An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998     

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.