Modification of GerQ reveals a functional relationship between Tgl and YabG in the coat of Bacillus subtilis spores

Here we describe the functional relationship between YabG and transglutaminase (Tgl), enzymes that modify the spore coat proteins of Bacillus subtilis. In wild-type spores at 37 degrees C, Tgl mediates the crosslinking of GerQ into higher molecular mass forms; however, some GerQ multimers are found in tgl mutant spores, indicating that Tgl is not essential. Immunoblotting showed that spores isolated from a yabG mutant after sporulation at 37 degrees C contain only very low levels of GerQ multimers. Heat treatment for 20 min at 60 degrees C, which maximally activates the enzymatic activity of Tgl, caused crosslinking of GerQ in isolated yabG spores but not in tgl/yabG double-mutant spores. In addition, the germination frequency of the tgl/yabG spores in the presence of l-alanine with or without heat activation at 60 degrees C was lower than that of wild-type spores. These findings suggest that Tgl cooperates with YabG to mediate the temperature-dependent modification of the coat proteins, a process associated with spore germination in B. subtilis.     

Mechanism of p53-dependent apoptosis induced by 3-methylcholanthrene: involvement of p53 phosphorylation and p38 MAPK.

Polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) cause untoward effects including carcinogenesis. Here we investigated the effect of MC on apoptosis. MC induced apoptosis, preceded by serine 15 phosphorylation and accumulation of p53. MC failed to cause apoptosis in p53-deficient MG63 cells, whereas ectopic expression of p53 in MG63 cells restored the response to MC. Therefore, MC-induced apoptosis was dependent on p53. MC also activated p38 mitogen-activated protein kinase (MAPK) at 16-24 h. Accumulation of p53 and p53 phosphorylated at serine 15 was not changed by SB203580, a specific inhibitor of p38 MAPK or overexpression of a dominant negative mutant of p38 MAPK at 8 h after MC treatment, whereas the accumulation was suppressed at 24 h. These results suggest that MC induces accumulation and phosphorylation of p53 via a p38 MAPK-independent (early) and p38 MAPK-dependent (late) pathway. SB203580 repressed MC-induced apoptosis. MC induced p38 MAPK activation in p53 expressing cells but not in p53-deficient cells, indicating that the p38 MAPK activation was dependent on early p53 activation. The current study shows that both p53 and p38 MAPK activation are required for MC-induced apoptosis and provides a novel model of a functional regulation between p53 and p38 MAPK in chemical stress-induced apoptosis.     

Cloning of a novel gene yrbB, encoding a protein located in the spore integument of Bacillus subtilis

A DNA fragment (2.7 kbp) containing three deduced open reading frames, orf1, orf2 and orf3 (partial sequence), was isolated from the genomic library of Bacillus subtilis using an antiserum raised against spore integument, and was sequenced. orf2 was 519 nucleotides long and encoded a protein of 172 amino acids with a predicted molecular size of 19552, corresponding to the protein which reacted with the antiserum. Immunoelectron microscopic observation indicated that YrbB, the product of orf2 , was located within the spore integument, mainly in the cortex layer with a part in the inner region of the coat layer.     

Structural differences in the motor domain of temperature-associated myosin heavy chain isoforms from grass carp fast skeletal muscle

We determined coding sequences for three types of grass carp myosin subfragment-1 (S1) heavy chain by extending 5′-regions of the three known genes encoding light meromyosin isoforms (10 °C, intermediate and 30 °C types). The primary structures of these three S1 heavy chain isoforms showed 81.4%, 81.2%, and 97.8% identities between the 10 °C and intermediate types, between the 10 °C and 30 °C types, and between the intermediate and 30 °C types, respectively. Isoform-specific differences were clearly observed between the 10 °C type and the other two types in 97 amino acid residues. Furthermore, among these amino acid mutations, 51 mutations occurred at the conserved residue sites of S1 heavy chain from fish and homoiotherm. Additionally, the 10 °C type showed striking differences compared with the other two types in the two surface loops, loop 1 located near the ATP-binding pocket and loop 2, which is one of the actin-binding sites, suggesting that such structural differences possibly affect their motor functions. Interestingly, this 10 °C-type myosin heavy chain isolated from adult grass carp skeletal muscle was surprisingly similar to the embryonic fast-type myosin heavy chain from juvenile silver carp in the structure of S1 heavy chain, indicating that it may also function as embryonic fast-type myosin heavy chain in juvenile stage.     

Solubilization of coat protein from Bacillus thiaminolyticus spores.

Solubilization of spore coat protein of Bacillus thiaminolyticus was investigated using various reagents, and partial characterization of solubilized protein was carried out. Five per cent of the sodium dodecyl sulphate (SDS) treatment was the most effective for solubilization of coat protein, and 5% SDS + 8 M urea and 0.06 N NaOH were also useful. Acrylamide gel disc electrophoresis indicated that the SDS-soluble fraction mainly consists of a single band of protein and its molecular weight was estimated at about 15,000. The SDS+ urea-soluble fraction comprised two proteins with a molecular weight of 14,500 and 32,000, and an alkali-soluble fraction of 12,000 and 25,000 respectively.     

The ultrastructure of the otolithic membrane and otolith in the juvenile mummichog,Fundulus heteroclitus

The sagitta otolithic membrane of Fundulus heteroclitus consists of two different zones. A structured zone (gelatinous layer), which usually exhibits a reticulated or honeycomb-like architecture, is composed of tightly arranged fibrous material and covers only the sensory region of the macula. The gelatinous layer extends from the otolith surface to the tips of the sensory hairs, and probably functions primarily as a mechanoreceptor. The arrangement of this zone is closely associated with specific overlying structural features of the otolith surface and may also influence the pattern of mineral deposition to some degree. A nonstructured zone (subcupular meshwork) consists of fibers in very loose networks and covers both sensory and nonsensory regions of the macula. Over the sensory region, some of this fibrous material extends from the epithelial surface, through pores in the gelatinous layer, to the surface of the overlying otolith. In the nonsensory region, fibers of the subcupular meshwork are relatively more numerous and extend around the peripheral margin of the otolith. Evidence is presented which suggests that the fibrous material of the subcupular meshwork is incorporated into the otolith as an organic matrix constituent. New aspects on the ultrastructure of the otolith are presented and discussed.