Genes involved in SkfA killing factor production protect a Bacillus subtilis lipase against proteolysis

Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.     

Aberrant glycosylation of alpha-dystroglycan causes defective binding of laminin in the muscle of chicken muscular dystrophy

Dystroglycan is a central component of dystrophin-glycoprotein complex that links extracellular matrix and cytoskeleton in skeletal muscle. Although dystrophic chicken is well established as an animal model of human muscular dystrophy, the pathomechanism leading to muscular degeneration remains unknown. We show here that glycosylation and laminin-binding activity of alpha-dystroglycan (alpha-DG) are defective in dystrophic chicken. Extensive glycan structural analysis reveals that Galbeta1-3GalNAc and GalNAc residues are increased while Siaalpha2-3Gal structure is reduced in alpha-DG of dystrophic chicken. These results implicate aberrant glycosylation of alpha-DG in the pathogenesis of muscular degeneration in this model animal of muscular dystrophy.     

Characterization of the DNA and structural proteins of entomopoxviruses from Melanoplus sanguinipes,Arphia conspirsa, and Phoetaliotes nebrascensis (Orthoptera)

Entomopoxvirus (EPV) occlusion bodies were isolated from virus infected nymphs of the grasshoppers Melanoplus sanguinipes, Arphia conspirsa, and Phoetaliotes nebrascensis. Separation of the viral structural proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave unique protein patterns for each of the three viruses. An occlusion body protein of approximately 100,000 MW was isolated from each virus. Cleavage of viral DNA with HinddIII and BamHI restriction endonucleases and separation of the fragments by agarose gel electrophoresis gave different DNA fragment patterns for each of the three entomopoxviruses. Molecular weight estimates of 120 × 10⁶ for M. sanguinipes EPV DNA, 129 × 10⁶ for A. conspirsa EPV DNA, and 125 × 10⁶ for P. nebrascensis EPV DNA were calculated from the sizes of the viral DNA fragments. Approximately 55% base sequence homology was detected by Southern hybridization of α-³²P-labeledM. sanguinipes EPV DNA with P. nebrascensis DNA. No base sequence homology was detected by Southern hybridization of labeled M. sanguinipes EPV DNA to Othnonius batesi EPV DNA (Coleoptera), Amsacta moorei EPV DNA (Lepidoptera), Euxoa auxiliaris EPV DNA (Lepidoptera), and vaccinia virus DNA fragments.     

Interactions between homopolymeric amino acids (HPAAs)

Many human proteins contain consecutive amino acid repeats, known as homopolymeric amino acid (HPAA) tracts. Some inherited diseases are caused by proteins in which HPAAs are expanded to an excessive length. To this day, nine polyglutamine-related diseases and nine polyalanine-related diseases have been reported, including Huntington's disease and oculopharyngeal muscular dystrophy. In this study, potential HPAA-HPAA interactions were examined by yeast two-hybrid assays using HPAAs of approximately 30 residues in length. The results indicate that hydrophobic HPAAs interact with themselves and with other hydrophobic HPAAs. Previously, we reported that hydrophobic HPAAs formed large aggregates in COS-7 cells. Here, those HPAAs were shown to have significant interactions with each other, suggesting that hydrophobicity plays an important role in aggregation. Among the observed HPAA-HPAA interactions, the Ala28-Ala29 interaction was notable because polyalanine tracts of these lengths have been established to be pathogenic in several polyalanine-related diseases. By testing several constructs of different lengths, we clarified that polyalanine self-interacts at longer lengths (>23 residues) but not at shorter lengths (six to approximately 23 residues) in a yeast two-hybrid assay and a GST pulldown assay. This self-interaction was found to be SDS sensitive in SDS-PAGE and native-PAGE assays. Moreover, the intracellular localization of these long polyalanine tracts was also observed to be disturbed. Our results suggest that long tracts of polyalanine acquire SDS-sensitive self-association properties, which may be a prerequisite event for their abnormal folding. The misfolding of these tracts is thought to be a common molecular aspect underlying the pathogenesis of polyalanine-related diseases.     

Participation of CD14 in the Phagocytosis of Smooth-Type Salmonella typhimurium by the Macrophage-Like Cell Line,J774.1

The role of CD14 in the phagocytosis and killing of microorganisms was investigated using macrophage-like cell lines, CD14-positive J774.1 cells and CD14-negative mutant J7.DEF3 cells derived from J744.1 cells. The cells were infected with Salmonella typhimurium organisms of the smooth (S)-form LT2, mutant rough (R)-form TV148 or Staphylococcus aureus 248βH. At 30 or 180 min incubation, the cells were washed and disrupted. Colony-forming units (CFUs) liberated from the disrupted cells were determined by quantitative cultivation, and the phagocytic index and killing rate were calculated. Both the phagocytic index and killing rate of J774.1 cells against LT2 organisms were greater than those of J7.DEF.3 cells. However, the index and rate of J774.1 cells against TV148 and 248βH organisms were similar to those of the J7.DEF.3 cells. The phagocytosis of LT2 organisms by J774.1 cells was partially inhibited by S-form LPS (S-LPS) and anti-CD14 antibody, but not by R-chemotype LPS (R-LPS). These results suggest that CD14 participates in the phagocytosis of S-form Salmonella.     

Characteristics of postural sway in older adults standing on a soft surface

Postural responses to challenging situations were studied in older adults as they stood on a foam surface. The experiment was designed to assess the relative contributions made by visual and somatosensory information to the correction of postural sway. Twenty-four subjects, aged 56-83, stood for 20 s on a 1) firm or 2) foam surface with 1) the eyes open or 2) the eyes closed. Centre-of-pressure trajectories under the subjects' feet were measured by using a force platform. A repeated-measure two-way MANCOVA (two surfaces vs. two vision conditions) showed a significant main effect for the surface, but not for the vision. No covariate effect for age was found. Anterior-posterior sway increased in the subjects who were merely standing on the foam surface independent of the vision condition. Medial-lateral sway dramatically increased if the subjects stood on the foam surface with their eyes closed, but not if they stood with their eyes open. These results indicate that older adults rely more on visual information to correct mediolateral postural sway. It appears that the deterioration in visual acuity that occurs with aging may increase the risk of sideway falls, particularly in challenging situations, e.g., when standing on irregular or soft surfaces.     

Electrical perception of the 'death message' in Chara: analysis of K+-sensitive depolarization

Wounding electrical responses were studied in Chara corallina. Specimens comprising two adjoining internodal cells were prepared. When one cell (victim cell) was cut, the other cell (receptor cell) generated four kinds of depolarization: (i) rapid depolarization; (ii) long-lasting depolarization; (iii) action potentials; and (iv) small spikes. In the present study, attention was focused on the long-lasting depolarization. A decrease in the electrical resistance suggested activation of ion channel(s). The duration of the depolarization was sensitive to the external ions. K(+) significantly prolonged the depolarization. On the other hand, Ca(2+), Mg(2+) and Na(+) had a tendency to shorten the duration prolonged by K(+). When a nodal end was continuously flushed with a medium lacking K(+), the depolarization was significantly shortened. Treatment of the nodal end with artificial cell sap for 2 min induced a long-lasting depolarization similar to that induced by cutting the victim cell. These findings suggested the involvement of K(+) released from the victim cell in generating the long-lasting depolarization by the receptor cell.     

Soymorphin-5, a soy-derived μ-opioid peptide, decreases glucose and triglyceride levels through activating adiponectin and PPARα systems in diabetic KKAy mice

Soymorphin-5 (YPFVV) derived from soybean β-conglycinin β-subunit is a μ-opioid agonist peptide having anxiolytic-like activity. Here, we show that soymorphin-5 improves glucose and lipid metabolism after long-term oral administration to KKAy mice, a type 2 diabetes model animal. Soymorphin-5 inhibited hyperglycemia without an increase in plasma insulin levels in KKAy mice. Soymorphin-5 also decreased plasma and liver triglyceride (TG) levels and liver weight, suggesting that soymorphin-5 improved lipid metabolism. Soymorphin-5 increased plasma adiponectin concentration and liver mRNA expression of AdipoR2, a subtype of adiponectin receptor that is involved in stimulating the peroxisome proliferator-activated receptor (PPAR)α pathway and fatty acid β-oxidation. The expressions of the mRNA of PPARα and its target genes acyl-CoA oxidase, carnitine palmitoyltransferase 1 A, and uncoupling protein-2, in the liver were also increased after oral administration of soymorphin-5. Furthermore, des-Tyr-soymorphin-5 (PFVV) without μ-opioid and anxiolytic-like activities did not decrease blood glucose levels in KKAy mice. These results suggest that μ-opioid peptide soymorphin-5 improves glucose and lipid metabolism via activation of the adiponectin and PPARα system and subsequent increases of β-oxidation and energy expenditure in KKAy mice.