INFLUENCE OF TEMPERATURE AND HYDROGEN ION CONCENTRATION UPON THE SPORE CYCLE OF BACILLUS SUBTILIS

1. At 5°C. no germination took place. 2. At 25°C. and at 37°C. germination occurs if the hydrogen ion concentration of the broth is kept between pH 5 and pH 10, but not at higher or lower pH values. 3. The completion of the spore cycle likewise requires a hydrogen ion concentration between pH 5 and pH 10. 4. The spores can germinate when the pH value is 10, although after germination the vegetative cells multiply only to a very slight extent and soon pass into spores. 5. The slight growth and multiplication of vegetative cells in broth of pH 10 suggest that the formation of endospores in this medium must be caused largely by the unfavorable reaction of the medium rather than by the accumulation of metabolic products. 6. Automatic adjustment of the medium seems to play a rôle in the completion of the spore cycle. 7. The results are not only of theoretical importance but they have a practical application to the preservation of food by canning and by other methods.     

Purification of the Ca2+-and Mg2+-requiring ATPase from rat brain synaptic plasma membrane.

A Ca2+-ATPase (Ca2+- and Mg2+-requiring ATPase) was purified from a synaptic plasma-membrane fraction of rat brain. This enzyme had properties similar to those of plasma-membrane Ca2+-ATPases from other organs: its splitting of ATP was dependent on both Ca2+ and Mg2+, it bound in a Ca2+-dependent fashion to calmodulin-Sepharose and it cross-reacted with specific antibodies raised against human erythrocyte-membrane Ca2+-ATPase. It had an apparent Mr of 138 000, similar to those of plasma-membrane ATPases from human erythrocyte and from dog heart sarcolemma. Previous high-Ca2+-affinity ATPases observed in brain had Mr 100 000; in at least one case, such an ATPase probably represented a different type of enzyme, derived from coated vesicles.     

Chromosomal Localization of the Human and Mouse Hyaluronan Synthase Genes

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designatedHAS1, HAS2,andHAS3in humans andHas1, Has2,andHas3in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to theStreptococcus pyogenesHA synthase, HasA. Furthermore, expression of any oneHASgene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the threeHASgenes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes.HAS1was localized to the human chromosome 19q13.3–q13.4 boundary andHas1to mouse Chr 17.HAS2was localized to human chromosome 8q24.12 andHas2to mouse Chr 15.HAS3was localized to human chromosome 16q22.1 andHas3to mouse Chr 8. The map position forHAS1reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17.HAS2mapped outside the predicted critical region delineated for the Langer–Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.     

Distribution of alginate and cellulose and regulatory role of calcium in the cell wall of the brown alga <Emphasis Type="Italic">Ectocarpus siliculosus</Emphasis> (Ectocarpales,Phaeophyceae)

Main conclusion

This work investigated a correlation between the three-dimensional architecture and compound–components of the brown algal cell wall. Calcium greatly contributes to the cell wall integrity.Brown algae have a unique cell wall consisting of alginate, cellulose, and sulfated polysaccharides. However, the relationship between the architecture and the composition of the cell wall is poorly understood. Here, we investigated the architecture of the cell wall and the effect of extracellular calcium in the sporophyte and gametophyte of the model brown alga, Ectocarpus siliculosus (Dillwyn) Lyngbye, using transmission electron microscopy, histochemical, and immunohistochemical studies. The lateral cell wall of vegetative cells of the sporophyte thalli had multilayered architecture containing electron-dense and negatively stained fibrils. Electron tomographic analysis showed that the amount of the electron-dense fibrils and the junctions was different between inner and outer layers, and between the perpendicular and tangential directions of the cell wall. By immersing the gametophyte thalli in the low-calcium (one-eighth of the normal concentration) artificial seawater medium, the fibrous layers of the lateral cell wall of vegetative cells became swollen. Destruction of cell wall integrity was also induced by the addition of sorbitol. The results demonstrated that electron-dense fibrils were composed of alginate-calcium fibrous gels, and electron negatively stained fibrils were crystalline cellulose microfibrils. It was concluded that the spatial arrangement of electron-dense fibrils was different between the layers and between the directions of the cell wall, and calcium was necessary for maintaining the fibrous layers in the cell wall. This study provides insights into the design principle of the brown algal cell wall.

     

Identification of tranilast-binding protein as 36-kDa microfibril-associated glycoprotein by drug affinity chromatography, and its localization in human skin

To elucidate the molecular mechanism involved in the suppression of keloids and hypertrophic scars by tranilast, we investigated the target protein of tranilast in bovine skin and aorta. A specific tranilast-binding protein was isolated from both tissues by drug affinity chromatography and was identified as 36-kDa microfibril-associated glycoprotein (36-kDa MAGP). Binding of 36-kDa MAGP to tranilast seemed to be specific since 36-kDa MAGP could be eluted from the drug affinity column by tranilast itself and also binding of 36-kDa MAGP to other anti-allergy drugs (amlexanox and cromolyn) is significantly weaker than that to tranilast. Light and electron microscopic immunohistochemistry detected the protein at the periphery of elastic fibers in normal human skin. In hypertrophic scar tissue, however, 36-kDa MAGP was located on small bundles of microfibrils. These findings provide support for the concept that elastogenesis occurs in scar tissue and 36-kDa MAGP might be one of the targets for tranilast.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

PYRENOID FORMATION ASSOCIATED WITH THE CELL CYCLE IN THE BROWN ALGA,SCYTOSIPHON LOMENTARIA (SCYTOSIPHONALES,PHAEOPHYCEAE)1

Vegetative cells of the brown alga Scytosiphon lomentaria (Lyngbye) Link characteristically have only one chloroplast with a prominent protruding pyrenoid, whereas zygotes have both paternal and maternal chloroplasts. In zygotes, before cell and chloroplast division, each chloroplast has an old and a new pyrenoid. In this study, we raised a polyclonal antibody to RUBISCO and examined the distribution of RUBISCO by immunofluorescence microscopy, focusing on new pyrenoid formation in vegetative cells of gametophytes and zygotes in Scytosiphon. In interphase, only one old pyrenoid was positively indicated by anti‐RUBISCO antibody in vegetative cells of gametophytes. From mid‐S phase, small fluorescence aggregates reflecting RUBISCO localization started to appear at stroma positions other than adjacent to the old protruding pyrenoid. The fluorescent spots eventually coalesced into a protrusion into the adjacent cytoplasm. We also used inhibitors to clarify the relationship between the cell cycle and new pyrenoid formation, using zygotes after fertilization. When DNA replication was blocked by aphidicolin, new pyrenoid formation was also inhibited. Washing out aphidicolin permitted new pyrenoid formation with the progression of the cell cycle. When mitosis was prolonged by nocodazole, which disrupted the spindle microtubules, the fluorescent masses indicating RUBISCO localization continued to increase when compared with pyrenoid formation in untreated zygotes. During treatment with chloramphenicol, mitosis and cytokinesis were completed. However, there was no occurrence of new RUBISCO localization within the chloroplast stroma beyond the old pyrenoid. From these observations, it seems clear that new pyrenoid formation in the brown alga Scytosiphon depends on the cell cycle.     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Metabolic activities of partially degenerated hypertrophic chondrocytes: gene expression of hyaluronan synthases

Ultrastructural aspects of hypertrophic chondrocytes in hamster and mouse epiphysial cartilage were examined in relation to their metabolic activities. With the hypertrophic change, cytoplasmic vacuolization proceeded leaving the partially intact endoplasmic reticulum (ER). In the hypertrophic cells, cytoplasmic hyaluronan was stained with the biotinylated hyaluronan-binding region (b-HABR) of aggrecan, and mRNAs of hyaluronan synthase (Has 1, Has 2 and Has 3) were detected by in situ hybridization. When the epiphysial cartilage was cultured in the presence of 35S, 3H-GlcNAc, 3H-proline or 14C-palmitic acid, vacuolated late hypertrophic chondrocytes were labeled with these radioactive precursors. The evidence indicates that late hypertrophic chondrocytes are metabolically active, which appears to be essential for the enlargement of chondrocytes.     

Influence of lactic acid bacteria on longevity of Caenorhabditis elegans and host defense against salmonella enterica serovar enteritidis

This study aimed to develop a convenient model to investigate the senescence of host defenses and the influence of food and nutrition. A small soil nematode, Caenorhabditis elegans, was grown for 3 days from hatching on a lawn of Escherichia coli OP50 as the normal food source, and subsequently some of the nematodes were fed lactic acid bacteria (LAB). The life spans of worms fed LAB were significantly longer than the life spans of those fed OP50. To investigate the effect of age on host defenses, 3- to 7-day-old worms fed OP50 were transferred onto a lawn of Salmonella enterica serovar Enteritidis for infection. The nematodes died over the course of several days, and the accumulation of salmonella in the intestinal lumen suggested that the worms were infected. The 7-day-old worms showed a higher death rate during the 5 days after infection than nematodes infected at the age of 3 days; no clear difference was observed when the worms were exposed to OP50. We then investigated whether the LAB could exert probiotic effects on the worms' host defenses and improve life span. Seven-day-old nematodes fed LAB from the age of 3 days were more resistant to salmonella than worms fed OP50 until they were infected with salmonella. This study clearly showed that LAB can enhance the host defense of C. elegans and prolong life span. The nematode appears to be an appropriate model for screening useful probiotic strains or dietetic antiaging substances.