Phylogenetic study of the species within the family Streptomycetaceae.

Species of the genus Streptomyces, which constitute the vast majority of taxa within the family Streptomycetaceae, are a predominant component of the microbial population in soils throughout the world and have been the subject of extensive isolation and screening efforts over the years because they are a major source of commercially and medically important secondary metabolites. Taxonomic characterization of Streptomyces strains has been a challenge due to the large number of described species, greater than any other microbial genus, resulting from academic and industrial activities. The methods used for characterization have evolved through several phases over the years from those based largely on morphological observations, to subsequent classifications based on numerical taxonomic analyses of standardized sets of phenotypic characters and, most recently, to the use of molecular phylogenetic analyses of gene sequences. The present phylogenetic study examines almost all described species (615 taxa) within the family Streptomycetaceae based on 16S rRNA gene sequences and illustrates the species diversity within this family, which is observed to contain 130 statistically supported clades, as well as many unsupported and single member clusters. Many of the observed clades are consistent with earlier morphological and numerical taxonomic studies, but it is apparent that insufficient variation is present in the 16S rRNA gene sequence within the species of this family to permit bootstrap-supported resolution of relationships between many of the individual clusters.

The effects of extended-release niacin on carotid intimal media thickness, endothelial function and inflammatory markers in patients with the metabolic syndrome.

BACKGROUND: Niacin is an agent that significantly increases high-density lipoprotein cholesterol (HDL-C), but its effects on surrogate markers of atherosclerosis and inflammatory markers are less clear. We studied the effects of niacin on carotid intimal media thickness (IMT), brachial artery reactivity as well as markers of inflammation and the metabolic profile of patients with metabolic syndrome. METHODS AND RESULTS: Fifty patients with the metabolic syndrome (Adult Treatment Panel (ATP) III criteria) were randomised to either extended-release niacin (1000 mg/day) or placebo. After 52 weeks of treatment, there was a change of carotid IMT of +0.009 +/- 0.003 mm in the placebo group and -0.005 +/- 0.002 mm in the niacin group (p = 0.021 between groups). Endothelial function improved by 22% in the group treated with niacin (p < 0.001), whereas no significant changes were seen in the placebo group. High sensitivity C-reactive protein decreased by 20% in the group treated with niacin for 52 weeks (p = 0.013). Niacin increased HDL-C (p < 0.001) and decreased low-density lipoprotein cholesterol and triglycerides (p < 0.001) significantly, and there were no adverse effects on fasting glucose levels after 52 weeks of treatment. CONCLUSION: Extended-release niacin therapy effects a regression in carotid intimal medial thickness and improvement in metabolic parameters (increased HDL and reduced triglycerides). Furthermore, extended-release niacin may demonstrate an anti-atherogenic effect in the metabolic syndrome by improving endothelial function and decreasing vascular inflammation.

Thirty cases of bronchial asthma associated with exposure to pet hamsters.

BACKGROUND: The identification, isolation, and elimination of allergen(s) causing bronchial asthma are the most efficient form of treatment. The pet industry has diversified recently, increasing the risk of exposure of pet owners to many unknown antigens. We clinically studied the characteristics of asthma associated with exposure to pet hamsters. METHODS: The study group comprised 30 adults in whom the onset, recurrence, or exacerbation of asthma was triggered by contact with pet hamsters. Clinical characteristics such as sex, age, period required for symptom onset, species of hamster, treatment and disease course, smoking status, and hamster-specific IgE antibodies in serum were studied. RESULTS: The male: female ratio of the study group was 1:1.3, and mean age was 37.7 years. Patients with no previous history of asthma initially presented with cough, progressing to episodes of asthma. Asthmatic symptoms were associated with hamster contact and ranged in severity from mild to severe. Three patients required hospital admission for treatment. The mean period from the start of hamster exposure to the onset of asthmatic episodes was 15.7 months. Dwarf hamsters were responsible for most cases. The CAP-RAST score for hamster-specific IgE antibodies was 1 to 4 in 22 patients and 0 in 8 patients. Eight patients with a score of 1 or higher for hamster-specific IgE antibodies had a CAP-RAST score of 0 for mite antigen. In these patients, terminating hamster contact resulted in a rapid improvement in symptoms, with no need for further treatment. Twenty-three of the 30 subjects (76.7%) were smokers. CONCLUSION: Exposure to pet hamsters is an important risk factor for the onset, recurrence, or exacerbation of asthma. Smoking may also increase the risk of asthmatic symptoms in patients exposed to hamsters.

Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Cosecretion of prolactin and growth hormone by dispersed pituitary cells of the adult bullfrog, Rana catesbeiana.

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.

A 38 kb segment containing the cdc2 gene from the left arm of fission yeast chromosome II: sequence analysis and characterization of the genomic DNA and cDNAs encoded on the segment.

A genomic 38 kbp segment on the c1750 cosmid clone containing the cdc2 gene, located in the left arm of chromosome II from Schizosaccharomyces pombe, was sequenced. The segment was found to have five previously known genes, pht1, cdc2, his3, act1 and mei4. Among 11 coding sequences (CDSs) predicted by the gene finding software INTRON.PLOT., four CDSs, pi007, pi010, pi014 and pi016, had considerable similarity to 40S ribosomal protein, glycosyltransferase, cdc2-related protein kinase and alpha-1, 2-mannosyltransferase, respectively. Another unusually huge open reading frame (ORF) (pi011), consisting of 2233 amino acids, existed, having significant homology to alpha-amylase, granule-bound glycogen synthase and the Sz. pombe YS 1110 clone product at the N-terminal, middle and C-terminal regions, respectively. All the predicted 11 CDSs were experimentally analysed by RACE PCR. The sequencing of the RACE products revealed that there were two small overlaps at the 3' untranslated regions (UTRs) between pi004 and pi005 (17 bp) and between pi007 and pi008 (2 bp). The distances between 5' end of the 5'UTR and the putative translation initiation codon varied from 10 to 302 nucleotides (nt) among the nine CDSs successfully analysed by 5'-RACE. The expression level of each CDS on this clone was determined. Among the 16 genes on this clone, the previously determined genes, pht1, cdc2, his3 and act1, were found to be most highly expressed. Finally, cDNAs of all the newly identified genes were detected by RACE, proving the actual expression of these genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AB004534.

Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1.

The complete sequence of the genome of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, which optimally grows at 95 degrees C, has been determined by the whole genome shotgun method with some modifications. The entire length of the genome was 1,669,695 bp. The authenticity of the entire sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2,694 open reading frames (ORFs) were assigned. By similarity search against public databases, 633 (23.5%) of the ORFs were related to genes with putative function and 523 (19.4%) to the sequences registered but with unknown function. All the genes in the TCA cycle except for that of alpha-ketoglutarate dehydrogenase were included, and instead of the alpha-ketoglutarate dehydrogenase gene, the genes coding for the two subunits of 2-oxoacid:ferredoxin oxidoreductase were identified. The remaining 1,538 ORFs (57.1%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs suggested that a considerable member of ORFs were generated by sequence duplication. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 47 tRNA genes including 14 genes with intron structures. All the assigned ORFs and RNA coding regions occupied 89.12% of the whole genome. The data presented in this paper are available on the internet homepage (http://www.mild.nite.go.jp).

Platelet activating factor causes a rapid increase in activity of prior expressed tissue factor on monocyte surface membrane.

It is known that tissue factor (TF) activity depends on cell membrane phospholipids. However, the mechanism involved in the regulation of TF activity by the modulation of the phospholipids has not yet been described in detail. To determine whether some mediators regulate TF activity by such a mechanism, we investigated the effect of platelet activating factor (PAF). Addition of PAF to TF-expressed monocytes caused a rapid and marked increase in the activity, but no increase in the antigen. Kinetic analyses were performed on TF-expressed monocytes with or without the addition of PAF, and on purified TF. The former revealed that the activity enhancement by PAF was associated with reduced Km, with Vmax remaining unaltered. The latter showed that the additional phosphatidylserine produced greater TF activity in purified TF, with an alteration pattern of kinetic parameters similar to that observed in the addition of PAF. From these results, we conclude that PAF regulates TF activity at the cell surface by alteration of the phospholipid composition of the membrane, and not by fresh production of TF apoprotein. The role of PAF as described in this paper must be one of the major regulatory systems in TF activity.

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Enhancement by prolactin of the GnRH-induced release of LH from dispersed anterior pituitary cells of the bullfrog (Rana catesbeiana).

The response of enzymatically dispersed anterior pituitary cells of the bullfrog (Rana catesbeiana) to gonadotropin-releasing hormone (GnRH) was studied by monitoring the release of luteinizing hormone (LH) into the culture medium. The cells responded to GnRH by releasing LH according to the incubation time and to the GnRH concentration. The responsiveness to GnRH became less conspicuous as the cell density was reduced. Addition of prolactin (PRL) to the medium enhanced the responsiveness to the secretagogue, and addition of antiserum against PRL lowered the responsiveness to a certain extent. Immunohistochemical studies of sectioned pituitaries revealed that PRL cells most frequently located in contact with LH cells. The possibility that PRL acts directly on gonadotrophs to enhance their responsiveness to GnRH was suggested.

Stimulation by proopiomelanocortin-derived peptides of LH release by bullfrog dispersed anterior pituitary cells.

Labeling and immunoprecipitation experiments confirmed that three proopiomelanocortin (POMC)-derived peptides, the N-terminal peptide of POMC (NPP), joining peptide (JP) and adrenocorticotropic hormone (ACTH), were released by the bullfrog (Rana catesbeiana) anterior pituitary. The effects of these three peptides on luteinizing hormone (LH) release by bullfrog dispersed anterior pituitary cells were studied. NPP and ACTH, but not JP, enhanced LH release concentration-dependently. Approximately 6 hr elapsed before the gonadotrophs responded to NPP and ACTH by releasing LH, whereas their response to human GnRH (hGnRH) was faster, suggesting that the modes of action of these two peptides and hGnRH differ. These results raise the possibility that NPP and ACTH act as paracrine factors in the bullfrog pituitary to enhance LH release either directly or indirectly.

[Clinical study on a concomitant therapy with fluconazole and human recombinant granulocyte colony stimulating factor in the treatment of systemic fungal infections with hematological disorders].

The clinical efficacy and the safety of concomitant therapy with fluconazole and recombinant human granulocyte colony stimulating factor (rhG-CSF) was compared with fluconazole monotherapy in neutropenic patients with hematological disorders. The clinical efficacy rate was 73.5% (25/34) in the combination therapy and 48.1% (37/77) in monotherapy. The difference between the two is statistically significant. Side effects were not observed in the combination group, but laboratory abnormalities were found in 6 patients with an incident rate of 11%. The combination therapy with fluconazole and rhG-CSF may be selected as empiric therapy for systemic fungal infection associated with hematological disorders, since this combination therapy showed high efficacy and low incident of side effects. Some patients, however, did not show increased neutrophil counts in spite of rhG-CSF administration.

Release of alpha-subunit of glycoprotein hormones from the bullfrog pituitary: possible effect of alpha-subunit on prolactin cell function.

On the basis of our previous finding that prolactin (PRL) cells of the bullfrog (Rana catesbeiana) contain immunoreactive alpha-subunit of pituitary glycoprotein hormones, experiments were conducted to see whether the immunoreactive alpha-subunit is released from PRL cells. Cell immunoblot analysis revealed that approximately 10% of cells that released immunoreactive PRL also released immunoreactive alpha-subunit. Western blot analysis of the culture media revealed that a considerable amount of the free form of alpha-subunit was released from the dispersed anterior pituitary cells. Moreover, it was found that addition of alpha-subunit to the culture medium enhanced PRL release. The results raise the possibility that alpha-subunit secreted by PRL cells acts as an autocrine and/or paracrine factor.

Involvement of LDL receptor in the proliferation of vascular smooth muscle cells.

To study the involvement of the low-density lipoprotein (LDL) receptor in the growth of vascular smooth muscle cells (VSMC), we compared the proliferation of cultured VSMC from Watanabe heritable hyperlipidemic (WHHL) rabbits, which lack the LDL receptor, and VSMC from normal Japanese white rabbits in response to platelet derived growth factor (PDGF). The increase in the number of VSMC from WHHL rabbits in response to PDGF (10(-8) M) was significantly lower than that of VSMC from normal rabbits. PDGF stimulated the synthesis of DNA in VSMC from both normal rabbits and WHHL rabbits, but the response was significantly lower in the latter. To determine the involvement of the LDL receptor in the decreased mitogenic response of WHHL rabbit VSMC, we used an anti-LDL receptor monoclonal antibody (MAb) to normal rabbit VSMC; DNA synthesis of VSMC was stimulated by PDGF, but the effect was significantly blocked by the anti-LDL receptor MAb. Mitogen-activated protein (MAP) kinase activity in normal rabbit VSMC was increased by exposure to PDGF, but the effect was significantly suppressed in the presence of the MAb. The anti-LDL receptor MAb markedly inhibited LDL binding to the surface of normal rabbit VSMC. These results suggest that the LDL receptor influences the proliferation of VSMC and thus might be involved in the pathogenesis of atherosclerosis.

Analysis of lung surfactant in the metamorphosing bullfrog (Rana catesbeiana).

In bullfrog (Rana catesbeiana) tadpoles, the lung begins to function at an advanced stage of metamorphosis. As a preliminary step for investigation of the mechanisms involved in lung maturation, pulmonary surfactant was prepared from tadpoles at advanced stages of metamorphosis and its biochemical properties were analyzed. Surfactant phospholipid analysis revealed that the major constituent was phosphatidylcholine (PC), as examined in the animals at late climax (stage 24). Other detectable phospholipids were phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylglycerol, a marker lipid in mammalian surfactant. As in mammals, PC in the surfactant was rich in saturated fatty acids, about 50% of fatty acid moieties being palmitic acid. The content of surfactant PC in the lung increased moderately around mid-climax and markedly at the end of climax. The effect of antiserum against bullfrog prolactin (PRL) on the pulmonary surfactant was studied in climactic tadpoles. The content of surfactant PC in the lung of the antiserum-treated larvae was lower than that in the lung of the normal rabbit serum-injected larvae, whereas the content of PC in the whole lung did not differ between the antiserum-treated and control groups. The results suggest that synthesis of surfactant in the amphibian lung is enhanced as metamorphosis progresses and that PRL is involved in lung maturation.

Endogenous Substrates for Energy Metabolism in Spermatozoa of the Sea Urchins Arbacia lixula and Paracentrotus lividus.

Energy metabolism was examined in the spermatozoa of the sea urchins Arbacia lixula and Paracentrotus lividus, which belong to the orders Arbacioida and Echinoida respectively. P. lividus spermatozoa contained various phospholipids and cholesterol, and their endogenous triglyceride (TG) content was quite low. After dilution of dry sperm in artificial seawater, the level of phosphatidylcholine (PC) decreased rapidly, but other phospholipids remained at constant levels. In contrast to those of P. lividus, the spermatozoa of A. lixula contained TG as well as phospholipids and cholesterol. Following incubation of A. lixula spermatozoa in artificial seawater, TG decreased, but there were no concomitant changes in the levels of phospholipids. Trace amounts of glycogen were present in both species. High lipase activity was demonstrated in A. lixula spermatozoa, but in P. lividus spermatozoa lipase activity was low and phospholipase A2 activity was high. It is thus concluded that A. lixula spermatozoa obtain energy for swimming through oxidation of endogenous TG, whereas P. lividus spermatozoa use PC as a substrate for energy metabolism. This suggests that the system of energy metabolism in spermatozoa is different in the orders Arbacioida and Echinoida.

Regulation of Na-K-ATPase gene expression by aldosterone in vascular smooth muscle cells.

Na-K-adenosinetriphosphatase (ATPase) activity profoundly influences vascular cell excitability, contractility, and volume regulation. The recent finding of mineralocorticoid hormone receptors in vascular tissue suggests the possibility that Na-K-ATPase gene expression in vascular tissue is regulated by the mineralocorticoid aldosterone. In this study, we investigated Na-K-ATPase gene expression by aldosterone in cultured rat vascular smooth muscle cells (VSMC). Na-K-ATPase alpha 1- and beta 1-isoform mRNAs, but not alpha 2- and alpha 3-isoform mRNAs, were expressed in cultured rat VSMC. Aldosterone caused a 2.3-fold increase in the alpha 1 mRNA and a 4.7-fold increase in the beta 1 mRNA accumulation with peak elevations at 24 and 6 h, respectively. Aldosterone induced the alpha 1 mRNA expression at physiological concentrations (half-maximum effective concentration = 2-3 nM), consistent with the binding of aldosterone to mineralocorticoid hormone receptors. The augmented alpha 1 mRNA expression by aldosterone was associated with a twofold increase in the alpha 1-subunit protein accumulation. Pretreatment of VSMC with cycloheximide caused a 10-fold increase in the alpha 1 mRNA expression, and the aldosterone-mediated alpha 1 mRNA accumulation was not observed in the presence of cycloheximide. Transfection experiments with the luciferase reporter gene revealed that aldosterone response sequences are located within the 5'-flanking regions of the alpha 1-isoform gene. These data demonstrate that the mineralocorticoid aldosterone directly stimulates Na-K-ATPase gene expression and protein accumulation in VSMC.