Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

Highly alkaline pectate lyase Pel-4A from alkaliphilic Bacillus sp. strain P-4-N: its catalytic properties and deduced amino acid sequence

The gene for a highly alkaline pectate lyase, Pel-4A, from alkaliphilic Bacillus sp. strain P-4-N was cloned, sequenced, and overexpressed in Bacillus subtilis cells. The deduced amino acid sequence of the mature enzyme (318 amino acids, 34 805 Da) showed moderate homology to those of known pectate lyases in the polysaccharide lyase family 1. The purified recombinant enzyme had an isoelectric point of pH 9.7 and a molecular mass of 34 kDa, and exhibited a very high specific activity compared with known pectate lyases reported so far. The enzyme activity was stimulated 1.6 fold by addition of NaCl at an optimum of 100 mM. When Pel-4A was stored at 50°C for 60 h, striking stabilization by 100 mM NaCl was observed in a pH range from 5 to 11.5, whereas it was stable only around pH 11 in the absence of NaCl. Received: June 10, 2000 / Accepted: October 3, 2000     

Cloning of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from terpenoid antibiotic-producing Streptomyces strains

We have isolated a mutant lacking 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity from a terpenoid antibiotic (terpentecin) producer, Streptomyces griseolosporeus MF730-N6, which uses both the mevalonate and nonmevalonate pathways for the formation of isopentenyl diphosphate, by screening terpentecin non-producing mutants. Terpentecin is known to be synthesized via the mevalonate pathway. The gene encoding HMG-CoA reductase (hmgg) was cloned and identified by complementation of the mutant, using a self-cloning system developed in this study for strain MF730-N6. The corresponding hmgs gene for HMG-CoA reductase was also cloned from Streptomyces sp. KO-3988, which produces the terpenoid antibiotic furaquinocin. Sequence analysis of hmgg and hmgs showed that both genes encode polypeptides of 353 amino acids which are 84% identical to each other. A search of protein sequence databases revealed that both gene products were also similar to HMG-CoA reductases from a variety of other organisms, including Streptomyces sp. CL190 (hmgg is 89% and hmgs 85% identical to its CL190 homolog), sea urchin (40.3 and 40.5%), German cockroach (37.6 and 38.4%), and Camptotheca acuminata (39.7 and 40.8%). Received: 17 May 1999 / Accepted: 10 September 1999     

Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil

Free-living, aerobic, copiotrophic ultramicrobacteria (UMB) that passed through a 0.45 &mgr;m membrane filter and had a cell volume of less than 0.3 &mgr;m(3) were isolated from polluted urban soil by using both the direct plating method and the membrane-filter enrichment technique. The efficiency of recovering UMB from the soil was much higher in the latter method than in the former. All of the UMB isolates grew well with a doubling time of less than 6 h either in a complex nutrient medium or a chemically defined medium. The average cell volumes of the UMB isolates, as measured by scanning electron microscopy and epifluorescent microscopy with an image analysis, ranged from 0.07 to 0.22 &mgr;m(3). The cell size was larger at the exponential phase of growth than at the stationary growth stage in general. Ultrathin-section electron microscopy of representatives of the UMB isolates showed that they had complete cell wall structures like typical Gram-negative or -positive bacteria. Phenotypic studies and phylogenetic analyses on the basis of 16S rDNA sequences showed that the UMB isolates were classified into three major groups, the beta and gamma subdivisions of the Proteobacteria and the Actinobacteria (the high G+C DNA group of Gram-positives). However, none of these isolates were assigned to any previously known species. These results demonstrate that free-living, relatively fast-growing, copiotrophic UMB strains undescribed so far are widely distributed in terrestrial environments, including urban soil.     

Simple analysis of local anaesthetics in human blood using headspace solid-phase microextraction and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring

A simple method for analysis of five local anaesthetics in blood was developed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry–electron impact ionization selected ion monitoring (GC–MS–EI-SIM). Deuterated lidocaine (d₁₀-lidocaine) was synthesized and used as a desirable internal standard (I.S.). A vial containing a blood sample, 5 M sodium hydroxide and d₁₀-lidocaine (I.S.) was heated at 120°C. The extraction fiber of the SPME system was exposed for 45 min in the headspace of the vial. The compounds adsorbed on the fiber were desorbed by exposing the fiber in the injection port of a GC–MS system. The calibration curves showed linearity in the range of 0.1–20 μg/g for lidocaine and mepivacaine, 0.5–20 μg/g for bupivacaine and 1–20 μg/g for prilocaine in blood. No interfering substances were found, and the time for analysis was 65 min for one sample. In addition, this proposed method was applied to a medico–legal case where the cause of death was suspected to be acute local anaesthetics poisoning. Mepivacaine was detected in the left and right heart blood samples of the victim at concentrations of 18.6 and 15.8 μg/g, respectively.     

Fermentation of soybean meal withAspergillus usamii improves zinc availability in rats

Soybean meal was fermented withAspergillus usamii to improve zinc availability through the degradation of phytic acid. Rats fed a diet containing fermented soybean meal showed greater femoral zinc than did animals fed a diet containing regular soybean meal. Zinc solubility in the small intestine was higher in the rats fed fermented soybean meal than in the rats fed regular soybean meal. These results suggested that fermentation withAspergillus usamii improved zinc availability in dietary soybean meal, which was induced by the increase of zinc solubility in the small intestine. Adding the same amount of phytate that was contained in the regular soybean mealbased diet did not affect the amount of zinc present in rats fed a fermented soybean meal-based diet with sodium phytate. Phytase activity was found in fermented soybean meal, and this activity may degrade added phytate in fermented soybean meal-based diet.     

Low-specificity l-threonine aldolase of Pseudomonas sp. NCIMB 10558: purification, characterization and its application to β-hydroxy-α-amino acid synthesis

Low-specificity l-threonine aldolase, catalyzing the reversible cleavage/condensation reaction between l-threonine/l-allo-threonine and glycine plus acetaldehyde, was purified to homogeneity from Pseudomonas sp. NCIMB 10558. The enzyme has an apparent molecular mass of approximately 145 kDa and consists of four identical subunits with a molecular mass of 38 kDa. The enzyme, requiring pyridoxal- 5′-phosphate as a coenzyme, is strictly l-specific at the α position, whereas it can not distinguish between threo and erythro forms at the β position. Besides the reversible cleavage/condensation of threonine, the enzyme also catalyzes the reversible interconversion between glycine plus various aldehydes and l-β-hydroxy-α-amino acids, including l-β-(3,4-dihydroxyphenyl)serine, l-β-(3,4-met‐hylenedioxyphenyl)serine and l-β-phenylserine, providing a new route for the industrial production of these important amino acids. Received: 10 November 1997 / Received revision: 7 January 1998 / Accepted 30 January 1998