Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

Cytotoxic cycloartane-type triterpenes from Combretum quadrangulare

Seven novel cycloartane-type triterpenes were isolated from Combretum quadrangulare, and their structures were elucidated on the basis of spectral analysis. All these compounds were tested for their cytotoxicity against murine colon 26-L5 carcinoma cells. Methyl quadrangularate B (2) and methyl quadrangularate D (4) exhibited potent cytotoxicity having ED₅₀ values 9.54 and 5.42 μM, respectively.     

Bcl-2 prevents hypoxia/reoxygenation-induced cell death through suppressed generation of reactive oxygen species and upregulation of Bcl-2 proteins

The function of bcl-2 in preventing cell death is well known, but the mechanisms whereby bcl-2 functions are not well characterized. One mechanism whereby bcl-2 is thought to function is by alleviating the effects of oxidative stress upon the cell. To examine whether Bcl-2 can protect cells against oxidative injury resulting from post-hypoxic reoxygenation (H/R), we subjected rat fibroblasts Rat-1 and their bcl-2 transfectants b5 to hypoxia (5% CO2, 95% N2) followed by reoxygenation (5% CO2, 95% air). The bcl-2 transfectants exhibited the cell viability superior to that of their parent non-transfectants upon treatment with reoxygenation after 24-, 48-, or 72-h hypoxia, but not upon normoxic serum-deprivation or upon serum-supplied hypoxic treatment alone. Thus bcl-2 transfection can prevent cell death of some types, which occurred during H/R but yet not appreciably until termination of hypoxia. The time-sequential events of H/R-induced cell death were shown to be executed via (1) reactive oxygen species (ROS) production at 1-12 h after H/R, (2) activation of caspases-1 and -3, at 1-3 h and 3-6 h after H/R, respectively, and (3) loss of mitochondrial membrane potential (DeltaPsi) at 3-12 h after H/R. These cell death-associated events were prevented entirely except caspase-1 activation by bcl-2 transfection, and were preceded by Bcl-2 upregulation which was executed as early as at 0-1 h after H/R for the bcl-2 transfectants but not their non-transfected counterpart cells. Thus upregulation of Bcl-2 proteins may play a role in prevention of H/R-induced diminishment of cell viability, but may be executed not yet during hypoxia itself and be actually operated as promptly as ready to go immediately after beginning of H/R, resulting in cytoprotection through blockage of either ROS generation, caspase-3 activation, or DeltaPsi decline.     

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.     

Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88

MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.