Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans

Alkaliphilic Bacillus sp. AH-101 was characterized in terms of physiological and biochemical characteristics, and 16S rDNA sequence homology and DNA–DNA hybridization analyses were performed. Phylogenetic analysis of strain AH-101 based on comparison of 16S rDNA sequences revealed that this strain is closely related to Bacillus halodurans. DNA–DNA hybridization of AH-101 and related Bacillus reference strains showed that the highest level of DNA–DNA relatedness (88%) was found between strain AH-101 and the B. halodurans type strain (DSM497). Our findings demonstrate that strain AH-101 is a member of the species B. halodurans. Received: June 10, 1999 / Accepted: August 6, 1999     

Enzymatic properties and nucleotide and amino acid sequences of a thermostable beta-agarase from the novel marine isolate, JAMB-A94

A gene, agaA, for a novel beta-agarase from the marine bacterium JAMB-A94 was cloned and sequenced. The 16S rDNA of the isolate had the closest match, of only 94.8% homology, with that from Microbulbifer salipaludis JCM11542(T). The agaA gene encoded a protein with a calculated molecular mass of 48,203 Da. The deduced amino acid sequence showed 37-66% identity to those of known agarases in glycoside hydrolase family 16. A carbohydrate-binding module-like amino acid sequence was found in the C-terminal region. The recombinant enzyme was hyper-produced extracellularly when Bacillus subtilis was used as a host. The purified enzyme was an endo-type beta-agarase, yielding neoagarotetraose as the main final product. It was very thermostable up to 60 degrees C. The optimal pH and temperature for activity were around 7.0 and 55 degrees C respectively. The activity was not inhibited by EDTA (up to 100 mM) and sodium dodecyl sulfate (up to 30 mM).     

Two morphological types of cell appendages on a strongly adhesive bacterium, Acinetobacter sp. strain Tol 5

Two morphological types of appendages, an anchor-like appendage and a peritrichate fibril-type appendage, have been observed on cells of an adhesive bacterium, Acinetobacter sp. strain Tol 5, by use of recently developed electron microscopic techniques. The anchor extends straight to the substratum without branching and tethers the cell body at its end at distances of several hundred nanometers, whereas the peritrichate fibril attaches to the substratum in multiple places, fixing the cell at much shorter distances.     

Coordinated gastric and sphincter motility evoked by intravenous CCK-8 as monitored by ultrasonomicrometry in rats

Gastric and sphincter motility evoked by intravenous injection of CCK-8 were investigated in urethane-anesthetized rats. Digital ultrasonomicrometry was used to monitor pyloric (PYL), antral (ANT), corpus (COR), and lower esophageal sphincter (LES) movements while simultaneously measuring intragastric pressure (IGP) and, in some experiments, subdiaphragmatic intraesophageal pressure (sIEP). Intracrystal distances (ICD) were measured continuously between pairs of piezoelectric crystals affixed to the serosa of PYL, ANT, COR (circular and longitudinal), and LES. Consecutive intravenous injections of CCK-8 (0.3, 1, and 3 microg/kg) at 30-min intervals caused dose-dependent simultaneous tonic contractions of PYL and ANT, LES opening, and drops in IGP with peak changes at 3 microg/kg of -17.9 +/- 2.1, -7.7 +/- 2.5, 6.5 +/- 1.4, and -29.2 +/- 3.8%, respectively, whereas intravenous saline had no effect. Rhythmic contractile activity was inhibited by CCK-8. COR responses were not significantly different from vehicle controls for most metrics, and the direction of response for circular COR varied between preparations, although not for repeated trials in a single preparation. During the LES response to CCK-8, sIEP rose in parallel with drops in IGP, indicating formation of a common cavity. Recovery of LES ICD after intravenous CCK occurred more rapidly than recovery of PYL ICD, suggesting the importance of preventing simultaneous patency of gastroesophageal and gastroduodenal passages. The CCK(A) receptor antagonist devazepide (500 microg/kg intravenous) inhibited motion responses evoked by intravenous CCK-8. These data revealed CCK-8-induced gastric and sphincter activity consistent with retropulsion of gastric content.     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.     

In vivo visualization of subendocardial arteriolar response in renovascular hypertensive hearts

Time-sequential responses to endothelium-dependent and -independent vasodilators and angiotensin-converting enzyme (ACE) inhibitors were studied in the subendocardial arterioles (Endo) of canine renovascular hypertension (HT) compared with subepicardial arterioles (Epi; both <120 microm) by charge-coupled device intravital microscope. Vascular responses to acetylcholine, papaverine, and cilazaprilat were compared between normotensive (NT) and HT dogs [4 wk and 12 wk of HT (4wHT and 12wHT)]. The acetylcholine-induced vasodilation of Endo in both 4wHT and 12wHT was smaller than that of NT (both P < 0.01 vs. 4wHT and 12wHT), and that of Epi was smaller than that of NT only in 12wHT (P < 0.05). The papaverine-induced vasodilation of Endo, but not Epi, was impaired only in 12wHT (both P < 0.01 vs. NT and 4wHT). Vasodilation by cilazaprilat remained unchanged at 4wHT and 12wHT in both Epi and Endo. In conclusion, at the early stage, the endothelium-dependent response of Endo was impaired, whereas at the later stage, the endothelium-dependent and -independent responses of Endo and the endothelium-dependent response of Epi were impaired. However, the vasodilatory responses to the ACE inhibitor were maintained in both Endo and Epi of HT.     

Aichi virus leader protein is involved in viral RNA replication and encapsidation

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.     

Isolation of a highly copper-tolerant yeast, Cryptococcus sp., from the Japan Trench and the induction of superoxide dismutase activity by Cu2+

Thirteen yeast strains were isolated from deep-sea sediment samples collected at a depth of 4500 m to 6500 m in the Japan Trench. Amongst them, strain N6 possessed high tolerance against Cu²⁺ and could grow on yeast extract/peptone/dextrose/agar containing 50 mM CuSO₄. Analysis of the 18S rDNA sequence indicates strain N6 belongs to the genus Cryptococcus. In contrast, the type strain of C. albidus, a typical marine yeast Rhodotorula ingeniosa and Saccharomyces cerevisiae did not grow at high concentrations of CuSO₄. Superoxide dismutase (SOD) catalyzes the scavenging of superoxide radicals. The activity of SOD in cell extract of strain N6 was very weak (<1 mU g^–1 total protein) when the strain was grown in the absence of CuSO₄. However, the activity was stimulated (25.8 mU g^–1 total protein) when cells were grown with 1 mM CuSO₄ and further enhanced to 110 mU g^–1 total protein with 10 mM CuSO₄. Catalase activity was increased only 1.4 or 1.1-fold with 1 mM or 10 mM CuSO₄ in the growth medium, respectively. These results suggest that SOD may have a role in the defensive mechanisms against high concentrations of CuSO₄ in strain N6.     

Isolation and metabolic characteristics of previously uncultured members of the order aquificales in a subsurface gold mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.