Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.     

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Endotoxin contamination in wound dressings made of natural biomaterials

Contamination by endotoxin of nine kinds of wound dressings made of natural biomaterials (calcium alginate, collagen, chitin, and poly-L-leucine) was examined with the use of water extracts. By applying the Limulus amoebocyte lysate (LAL) test, high concentrations of endotoxin were detected in extracts from three kinds of products made of calcium alginate. These extracts evoked fever in rabbits and induced the release of a proinflammatory (pyrogenic) cytokine, interleukin-6 (IL-6), from human monocytic cells (MM6-CA8). The effects disappeared when the extracts were treated with endotoxin-removing gel column chromatography or with an endotoxin antagonist, B464, confirming that the contaminating pyrogen was endotoxin. A noteworthy finding was that one of the endotoxin-containing extracts showed very weak IL-6-inducibility in human monocytic cells in contrast to its high pyrogenicity to rabbits. The discrepancy could be explained based on differences between humans and rabbits in sensitivity to the endotoxin, because the extract showed higher proinflammatory-cytokine (TNF-alpha)-inducibility in rabbit whole-blood cells (WBCs) than human WBCs. The results suggest that the LAL test is a useful method of detecting endotoxin contamination in wound dressings and the MM6-CA8 assay is a good supplement to the LAL test for evaluating pyrogenicity in humans accurately.     

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