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PubMed1986

Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels.

Changes in cell shape are postulated to modulate gene expression during differentiation of a number of cell types, including rabbit synovial fibroblasts, which are inducible for expression of the zymogen form of the metalloendopeptidase, collagenase. In the work presented here, fibroblasts cultured on and within hydrated collagen gels were allowed to contract by release of the gels from the sides of the culture dish. Within 24 h of cell release, synthesis and secretion of procollagenase was initiated in the absence of any chemical manipulation. Fibroblasts grown in and on collagen also responded to 12-O-tetradecanoylphorbol-13-acetate and cytochalasin B with morphologic change and induced procollagenase. However, colchicine, which altered morphology to varying degrees in cells on plastic, on collagen, and within collagen gels, did not induce procollagenase expression. In all cases, the enzyme was induced only after reorganization of polymerized actin, rather than after a change in cellular morphology per se. As a first approach to identifying other aspects of the stimulated phenotype that could affect collagen turnover, the expression of collagen and endogenous metalloproteinase inhibitors in relation to procollagenase secretion was investigated. Collagen secretion by fibroblasts decreased when procollagenase secretion was induced by the pharmacologic agents, but not when cells were stimulated by contraction on or within collagen gels. The expression of two endogenous inhibitors was not coordinately regulated with induction of procollagenase. Therefore, the extracellular matrix and the cellular actin cytoskeleton may transduce signals that modulate the tissue remodeling phenotype of fibroblasts.

Actins↗
PubMed1990

Cleavage specificity of human skin type IV collagenase (gelatinase). Identification of cleavage sites in type I gelatin, with confirmation using synthetic peptides.

Type IV collagenase (gelatinase) readily cleaves denatured collagen into very small peptides. Large cyanogen bromide fragments (25 kDa) of type I collagen are degraded at the same rate as the complete alpha-chain. A number of the gelatinolytic cleavage sites of alpha 1(I)CB7 and alpha 1(I)CB8, representing 50% of the collagen alpha-chain, were determined by sequence analysis of product peptides. In addition to the expected cleavage between glycine and hydrophobic residues, several other cleavage sites were identified. These sites were Gly-Glu, Gly-Asn, and Gly-Ser. Basic residues were found adjacent to the cleavage site in several cases. Hexapeptides containing these unexpected cleavage sites were synthesized, and Km and kcat values were determined. All but one of the Km values were in the submillimolar range, and turnover numbers for the peptides uncharged at the carboxyl terminus were on the order of 10,000/h. Of particular significance was the finding that hydroxyproline occurs 5 residues from the cleavage site in all carboxyl-terminal product peptides and also occurs 5 residues from the cleavage site in seven of nine amino-terminal product peptides. A requirement for hydroxyproline may be of importance in determining the specificity of this enzyme for denatured collagenous substrates.

Amino Acid Sequence↗
PubMed2004

Temperature dependence of metabolic rates for microbial growth, maintenance, and survival.

Our work was motivated by discoveries of prokaryotic communities that survive with little nutrient in ice and permafrost, with implications for past or present microbial life in Martian permafrost and Europan ice. We compared the temperature dependence of metabolic rates of microbial communities in permafrost, ice, snow, clouds, oceans, lakes, marine and freshwater sediments, and subsurface aquifer sediments. Metabolic rates per cell fall into three groupings: (i) a rate, microg(T), for growth, measured in the laboratory at in situ temperatures with minimal disturbance of the medium; (ii) a rate, microm(T), sufficient for maintenance of functions but for a nutrient level too low for growth; and (iii) a rate, micros(T), for survival of communities imprisoned in deep glacial ice, subsurface sediment, or ocean sediment, in which they can repair macromolecular damage but are probably largely dormant. The three groups have metabolic rates consistent with a single activation energy of approximately 110 kJ and that scale as microg(T):microm(T):micros(T) approximately 10(6):10(3):1. There is no evidence of a minimum temperature for metabolism. The rate at -40 degrees C in ice corresponds to approximately 10 turnovers of cellular carbon per billion years. Microbes in ice and permafrost have metabolic rates similar to those in water, soil, and sediment at the same temperature. This finding supports the view that, far below the freezing point, liquid water inside ice and permafrost is available for metabolism. The rate micros(T) for repairing molecular damage by means of DNA-repair enzymes and protein-repair enzymes such as methyltransferase is found to be comparable to the rate of spontaneous molecular damage.

Bacteria↗
PubMed2004

Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing.

Movile Cave is an unusual groundwater ecosystem that is supported by in situ chemoautotrophic production. The cave atmosphere contains 1-2% methane (CH4), although much higher concentrations are found in gas bubbles that keep microbial mats afloat on the water surface. As previous analyses of stable carbon isotope ratios have suggested that methane oxidation occurs in this environment, we hypothesized that aerobic methane-oxidizing bacteria (methanotrophs) are active in Movile Cave. To identify the active methanotrophs in the water and mat material from Movile Cave, a microcosm was incubated with a 10%13CH4 headspace in a DNA-based stable isotope probing (DNA-SIP) experiment. Using improved centrifugation conditions, a 13C-labelled DNA fraction was collected and used as a template for polymerase chain reaction amplification. Analysis of genes encoding the small-subunit rRNA and key enzymes in the methane oxidation pathway of methanotrophs identified that strains of Methylomonas, Methylococcus and Methylocystis/Methylosinus had assimilated the 13CH4, and that these methanotrophs contain genes encoding both known types of methane monooxygenase (MMO). Sequences of non-methanotrophic bacteria and an alga provided evidence for turnover of CH4 due to possible cross-feeding on 13C-labelled metabolites or biomass. Our results suggest that aerobic methanotrophs actively convert CH4 into complex organic compounds in Movile Cave and thus help to sustain a diverse community of microorganisms in this closed ecosystem.

Carbon Radioisotopes↗
PubMed2003

The influence of Lentinus edodes (Shiitake mushroom) preparations on bacteriological and morphological aspects of the small intestine in piglets.

Among substances intended to replace growth promoting antibiotics in pig nutrition, non-digestible oligosaccharides or polysaccharides could be potential alternative compounds. Therefore, the influence of beta-1,3-1,6 glucans on bacteriological, biochemical and morphological aspects of the small intestine in weaned piglets was investigated. As sources of beta-glucans, Lentinan (extract of Lentinus edodes mycelium) or dried L. edodes mycelium were added to the diet. Four homogenous groups of 5 newly weaned piglets (4 weeks of age) received one of four diets: control diet (C), C supplemented with Avilamycin (50 mg/kg, positive control), C supplemented with 0.1% of Lentinan and C supplemented with 5% of dried L. edodes mycelium powder. A first group of 10 piglets was euthanized after 11 days and the remaining 10 on day 12 of the experiment. The gastrointestinal tract was divided in segments and samples taken from digesta (stomach, proximal and distal jejunum, caecum), mucosal scrapings (jejunum) and ring shaped tissue samples (1 cm) of proximal and distal jejunum. Bacterial counts were made with digesta and mucosal samples, and short-chain fatty acids (SCFA), lactic acid and ammonia concentrations were determined. Tissue samples of both jejunal sites were embedded in paraffin wax for morphometrical (villus length, crypt depth) and histological observations (numbers of intraepithelial lymphocytes (IEL), goblet cells, apoptotic enterocytes on villi, mitotic cells in crypts). Only the diet containing 5% of dried L. edodes consistently resulted in lower viable counts (ca. 1-2 log10 CFU) of total bacteria, E. coli, streptococci and lactic acid bacteria, and luminal and mucosal effects agreed very well. With this diet, acetate and butyrate concentrations in the distal jejunum were doubled, which is favourable in view of the trophic effect on enterocytes and colonocytes. Villus length (V) was increased with both diets containing beta-glucans while crypt depth (C) was not altered, but V/C was higher. IEL counts were decreased by both diets although bacterial numbers, which is only one parameter of bacterial load, were only diminished with the L. edodes feed. The three supplemented feeds lowered the number of apoptotic enterocytes on the villi, but these numbers were very low (control diet : 44 cells per 100 villi), making clear interpretation difficult. The mitotic index was slightly lower with the L. edodes feed, although not statistically significant. Decreased viable counts observed with the latter diet is a favourable effect as it is accepted that a lower bacterial load causes lower turnover rates of the intestinal epithelial cells, while there is also less competition for specific substrates. A higher V/C ratio, a smaller number of IEL in the epithelium and a lower apoptotic index also indicate slower turnover rate of the mucosa when Lentinan and L. edodes diets were fed. The inconsistent effects observed with Lentinan were probably due to the low amount added to the diet. It should be taken into account that the influence of L. edodes mycelium powder was more likely due to the presence of antibacterial compounds (eg. lenthionine, lentinamycin, terpenoids, polyphenols), rather than to an immunostimulating action of beta-glucans with increased release of IgA onto the mucosa surface.

Adaptation, Physiological↗
PubMed1993

A study on the mechanism of action of sceptrin, an antimicrobial agent isolated from the South Pacific sponge Agelas mauritiana.

The mechanism of action of sceptrin, an antimicrobial agent isolated from the sponge Agelas mauritiana, was investigated. Sceptrin has been reported to exhibit antibacterial and antifungal activities. In our studies, sceptrin demonstrated a bacteriostatic rather than bactericidal effect on exponentially growing Escherichia coli cells at the MIC. Under these conditions, the culture produced chains of cells, and incorporation of radio-labelled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3H-uridine into RNA was slightly inhibited. At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabelled precursors, and induced the formation of unusual spheroplasts. Peptidoglycan turnover in E. coli appeared to be stimulated by sceptrin as demonstrated by a release of diaminopimelic acid-containing high molecular weight material. Subsequent studies of the release of potassium ions from E. coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells. It is proposed that spheroplasts formation may reflect a cell wall effect that occurs subsequent to membrane damage.

Animals↗
PubMed1982

Messenger function of prostaglandins in cell to cell interactions and control of proteinase activity in the rheumatoid joint.

Destruction of joint structures in arthritis may result from failure of normal mechanisms controlling interactions among cells of the various tissues of the joint. Normal synovium in culture produces less prostaglandin E (PGE) and collagenase than rheumatoid. When rheumatoid synovium is dissociated into cells, the adherent cell cultures rapidly lose the ability to synthesize large amounts of PGE and collagenase and become indistinguishable from normal synovial cells. A mononuclear cell factor (MCF) derived from supernatant media of cultured human blood mononuclear cells and a 'synovial factor(s)' (SF) from cultures of either normal or rheumatoid synovial fragments both stimulate production of PGE and proteinase by cells derived from human synovium, cartilage and bone. The activities of factors which may be present in these stimulatory supernatants may be unmasked in vitro when they are removed from the normal control present in vivo. Normal synovium probably contains cells which, with the appropriate stimulus, may be recruited to participate in joint tissue degradation. Normal connective tissue turnover may also be controlled by a neutral metallo-proteinase inhibitor (TIMP), which is produced in considerable amounts by normal synovium, but which cannot be detected in cultures of rheumatoid synovium. While corticosteroids inhibit the production and action of MCF and SF, they stimulate production of TIMP by normal or rheumatoid synovial tissue in vitro and may contribute to the endogenous control mechanisms. PGE may also have a modulatory role in these cellular interactions.

Arthritis, Rheumatoid↗
PubMed2004

Microcosm studies for neutralization of hypolimnic acid mine pit lake water (pH 2.6).

Ten microcosms of 0.088 m3 water volume (0.3 m i.d. and 1.20 m height) were designed for neutralization studies representing hypolimnic ecosystem models for acid mine pit lakes. Sediment and water were collected from an acid lignite mine pit lake (Brandenburg, Germany) and filled into the microcosms. To determine the efficacy of controlled in situ organic carbon amendments as a possible neutralization method, sediment and water were treated with ethanol and Carbokalk with and without wheat straw. The water chemistry was monitored for 1 yr. At start-up and end of the experiments, the sedimentwas characterized. Iron and sulfate were removed with varying intensity from the water phase as a result of microbial iron and sulfate reduction together with a subsequent precipitation of unsoluble sulfide minerals to the sediment. The pH rose, and alkalinity generation and bacterial growth were observed. Neutralization rates were calculated using equivalents of accumulated total reduced inorganic sulfur together with the nonsulfidic reactive ferrous iron in the sediment. In the treated microcosms, the neutralization rates were between 6 and 15 equiv m(-2) a(-1). Carbokalk was most effective in stimulating growth of sulfate-reducing bacteria and probably also served as inoculum. With Carbokalk together with wheat straw, the pH increased from 2.6 to around 6.5 within the whole microcosm. The critical revision of the results indicates that the application of Carbokalk (approximately 3.9 kg m(-2)) together with the application of wheat straw (approximately 9.3 kg m(-2)) is most suitable for further experiments in outdoor enclosures (mesocosms). For that case, the prediction of the water quality for a lake water column after multiple lake turnover events is presented based on batch reaction simulation using the geochemical model PHREEQC.

Carbon↗
PubMed2006

Impact of water table depth on forest soil methane turnover in laboratory soil cores deduced from natural abundance and tracer 13C stable isotope experiments.

We investigated turnover of methane (CH4) in soils from a poorly drained UK forest. In situ, this forest exhibited a negligible soil-atmosphere CH4 flux, whereas adjacent grassland plots were sources of CH4. We hypothesised that the forest plots exhibited reduced anaerobic CH4 production through water-table draw down. Consequently, we exposed soil cores from under oak to high and low water-table conditions in the laboratory. Methane fluxes increased significantly in the high water-table (1925+/-1702 mug CH4 m(-2) h(-1)) compared to the low one (-3.5+/-6.8 microg CH4 m(-2) h(-1)). Natural abundance delta13C values of CH4 showed a strong depletion in high water-table cores (-56.7+/-2.9 per thousand) compared to methane in ambient air (-46.0 per thousand) indicative of methanogenic processes. The delta13C values of CH4 from low water-table cores (delta13C-46.8+/-0.2 per thousand) was similar to ambient air and suggested little alteration of headspace CH4 by the soil microbial community. In order to assess the CH4 oxidizing activity of the two treatments conclusively, a 13CH4 spike was added to the cores and 13CO2 production was measured as the by-product of CH4 oxidation. 13CH4 oxidation rates were 57.5 (+/-12.7) and 0.5 (+/-0.1) microg CH4 m(-2) h(-1) for high and low water-tables, respectively. These data show that the lower water-table hydrology treatment impacted methanogenic processes without stimulating methanotrophy.

Carbon Isotopes↗
PubMed2000

Pretanned leather shavings in a supplement mixture for steers: I. In situ and in vitro disappearance, ruminal fermentation, and organic matter, nitrogen, and fiber digestion.

Two digestion studies were conducted to evaluate the use of pretanned leather shavings as a component of a protein supplement. In Exp. 1, the in situ and in vitro disappearance of pretanned leather shavings and soybean meal was evaluated. Results revealed that less than 18.4% of the pretanned leather shavings was solubilized and disappeared when exposed to McDougall's buffer for 48 h, but there was 90.0% disappearance with 48-h exposure to a .1 N HCl/pepsin treatment and 97.0% disappearance with exposure to a two-stage digestion. In situ disappearance following 72 h in the rumen allowed 6.8% disappearance. Thus, leather shavings seem to be relatively indigestible in the rumen, but postruminal digestion may be possible. In Exp. 2, six Angus x Holstein steers, fitted with ruminal and duodenal cannulas, were used in a replicated 3 x 3 Latin square to evaluate ruminal and digestion effects of the following supplements combined with fescue hay at 1.7% of BW (DM basis): no supplementation (control); supplementation intraruminally with soybean meal at .07% of BW (as-fed basis); and supplementation intraruminally with a combination of soybean meal and pretanned leather shavings (17:8 ratio) at .05% of BW (isonitrogenous to soybean meal; as-fed basis). Ruminal fluid passage rate was greater and fluid turnover time was shorter in steers fed leather shavings than in those fed soybean meal (P = .10). Ruminal pH was lower (P = .04) for supplemented steers than for control steers and ruminal NH3 N concentration was greater (P = .01) in steers fed soybean meal than in those fed leather shavings. Total VFA concentration was increased (P = .02) by supplementation. Supplementation with soybean meal increased (P < .05) ruminal molar proportions of butyrate, valerate, and isovalerate compared with leather shavings. Duodenal OM flow and OM disappearing in the intestines were increased by supplementation (P < .10), but not by the type of supplement fed (P > .10). Ruminal digestion of OM and total tract OM digestion were unaffected (P > .10) by supplementation and the type of supplement fed. Flow and digestion of NDF were unaffected (P > .10) by the treatments. Flow of N and the quantity of N disappearing in the intestines were increased (P < .05) by supplementation but did not differ (P > .10) between supplementation groups. Microbial N flow, N utilization for net microbial protein synthesis, and ruminal N disappearance were unaffected (P > .10) by supplementation and the type of supplement provided. Combining pretanned leather shavings with soybean meal seemed to have no deleterious effects on digestion or fermentation and to allow for escape of some N to the lower tract.

Animal Feed↗
PubMed2004

In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor.

OBJECTIVES: Production of beta-lactamases is the main mechanism of beta-lactam resistance in Gram-negative bacteria. Despite the current use of clavulanic acid, sulbactam and tazobactam, the prevalence of class A and class C enzymes is increasing worldwide, demanding new beta-lactamase inhibitors. Here we report the antimicrobial properties of AVE1330A, a representative of a novel class of bridged bicyclico[3.2.1]diazabicyclo-octanones in combination with ceftazidime. MATERIALS AND METHODS: IC(50) and kinetic parameters of the hydrolysis reaction were used to characterize beta-lactamase inhibition by AVE1330A. MICs for >600 strains were determined with the combination ceftazidime/AVE1330A at a fixed ratio of 4:1. RESULTS: IC(50)s of AVE1330A for TEM-1 and P99 enzymes were 0.0023 mg/L (8 nM) and 0.023 mg/L (80 nM), compared with 0.027 mg/L (130 nM) and 205.1 mg/L (1 x 10(6) nM) of clavulanic acid and 0.013 mg/L (40 nM) and 1.6 mg/L (5000 nM) of tazobactam. A highly stable covalent complex led to a low turnover of AVE1330A. MICs of ceftazidime/AVE1330A for Enterobacteriaceae were at least eight-fold lower than those of ceftazidime alone. All of the Escherichia coli, Klebsiella pneumoniae, Citrobacter and Proteus mirabilis strains, including ceftazidime-resistant isolates, were inhibited at 4-8 mg/L. Only 2 mg/L were required to inhibit other Proteeae, Enterobacter, Salmonella and Serratia. CONCLUSION: The combination of ceftazidime with AVE1330A exhibited broad-spectrum activity against Ambler class A- and class C-producing Enterobacteriaceae.

Azepines↗
PubMed1976

Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei.

Dihydrofolate reductase has been purified from a methotrexate-resistant strain of Lactobacillus casei NCB 6375. By careful attention to growth conditions, up to 2.5 g of enzyme is obtained from a 400 litre culture. The purification procedure, involving poly-ethyleneimine treatment, DEAE-cellulose chromatography and affinity chromatography on methotrexate-aminohexyl-Sepharose, operates on the gram scale, with overall yields of 50-60%. Elution of the affinity column by reverse (upward) flow was used, as it led to recovery of the enzyme in a much smaller volume. The enzyme obtained appears to be more than 98% pure, as judged by gel electrophoresis, isoelectric focusing, and gel filtration. It has a mol.wt. of approx. 17900 and a turnover number of 4s-1 (50mM-triethanolamine/400mM-KCl, pH 7.2, 25 degrees C) with dihydrofolate and NADPH as substrates. The turnover number for folate is 0.02s-1. Michaelis constants for a variety of substrates have been measured by using a new fluorimetric assay (0.36 muM-dihydrofolate; 0.78 muM-NADPH), and binding constants determined by using the quenching of protein fluorescence (dihydrofolate, 2.25 X 10(6)M-1; NADPH, greater than 10(8)M-1). The pH/activity profile shows a single maximum at pH 7.3; at this pH, marked activation by 0.5M-NaCl is observed.

Amino Acids↗
PubMed2005

Investigation of the mechanism of resistance to third-generation cephalosporins by class C beta-lactamases by using chemical complementation.

The widespread use of antibiotics to treat bacterial infections has led to the continuing challenge of antibiotic resistance. For beta-lactam antibiotics, the most common form of resistance is the expression of beta-lactamase enzymes, which inactivate the antibiotics by cleavage of the beta-lactam core. In this study, chemical complementation, which is a general method to link the formation or cleavage of a chemical bond to the transcription of a reporter gene in vivo, was employed in combination with combinatorial mutagenesis to study the mechanism by which the class C beta-lactamase P99 might evolve resistance to the commonly administered third-generation cephalosporin cefotaxime. The chemical complementation system was first shown to be able to distinguish between the wild-type (wt) class C beta-lactamase P99 and the clinically isolated extended-spectrum class C beta-lactamase GC1 in the presence of cefotaxime. The system was then employed to evaluate the activity of mutants of wt P99 towards cefotaxime. A number of single-point mutations at position 221 (Tyr in wt P99) were identified that conferred resistance towards inhibition by cefotaxime, with as much as a 2000-fold increase in k(cat) and a 100-fold increase in k(cat)/K(M) (k(cat)=the rate of catalysis; K(M)=the Michaelis constant), as compared to those of the wt enzyme. Finally, the chemical complementation system was employed in a high-throughput screen to identify a number of mutants of P99 that have multiple mutations around the substrate-binding pocket that increase resistance towards cefotaxime inhibition. The catalytic turnover of cefotaxime by the most active mutant identified was 5500 times higher than that of the wt P99. The resistant mutants suggest a mechanism by which a number of mutations can confer resistance by increasing the flexibility of the Omega loop and altering the positioning of residue 221. Thus, as illustrated in this study, chemical complementation has the potential to be used as a high-throughput screen to study a wide range of enzyme-drug interactions.

Anti-Bacterial Agents↗
PubMed2002

Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site.

Understanding the basic principles that govern RNA binding by aminoglycosides is important for the design of new generations of antibiotics that do not suffer from the known mechanisms of drug resistance. With this goal in mind, we examined the binding of kanamycin A and four derivatives (the products of enzymic turnovers of kanamycin A by aminoglycoside-modifying enzymes) to a 27 nucleotide RNA representing the bacterial ribosomal A site. Modification of kanamycin A functional groups that have been directly implicated in the maintenance of specific interactions with RNA led to a decrease in affinity for the target RNA. Overall, the products of reactions catalyzed by aminoglycoside resistance enzymes exhibit diminished binding to the A site of bacterial 16S rRNA, which correlates well with a loss of antibacterial ability in resistant organisms that harbor these enzymes.

Anti-Bacterial Agents↗
PubMed1991

Evolved neomycin phosphotransferase from an isolate of Klebsiella pneumoniae.

A new aminoglycoside resistance gene (aphA1-IAB) confers high-level resistance to neomycin. The sequence of aphA1-IAB is closely related to aphA1 found in the transposons Tn4352, Tn903 and Tn602. For example, aphA1-IAB differs from aphA1-903 at five nucleotides that result in four amino acid replacements. The enzyme encoded by aphA1-IAB has a significantly higher turnover number with neomycin, kanamycin and G418 as substrates than does the aphA1-903 enzyme. A parsimonious phylogenetic tree suggests that aphA1-IAB evolved from an ancestral form that is closely related or identical to the aphA1 found in Tn903. The excess of replacement substitutions over silent substitutions in aphA1-IAB, as well as its convergence toward aphA3 from Staphylococcus aureus, is indicative of selective evolution. Our hypothesis to explain these results is that aphA1-IAB evolved under the selective pressure of neomycin use in relatively recent times.

Amino Acid Sequence↗
PubMed1980

Enhanced biosynthesis of human skin collagenase in fibroblast cultures from recessive dystrophic epidermolysis bullosa.

Using a sensitive, specific immunoprecipitation method, the biosynthesis of human skin collagenase was studied in fibroblast cultures from patients with recessive dystrophic epidermolysis bullosa. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized immunoprecipitates showed two 3H-labeled procollagenase species that comigrated with those harvested from control cultures. Recessive dystrophic epidermolysis bullosa cultures accumulated increased amounts of collagenase. Both the initial rate of accumulation of intracellular enzyme and the rate of secretion were enhanced, suggesting that excessive accumulation is related to increased synthesis. Because the turnover of labeled collagenase was unaltered, the accumulation could not be attributed to diminishing enzyme degradation. No preferential incorporation of [3H]leucine into recessive dystrophic epidermolysis bullosa collagenase occurred. Furthermore, the mutant cultures displayed no alteration in total protein synthesis, the intracellular leucine pool, or the growth kinetics of the cells. Cells from a patient with dominant epidermolysis bullosa did not show enhanced accumulation of collagenase. The levels of collagenase synthesized in vitro correlated with those observed previously in vivo in recessive dystrophic epidermolysis bullosa patients, suggesting that this biochemical trait is pathogenetically significant in the disorder.

Cells, Cultured↗
PubMed2006

Heterotrophic Archaea dominate sedimentary subsurface ecosystems off Peru.

Studies of deeply buried, sedimentary microbial communities and associated biogeochemical processes during Ocean Drilling Program Leg 201 showed elevated prokaryotic cell numbers in sediment layers where methane is consumed anaerobically at the expense of sulfate. Here, we show that extractable archaeal rRNA, selecting only for active community members in these ecosystems, is dominated by sequences of uncultivated Archaea affiliated with the Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group, whereas known methanotrophic Archaea are not detectable. Carbon flow reconstructions based on stable isotopic compositions of whole archaeal cells, intact archaeal membrane lipids, and other sedimentary carbon pools indicate that these Archaea assimilate sedimentary organic compounds other than methane even though methanotrophy accounts for a major fraction of carbon cycled in these ecosystems. Oxidation of methane by members of Marine Benthic Group B and the Miscellaneous Crenarchaeotal Group without assimilation of methane-carbon provides a plausible explanation. Maintenance energies of these subsurface communities appear to be orders of magnitude lower than minimum values known from laboratory observations, and ecosystem-level carbon budgets suggest that community turnover times are on the order of 100-2,000 years. Our study provides clues about the metabolic functionality of two cosmopolitan groups of uncultured Archaea.

Anaerobiosis↗
PubMed1967

Response of intracellular proteolysis to alteration of bacterial protein and the implications in metabolic regulation.

An assessment has been made of the extent to which the breakdown of microbial cellular proteins is regulated by their metabolic state or function. For this purpose, a number of agents and conditions that alter the synthesis, structure, or utility of cellular protein were examined for the effect on their lability. In Escherichia coli, 5-fluorouracil, p-fluorophenylalanine, norleucine, canavanine, thienylalanine, and puromycin, which engender nonfunctional cellular protein en masse, and ultraviolet irradiation increase the breakdown rate of proteins synthesized in their presence as much as two- to threefold without altering the general capacity for proteolysis. The effects are complicated by, but experimentally distinguishable from, secondary changes in proteolysis that accompany growth inhibition. In contrast, no potentiation of proteolysis is elicited by the presence of suppressor genes, by the administration of heat, or by the biosynthetic alterations attending large changes in the conditions of cultivation or by those attending bacteriophage infection. Thus, although mass perturbations in protein conformation are catabolically distinguishable, the more individual and limited conformational modifications that might occur in disuse do not appear to be the primary determinants of the protein turnover rate. In Bacillus subtilis, turnover synthesis of protein during starvation is as susceptible to treatment with actinomycin D as that during growth. Treatment alters neither the rate of intracellular proteolysis nor the catabolic pattern of the modicum of proteins that are still synthesized. It is concluded that there is no correlation between metabolic stability of protein and the stability of its messenger ribonucleic acid.

Alanine↗