Surface carbohydrates as recognition determinants in non-opsonic interactions and intracellular viability of group B Streptococcus strains in murine macrophages

Mononuclear cells have been found to play a key role in phagocytosis and eventual killing of group B streptococci (GBS). The rich array of sugars on bacterial surface plus the presence of membrane-associated lectin-receptors on the macrophage suggests that this is a likely means for GBS recognition by these host defense cells. Macrophages have been shown to bind GBS in the absence of serum components. However, participation of carbohydrate moieties in GBS intracellular survival had not been completely elucidated. The aim of this study was to assess the involvement of sugars on adherence and intracellular viability in murine macrophages of GBS serotypes Ia (85147 and 90222 strains), III (80340 and 90356 strains) and V (88641 and 90186 strains) isolated from assymptomatic carriers and patients, respectively. Most isolates showed higher adherence within 2-h incubation. Only 90222-Ia strain exhibited progressive adherence rate until 12-h incubation. All strains showed intracellular viability during first 0.5-h of incubation. Except for 90186-V strain that survived only for 2 h, strains of all serotypes tested were found to survive 24 h into macrophages. Treatments of bacteria by glycosidases inhibited macrophage interaction with GBS strains at varied levels. Neuraminidase inhibited 90-97% adherence and 100% intracellular survival of GBS strains (P<0.0001). Host cell treatments with Rhamnose, N-acetyl-D-glucosaminidase and Fucose (5 mg/ml) inhibited adherence and intracellular viability of GBS strains at varied levels. Removal of GlcNAc residues of invasive GBS isolates enhanced intracellular viability, suggesting that GlcNAc residues may act by intercepting the expression of hidden receptors probably related with invasiveness and survival within macrophages. Lastly, our results demonstrate involvement of sialic acid specific receptors on macrophages and lectinophagocytosis in non-opsonic interaction and survival of GBS invasive isolates.     

Local skin response in mice induced by a single intradermal injection of bacterial lipopolysaccharide and lipid A.

Dermal inflammation and hemorrhagic necrosis induced by bacterial lipopolysaccharide (LPS) and lipid A were studied in mice. In ddY mice, a single intradermal injection of Salmonella typhimurium S-form LPS and lipid A into the abdominal dermis elicited an edematous change due to an increase in local vascular permeability 12 h postinjection, followed by hemorrhagic necrosis from 24 to 72 h. This skin reaction was also induced in a dose-dependent manner by S-form LPS, R-mutant LPS, and lipid A of S. typhimurium and Escherichia coli, but not by polysaccharide from Salmonella S-form LPS. The dermal inflammation-inducing activities of LPS and lipid A were roughly in the following order (from highest to lowest): Re-form LPS, Rc-form LPS and lipid A, Ra-form LPS, and S-form LPS. These results suggest that the lipid A portion of the LPS molecule is responsible for the skin reaction. In C3H/HeN mice, Re-form LPS and lipid A induced the same intensity of skin reaction as that in ddY mice. In C3H/HeJ mice, which have a low response to LPS, Re-LPS and lipid A did not induce any hemorrhagic response but showed a distinct edematous change. Although hemorrhagic necrosis and edematous changes could be explained by quantitative differences in skin lesions, the other possible explanation is that hemorrhagic necrosis and the increase in local vascular permeability are induced by different mechanisms, only one of which depends on the regulation of the lps gene.     

Meu10 is required for spore wall maturation in Schizosaccharomyces pombe

BACKGROUND: Many genes are meiosis and/or sporulation-specifically transcribed during this process. Isolation and analysis of these genes might help us to understand how meiosis and sporulation are regulated. For this purpose, we have isolated a large number of cDNA clones from Schizosaccharomyces pombe whose expression is up-regulated during meiosis. RESULTS: We have isolated meu10+ gene, which encodes 416 amino acids and bears homology to SPS2 of Saccharomyces cerevisiae. A strain whose meu10+ gene has been deleted forms no viable spores. Thin-section electron micrographs showed that the meu10Delta strain has abnormally formed spore walls, and then they disrupt, allowing cytoplasmic material to escape. The Meu10-GFP fusion protein is localized to the spore periphery, thereafter returned to the cytoplasm after sporulation. Meu10-GFP localization to the spore wall was almost normal in the bgs2Delta or chs1Delta mutants that lack 1,3-beta-glucan or chitin, respectively. In contrast, 1,3-beta-glucan is abnormally localized in meu10Delta cells. Meu10 has an N-terminal domain with homology to the mammalian insulin receptor and a C-terminal domain with a transmembrane motif. Mutants whose N-terminal or C-terminal domain was truncated were severely defective for sporulation. CONCLUSIONS: Meu10 is a spore wall component and plays a pivotal role in the formation of the mature spore wall structure.     

Characterization of CO3Ap-collagen sponges using X-ray high-resolution microtomography

For reconstruction and regeneration of hard tissues, scaffold biomaterials with large size pores and high porosity are important, in addition to their roles as supporting frames. To develop a new biodegradable scaffold biomaterial, CO3Ap, which has crystallinity and a chemical composition similar to bone, was synthesized at pH 7.4 and 60 degrees C. Then, the CO3Ap was mixed with a neutralized collagen gel and the CO3Ap-collagen mixtures with different kinds of CO3Ap contents and porosity were lyophilized into sponges. Scanning electron micrography (SEM) observation of CO3Ap-collagen sponges showed favorable pores for cell invasion. Approximately 50-300 microm size pores appeared to continue through the bulk. Higher magnification of the sponge showed a better adhesion between CO3Ap crystals and collagen. X-ray high-resolution microtomography revealed a clear image of the 3D structure of the sponges. The porosity of 0, 70 and 90%(w/w) CO3Ap-collagen sponges was 79.2 +/- 2.8%, 72.6 +/- 2.4% and 48.9 +/- 6.1%, respectively. The 70%(w/w) CO3Ap-collagen sponge appeared to be the most favorable biomaterial from the viewpoint of natural bone properties. Mouse osteoblast MC3T3-E1 cells were cultured in alphaMEM with 10% FCS for 2 weeks. Hematoxylin-eosin staining confirmed osteoblast cells invaded well into the CO3Ap-collagen sponge. These sponges are expected to be used as hard tissue scaffold biomaterials for therapeutic uses.     

Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene (DRD4) with schizophrenia and personality traits

The human dopamine D4 receptor (DRD4) is of major interest in molecular studies of schizophrenia and personality traits. We examined the association of schizophrenia and polymorphisms in the upstream region of the DRD4 gene (−768G>A in the negative modulator region; −521C>T, −376C>T, and −291C>T in the cell type-specific promoter region; and −616C>G between the two regions) in 208 schizophrenic patients and 210 normal controls. No significant difference in genotype and allele frequencies was observed between the two groups, indicating that these polymorphisms do not make a major contribution to the pathogenesis of schizophrenia. We also studied the association of polymorphisms in the upstream region and a 48-bp repeat polymorphism in exon III of the DRD4 gene with personality traits in 173 Japanese individuals who completed the temperament and character inventory (TCI). The −768G>A polymorphism was significantly associated with reward dependence (P = 0.044), while no significant association was observed between novelty seeking and polymorphisms in the upstream region or the exon III repeat polymorphism of the DRD4 gene. Received: August 28, 2000 / Accepted: October 25, 2000     

Prevalence of asthma in adults in Menda town rural-mountain area in Japan]

Several reports have suggested that the prevalence of asthma in adults is currently increasing. However, recent prevalence of asthma has not reported in Japan, especially in rural-mountain areas. To investigate the prevalence of asthma in adults in Japan, we conducted clinical epidemiological research on 5066 inhabitants of Menda town, in a rural-mountain area of Japan. The study population comprised 98.7% of adults in the town, including senior high school students whose age were more than 15 years old. The prevalence of asthma among adults was 3.6%. The ratio of prevalence in males to prevalence in females was 1.44. Peaks prevalences were observed in the age ranges of 15-19 and > 70 years old in males, and 15-19, 40-49 and > 70 years old in females.     

p53蛋白的免疫亲和层析纯化

建立了ｐ５３单克隆抗体ｐＡｂ１８０１的免疫亲和层极法纯化ｐ５３蛋白，所纯化的ｐ５３蛋白经Ｗｅｓｔｅｒｎ Ｂｌｏｔ（ＥＣＬ法）检测证明：用此法从ｐ５３阳性的ＳＷ４８０细胞中分离到ｐ５３蛋白。银染显示ｐＨ２．０甘氨酸缓冲液比ｐＨ２．８的甘氨酸缓冲液洗脱效果好，这种方法的建立将为分离肿瘤细胞中引起ｐ５３蛋白功能失活和研究肿瘤发生机制提供一种有效途径。     

Argyrophilic nucleolar organizer regions in colorectal malignancy: application of typing by their density

Colorectal neoplasms obtained at colonoscopy were examined by argyrophilic nucleolar organizer region (AgNOR) staining to evaluate the usefulness of AgNOR as a discriminant for malignancy. AgNOR dots were divided into two kinds: 'structures' (larger and less-densely stained) corresponding to the nucleolus, and 'units' (smaller and densely stained) presumed to be true AgNOR within the structure. The number of structures per nucleus did not differ between the adenoma and carcinoma groups, whereas the number of units per nucleus showed a significant difference. However there were several cases showing an overlap between the adenoma and carcinoma groups, leading to a difficulty in deciding whether any given case was benign or malignant. Three types of AgNOR patterns were categorized based on the ratio of units to structure. Type I was defined as the unit being indistinguishable from the structure, Type II as each structure having one to five units, and Type III as at least one structure having six or more units, irrespective of total number of units per nucleus. The colorectal lesions in which more than half of the neoplastic cells showed Type III coincided well with carcinomas histologically diagnosed, with the exception of adenomas with severe atypia. Labeling index of proliferating cell nuclear antigen (PCNA LI) differed between the adenoma and carcinoma groups with a considerable extent of overlap, and correlated to some extent with the AgNOR values. These results showed that the AgNOR staining was useful for determining malignancy and its usefulness appeared superior to PCNA LI.