Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58.

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth.

Purification and characterization of vanillin dehydrogenases from alkaliphile Micrococcus sp. TA1 and neutrophile Burkholderia cepacia TM1.

Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP(+) as a coenzyme for its activity, but that from strain TM1 required NAD(+). These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers.

Site-specific and asymmetric hydrolysis of prochiral 2-phenyl-1,3-propanediol diacetate by a bacterial esterase from an isolated strain.

Bacillus cereus 809A and Burkholderia sp. 711C were isolated from soil. These strains demonstrate hydrolysis activity towards prochiral 2-phenyl-1,3-propanediol diacetate and accumulated the corresponding chiral monoacetates into the reaction mixture. When 2-phenyl 1,3-propanediol diacetate was used as a substrate, the produced monoacetates with Burkholderia sp. 711C were obtained in a racemic form but that produced by Bacillus cereus 809A showed an excess of the (S)-form. The resting cell reaction revealed that for Bacillus cereus 809A, there was an enrichment of one of the enantiomers of the monoacetate such that the enantiomeric excess (e.e.) of the (S)-form was over 95%. The purified enzyme from Bacillus cereus 809A hydrolyzed diacetate to monoacetate, and the e.e. value of the (S)-form increased by prolonged reaction in a way similar to the resting cell reaction. From N-terminal amino acids, this esterase is conserved in some strains of Bacillus for which the genomic sequences have been reported.

Formaldehyde uptake by Methylobacterium sp. MF1 and Acidomonas methanolica MB 58 with the different formaldehyde assimilation pathways.

Methylobacterium sp. MF1 (an obligate methylotrophic bacterium isolated newly by the authors) and Acidomonas methanolica MB58 (a facultative methylotrophic bacterium) uptake formaldehyde similarly. It was found that the former assimilated formaldehyde via the serine pathway whereas the latter did so via the ribulose-monophosphate pathway from the measurement of the key enzyme activities in each assimilation pathway. That is, hydroxy pyruvate reductase was detected in only the above-mentioned MF1 strain, but hexulose phosphate synthase (HPS) was not. The efficiencies of formaldehyde consumption by both strains under a continuous chemostat cultivation in the steady state were almost the same in spite of their different assimilation pathways. That is, the consumption efficiencies of the MF strain and the MB58 strain were ca. 1.2 g/L/d and ca. 1.8 g/L/d, respectively, under the experimental conditions. In the future, optimal continuous operating conditions will be investigated.

Production of sepedonin by Sepedonium chrysospermum NT-1 in submerged culture.

Strains of Sepedonium chrysospermum and the anamorph strain of Hypomyces chrysospermus (congruent with Apiocrea chrysosperma) were isolated and purified from parasitic filamentous fungi on the fruiting bodies of Boletaceae, such as the Gyroponus and Suillus genera in Japan, and identified from formations of conidia and chlamydospores. It is known that these strains produce sepedonin. S. chrysospermum NT-1 strain was selected from these strains and isolated. As the optimum medium (CY-1 medium), 0.1% yeast extract was added to the fruiting-body-forming medium (C medium) of Schizophyllum commune. After 8 days of growth on CY-1 medium, the yield of sepedonin was about 34 mg per 2 g of glucose added. This sepedonin seemed to inhibit the growth of various gram-positive and gram-negative bacteria, yeasts and molds.

Thermolabile variant, PHOX-S, of prophenol oxidase in Drosophila melanogaster.

The Phox(S) strain of Drosophila melanogaster is an electrophoretically slow variant found in a wild population at Victoria, Australia. Prophenol oxidase isoform A(1) from PHOX-S was purified and characterized biochemically and genetically. The purified fraction of A(1) from PHOX-S showed a homodimer with a molecular weight of the subunit of approximately 77 kDa. The Phox(S) strain was temperature sensitive in vivo in culture, and the purified protein was thermolabile in vitro. By the deletion mapping method, the Phox(S) locus was cytologically estimated to be at the location 55-A on the right arm of the second chromosome and 79.6 genetically. These data show that PHOX-S is an electrophoretic variant of MOX and that PHOX-S is the first thermolabile protein found in invertebrate prophenol oxidase.

Formaldehyde-limited cultivation of a newly isolated methylotrophic bacterium, Methylobacterium sp. MF1: enzymatic analysis related to C1 metabolism.

Formaldehyde is a highly toxic compound to most living organisms. We have isolated a bacterial strain that is able to efficiently degrade formaldehyde and use it as a sole carbon source. The isolated strain was identified as Methylobacterium sp. MF1, which could grow on formaldehyde and methanol. Methylobacterium sp. MF1 was grown in batch culture using 1.2 g/l formaldehyde as a sole carbon source, which was all consumed within 200 h. In order to decompose formaldehyde more efficiently, formaldehyde-limited chemostat cultivation of Methylobacterium sp. MF1 was investigated. Formaldehyde was consumed at 1.7 g/l/d when the dilution rate was 0.012 h(-1). Under these conditions, the cell turbidity (OD610) reached 2.0. Furthermore, when the initial turbidity was adjusted to 3.0 using methanol-grown cells, continuous cultivation could be started at an initial dilution rate of 0.008 h(-1). Using these conditions, consumption of formaldehyde could be continued for at least 600 h. The enzyme activities of cells growing as a chemostat culture, using methanol or formaldehyde as a sole carbon source, were compared to that of C1 metabolism. No difference was detected in the enzyme activities for the oxidation and assimilation of C1 compounds between the two cell-free extracts. Furthermore, methanol dehydrogenase activity was detected at the same level when formaldehyde was used as a sole carbon source. These results suggest that the resistance to the toxic effects of formaldehyde exhibited by Methylobacterium sp. MF1 is related to factors other than C1 metabolism.

Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid.

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.

E. coli expression of TIMP-4 and comparative kinetic studies with TIMP-1 and TIMP-2: insights into the interactions of TIMPs and matrix metalloproteinase 2 (gelatinase A).

The inhibitory properties of TIMP-4 for matrix metalloproteinases (MMPs) were compared to those of TIMP-1 and TIMP-2. Full-length human TIMP-4 was expressed in E. coli, folded from inclusion bodies, and the active component was purified by MMP-1 affinity chromatography. Progress curve analysis of MMP inhibition by TIMP-4 indicated that association rate constants (k(on)) and inhibition constants (K(i)) were similar to those for other TIMPs ( approximately 10(5) M(-)(1) s(-)(1) and 10(-)(9)-10(-)(12) M, respectively). Dissociation rate constants (k(off)) for MMP-1 and MMP-3 determined using alpha(2)-macroglobulin to capture MMP dissociating from MMP-TIMP complexes were in good agreement with values deduced from progress curves ( approximately 10(-)(4) s(-)(1)). K(i) and k(on) for the interactions of TIMP-1, -2, and -4 with MMP-1 and -3 were shown to be pH dependent. TIMP-4 retained higher reactivity with MMPs at more acidic conditions than either TIMP-1 or TIMP-2. Molecular interactions of TIMPs and MMPs investigated by IAsys biosensor analysis highlighted different modes of interaction between proMMP-2-TIMP-2 (or TIMP-4) and active MMP-2-TIMP-2 (or TIMP-4) complexes. The observation that both active MMP-2 and inactive MMP-2 (with the active site blocked either by the propeptide or a hydroxamate inhibitor) have essentially identical affinities for TIMP-2 suggests that there are two TIMP binding sites on the hemopexin domain of MMP-2: one with high affinity that is involved in proMMP-2 or hydroxamate-inhibited MMP-2; and the other with low affinity involved in formation of the complex of active MMP-2 and TIMP-2. Similar models of interaction may apply to TIMP-4. The latter low-affinity site functions in conjunction with the active site of MMP-2 to generate a tight enzyme-inhibitor complex.

Estado atual da lesäo periférica de células gigantes / Current status of periferal giant cell lesions

Através da revisäo bibliográfica pertinente a lesäo periférica de células gigantes, foram discutidos e analisados seus aspectos clínicos e histopatológicos. Baseados nisso é dado o atual estado desta lesäo