Ser(221) phosphorylation, plasma catecholamines, and leg glucose and net FA uptake were significantly higher in the glycogen-depleted than in the glycogen-loaded state without apparent differences in muscle high-energy phosphates. Thus exercise in the glycogen-depleted state elicits an enhanced uptake of circulating fuels that might be associated with elevated muscle AMPK activation. It is concluded that muscle AMPK activity and ACCbeta Ser(221) phosphorylation at rest and during exercise are sensitive to the fuel status of the muscle. During exercise, this dependence may in part be mediated by humoral factors.     

Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37 degrees C in a chelating buffer. The epithelium was then harvested and cultivated under three different conditions: 1) on a collagen coating, 2) embedded in a collagen gel, or 3) embedded in a gel put on a porous well insert. The effect of conditioned medium (CM), insulin, transferrin, selenium, and the oxygen content was assessed. Viability was tested by the metabolic dimethylthiazol-diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21 degrees C for 75 min gave an optimal yield of 3 x 10(6) (2.0-3.8 x 10(6)) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put on a porous membrane was superior to the other methods applied [P < 0.003; median viability 71% (62-100%) compared with preculture values] after 24 h, which was a 160% increase in viability compared with coat-cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects. A simple method is presented, which makes cultivation of colonocytes obtained at endoscopy possible for up to 72 h.     

Dysfunctional MreB inhibits chromosome segregation in Escherichia coli

The mechanism of prokaryotic chromosome segregation is not known. MreB, an actin homolog, is a shape-determining factor in rod-shaped prokaryotic cells. Using immunofluorescence microscopy we found that MreB of Escherichia coli formed helical filaments located beneath the cell surface. Flow cytometric and cytological analyses indicated that MreB-depleted cells segregated their chromosomes in pairs, consistent with chromosome cohesion. Overexpression of wild-type MreB inhibited cell division but did not perturb chromosome segregation. Overexpression of mutant forms of MreB inhibited cell division, caused abnormal MreB filament morphology and induced severe localization defects of the nucleoid and of the oriC and terC chromosomal regions. The chromosomal terminus regions appeared cohered in both MreB-depleted cells and in cells overexpressing mutant forms of MreB. Our observations indicate that MreB filaments participate in directional chromosome movement and segregation.     

An alpha-proteobacterium converts linear alkylbenzenesulfonate surfactants into sulfophenylcarboxylates and linear alkyldiphenyletherdisulfonate surfactants into sulfodiphenylethercarboxylates

The surfactant linear alkylbenzenesulfonate (LAS; 0.5 mM) or linear monoalkyldiphenyletherdisulfonate (LADPEDS; 0.5 mM) in salts medium was easily degraded in laboratory trickling filters, whereas carbon-limited, aerobic enrichment cultures in suspended culture with the same inocula did not grow. We took portions of the trickling filters which degraded LADPEDS, shook the organisms from the solid support (polyester), and found that growth in suspended culture in LADPEDS-salts medium occurred only in the presence of some solid support (polyester fleece or glass wool), though little biomass was immobilized on the support. The end products in suspended culture were identical with those from the trickling filters. There was low plating efficiency of LADPEDS-grown cultures on complex medium, and no picked colony or mixture of colonies grew in LADPEDS-salts-glass wool medium. However, selective plates containing LADPEDS-salts medium solidified with agarose yielded LADPEDS-dependent, pinpoint colonies which could be picked singly and subcultured in selective liquid medium. Isolate DS-1 was a bacterium which showed 93% sequence homology (16S ribosomal DNA) to its nearest phylogenetic neighbor, an alpha-proteobacterium. Strain DS-1 grew heterotrophically in LADPEDS-salts-glass wool medium and converted the set of aryl-substituted alkanes to the corresponding aryl-substituted carboxylic acids of shorter chain length. Similarly, strain DS-1 grew heterotrophically with commercial LAS, converting it to a set of sulfophenylcarboxylates. Growth with a single isomer of LAS [3-(4-sulfophenyl)dodecane] was concomitant with excretion of 4-(4-sulfophenyl)hexanoate, which was identified by matrix-assisted laser desorption ionization mass spectrometry. The growth yield (6.4 g of protein/mol of C) indicated mass balance, which, with the specific growth rate (0.05 h(-1)), indicated a specific utilization rate of LAS of 2.2 mkat/kg of protein.     

Mass spectrometry unravels disulfide bond formation as the mechanism that activates a molecular chaperone

The heat shock protein Hsp33 is a very potent molecular chaperone with a distinctive mode of functional regulation; its activity is redox-regulated. In its reduced form all six cysteinyl residues of Hsp33 are present as thiols, and Hsp33 displays no folding helper activity. Exposure of Hsp33 to oxidizing conditions like H(2)O(2), however, rapidly converts Hsp33 into an efficient molecular chaperone. Activated Hsp33 binds tightly to refolding intermediates of chemically denatured luciferase and suppresses efficiently their aggregation in vitro. Matrix-assisted laser desorption/ionization-mass spectrometry peptide mapping in combination with in vitro and on target protein chemical modification showed that this activation process of Hsp33 is accompanied by the formation of two intramolecular disulfide bonds within Hsp33: Cys(232)-S-S-Cys(234) and Cys(265)-S-S-Cys(268). Cys(141), although not involved in disulfide bond formation, was found highly reactive toward chemical modifications. In contrast, Cys(239) is readily accessible under reducing conditions but becomes poorly accessible though still reduced when Hsp33 is in its active state. This indicates a significant conformational change during the activation process of Hsp33. Mass spectrometry, thus, unraveled a novel molecular mechanism by which alteration of the disulfide bond structure, as a result of changes in the cellular redox potential, results in the activation of a molecular chaperone.     

Immediate localized CDKN1A (p21) radiation response after damage produced by heavy-ion tracks

Using confocal microscopy on immunofluorescence-stained cells, we have investigated the response of CDKN1A (p21), one of the key proteins involved in the DNA damage response pathway, after irradiation with accelerated lead or chromium ions. Each traversal of an accelerated ion leads to the formation of a single, bright focus of the CDKN1A protein in the nuclei of human fibroblasts within 2 min after irradiation at 4 degrees C. This immediate, localized CDKN1A response is specific for particle irradiation with a high linear energy transfer (LET), whereas X irradiation, after a period of induction, yields a diffusely spread pattern, in line with the differences in the microscopic dose deposition pattern of both radiation types. The particle-induced CDKN1A foci persist for several hours until they become diffuse and vanish. These findings suggest that CDKN1A accumulates at the sites of primary DNA damage, possibly mediated by the interaction with proteins involved in DNA repair. Here, for the first time, an immediate biological response confined to the radial extension of low-energy particle tracks ( approximately 1 micrometer) is directly visualized and correlated to ion traversals. This indicates that particle irradiation represents an ideal tool to study the processing of biological damage induced in defined subnuclear regions.     

Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man₈GlcNAc₂ oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man₆GlcNAc₂, Man₇GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides, whereas Man₈GlcNAc₂ and, to a lesser extent, Man₅GlcNAc₂ oligosaccharides supported degradation. These results suggest a role for the Man₈GlcNAc₂ oligosaccharide in the degradation process. They may indicate the presence of a Man₈GlcNAc₂-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.