The phylogenic position of Peptococcus niger based on 16S rRNA sequence studies

A 1330 base-pair fragment of a 16S rRNA gene has been amplified, cloned and sequenced. Comparison to other 16S rRNA sequences of eubacteria showed that P. niger represents a deep branch within the subdivision "Gram-positive with Gram-negative cell walls". It is not related to peptostreptococci, representatives of this genus studied so far are more closely related to clostridia.     

Peptostreptococcus barnesae sp. nov., a Gram-positive,anaerobic, obligately purine utilizing coccus from chicken feces

Anaerobic, Gram-positive cocci were obtained from chicken feces by direct isolation, which grew on the purines uric acid, xanthine, 6,8-dihydroxypurine, guanine, and hypoxanthine. Adenine and glycine were fermented, but not as readily. Acetate, formate, ammonia, and CO₂ were products. The isolated strains were nutritionally non-fastidious, however, they required selenite, molybdate, and tungstate as micronutrients. The cells were spherical and 0.5–0.9 m in diameter. The addition of bile salts enhanced the growth rate in most cases. The organisms proved to be quite resistant to lysis. The guanosine-plus-cytosine (G+C) content of their deoxyribonucleic acid was 33.6 to 34.8 mol%. The peptidoglycan was of the same structure (Gly-Lys-d-Asp) as reported for the anaerobic cocci of Hare group IX. However, the latter strains could only utilize glycine, not purines. Therefore, it is proposed to form a new species, Peptostreptococcus barnesae sp. nov.This paper is dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

Enhancement of spontaneous and lymphokine activated human macrophage cytotoxicity by hyperthermia

Human macrophages grown on hydrophobic teflon membranes from blood-born monocytes were incubated at hyperthermic temperatures for various time periods and then tested for their ability to inhibit the growth of an allogeneic lymphoma cell line (U 937). Incubation at 40.5 degrees C greatly enhanced macrophage cytotoxicity. This effect of hyperthermia developed slowly with an optimal incubation period of 48 h. In addition, lymphokine activation of macrophages for cytotoxicity appeared to be more effective at elevated temperatures.     

Purification of NADPH-dependent electron-transferring flavoproteins and N-terminal protein sequence data of dihydrolipoamide dehydrogenases from anaerobic, glycine-utilizing bacteria.

Three electron-transferring flavoproteins were purified to homogeneity from anaerobic, amino acid-utilizing bacteria (bacterium W6, Clostridium sporogenes, and Clostridium sticklandii), characterized, and compared with the dihydrolipoamide dehydrogenase of Eubacterium acidaminophilum. All the proteins were found to be dimers consisting of two identical subunits with a subunit Mr of about 35,000 and to contain about 1 mol of flavin adenine dinucleotide per subunit. Spectra of the oxidized proteins exhibited characteristic absorption of flavoproteins, and the reduced proteins showed an A580 indicating a neutral semiquinone. Many artificial electron acceptors, including methyl viologen, could be used with NADPH as the electron donor but not with NADH. Unlike the enzyme of E. acidaminophilum, which exhibited by itself a dihydrolipoamide dehydrogenase activity (W. Freudenberg, D. Dietrichs, H. Lebertz, and J. R. Andreesen, J. Bacteriol. 171:1346-1354, 1989), the electron-transferring flavoprotein purified from bacterium W6 reacted with lipoamide only under certain assay conditions, whereas the proteins of C. sporogenes and C. sticklandii exhibited no dihydrolipoamide dehydrogenase activity. The three homogeneous electron-transferring flavoproteins were very similar in their structural and biochemical properties to the dihydrolipoamide dehydrogenase of E. acidaminophilum and exhibited cross-reaction with antibodies raised against the latter enzyme. N-terminal sequence analysis demonstrated a high degree of homology between the dihydrolipoamide dehydrogenase of E. acidaminophilum and the electron-transferring flavoprotein of C. sporogenes to the thioredoxin reductase of Escherichia coli. Unlike these proteins, the dihydrolipoamide dehydrogenases purified from the anaerobic, glycine-utilizing bacteria Peptostreptococcus glycinophilus, Clostridium cylindrosporum, and C. sporogenes exhibited a high homology to dihydrolipoamide dehydrogenases known from other organisms.     

5-Hydroxymethylcytosine is an essential intermediate of active DNA demethylation processes in primary human monocytes

Background

Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases.

Results

We previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes.

Conclusions

The work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.     

Catalytic and molecular properties of the quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase from Ralstonia eutropha strain Bo

The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparent k(cat)/K(m) and K(i) values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k(cat)/K(m) value of 788 x 10(4) M(-1) s(-1). The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.     

A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine

A cytochrome P450 and an iron-sulfur protein, whose expression was specifically induced during growth of Mycobacterium sp. strain HE5 on morpholine as the sole source of carbon, nitrogen, and energy were purified to apparent homogeneity. Due to the lack of enzymatic activity, carbon monoxide difference spectra and determination of the acid-labile sulfur, respectively, were used to detect the proteins during purification. The cytochrome P450, designated P450mor, was characterized as a monomer with an apparent molecular mass of 44.7 kDa. The amino acid sequence of an internal peptide comprising 19 amino acids was identical to the sequence derived from a gene encoding a cytochrome P450 from Mycobacterium smegmatis mc(2)155 suggested to be involved in the utilization of piperidine and pyrrolidine. The iron-sulfur protein was characterized as a ferredoxin exhibiting a molecular mass of 6.8 kDa and named Fdmor. An identity of 48-77% was obtained for the 30 N-terminal amino acids of Fdmor and the corresponding sequences of different 3Fe-4S-ferredoxins known to be involved in P450-dependent reactions. From these data we concluded that growth of Mycobacterium sp. strain HE5 on morpholine led to the expression of a cytochrome P450-dependent monooxygenase system composed of at least two different proteins.     

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.     

Selenium, the element of the moon, in life on earth

The present status of selenium biochemistry is reviewed with particular emphasis on biomedical problems related to the selenium status of humans and experimental animals. Historical milestones of selenium biochemistry starting from the identification of the first selenoenzymes up to the elucidation of prokaryotic and eukaryotic selenoprotein biosynthesis are compiled. Topical hypotheses on the biological role of selenium in general and of individual selenoproteins in respect to antioxidant defense, redox regulation of metabolic processes, thyroid function, spermatogenesis, oncogenesis, and atherogenesis are critically evaluated.