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.     

Cell surface laminin-like substances and laminin-related carbohydrates of rat ascites hepatoma AH7974 and its variants with different lung-colonizing potential

Rat ascites hepatoma AH7974 cells strongly expressed antilaminin antibody-reactive substances (laminin-like substances) andGriffonia simplicifolia isolectin B4 (GS)-reactive carbohydrate (alpha)-d-galactose; alpha-Gal) on their cell surface. The alpha-Gal expression was not apparently influenced by the pretreatment of cells with methanol. The cell membrane laminin-like substances had approximate molecular weights of 150, 62 and 56 kDa in denaturating reducing conditions, of which the 62 and 56 kDa bands were stained with GS. The cell membrane molecules bearing alpha-Gal were 62 and 56 kDa and were stained with antilaminin antibody. Therefore, the major molecules bearing alpha-Gal residues of AH7974 cell membrane are considered to be laminin-like substances. To determine the role of the substances in metastasis, we selected four cell lines (74AD, 74AD-f, 74FL, 74FL-a) from AH7974 in culture. 74AD and 74FL-a are adherent lines and 74AD-f and 74FL are floating lines. All of these cell lines strongly expressed laminin-like substances, but a marked difference was found in expression of alpha-Gal, which was most strongly expressed by 74FL, followed by 74AD, and rarely by 74AD-f and 74FL-a; the staining intensity was positively correlated with their experimental lung-colonizing potential. Cell membrane laminin-like substances were 200, 97, 62, 56 and 46 kDa and among them 62 and 56 kDa molecules were glycosylated with alpha-Gal. The pretreatment of 74FL cells with antilaminin antibody or with human type A serum (containing natural antibody to alpha-Gal epitope) depressed remarkably the lung-colonizing potential of the cells. These results suggest that the expression of 62 and 56 kDa laminin-like substances with alpha-Gal residues on tumor cell surfaces is one of the determinants associated with lung-colonizing potential of these cells.     

Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability

To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado- Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n- CGG]/[normal (CAG)n-GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in 'triplet repeat diseases'.      

Intergenerational instability of the CAG repeat of the gene for Machado- Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat

Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects intergenerational instability of the CAG repeat. The [expanded (CAG)n-CGG]/[normal (CAG)n- GGG] haplotypes were found to result in significantly greater instability of the CAG repeat compared to the [expanded (CAG)n- CGG]/[normal (CAG)n-CGG] or [expanded (CAG)nGGG]/[normal (CAG)n-GGG] haplotypes. Multiple stepwise logistic regression analysis revealed that the relative risk for a large intergenerational change in the number of CAG repeat units (< -2 or > 2) is 7.7-fold (95% CI: 2.5-23.9) higher in the case of paternal transmission than in that of maternal transmission and 7.4-fold (95% CI: 2.4-23.3) higher in the case of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes than in that of transmission from a parent with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The combination of paternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-GGG] haplotypes resulted in a 75.2-fold (95% CI: 9.0-625.0) increase in the relative risk compared with that of maternal transmission and the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] or [expanded (CAG)n- GGG]/[normal (CAG)n-GGG] haplotypes. The results suggest that an inter- allelic interaction is involved in the intergenerational instability of the expanded CAG repeat.      

Immunogenetic background of patients with autoimmune fatigue syndrome

We have previously reported that approximately 50% of children with chronic nonspecific complaints were positive for antinuclear antibodies (ANA), and that a novel autoantibody to a 62 kD protein (anti-Sa) was found in 40% of these ANA-positive patients. Therefore, we proposed a distinct disease entity termed autoimmune fatigue syndrome (AIFS). We hypothesized that if autoimmune mechanisms did play an important role in the pathogenesis of AIFS, it is possible that it is immunogenetically regulated as observed in other autoimmune disorders. In order to examine the immunogenetic background of AIFS patients, HLA-A, -B, -C, and -DR loci were analyzed serologically in 61 AIFS patients. AIFS was found to be positively associated with the class I antigen HLA-B61 and with the class II antigen HLA-DR9, with odds ratios of 2.77 (p = 0.015, Pcorr = 0.48) and 2.60 (p= 0.012, Pcorr = 0.17), respectively. A negative association was also found between AIFS and HLA-DR2 with odds ratio of 0.25 (p = 0.029, Pcorr = 0.041). When comparing anti-Sa positive AIFS patients with healthy controls, the odds ratios associated with HLA-B61, DR9, and DR2 were 3.42 (p = 0.021, Pcorr = 0.22), 3.96 (p = 0.0011, Pcorr = 0.015), and 0.16 (p = 0.0022, Porr = 0.031), respectively. Thus, the HLA associations observed in this study suggested that immunogenetic background might play a role in AIFS.     

A simple and rapid method for the determination of macrophage activating factor involving a new type of apparatus suitable for the measurement of macrophage chemiluminescence

A simple and rapid method for the determination of macrophage activating factor is described. A new type of apparatus suitable for the measurement of macrophage chemiluminescence was devised, and the effect of lymphokines on macrophage activities was studied by measuring phorbol myristate acetate-induced luminol-dependent chemiluminescence. An outstanding feature of the new apparatus is that the plastic dish used for the cell culture can be used as the vessel for the chemiluminescence reaction. When thioglycollate-elicited ICR mouse peritoneal macrophages were incubated with lymphokines, their ability to generate chemiluminescence increased rapidly, reaching a maximal level at about 4 h, and then it progressively decreased to the control level at 8 h. Although this increasing effect of lymphokines on macrophage chemiluminescence was short-lived, it could be seen at a relatively low concentration, at which lymphokine-mediated cytotoxic activity of macrophages was not observed.     

Expression of canine interferon-gamma by a recombinant vaccinia virus and its antiviral effect

A recombinant vaccinia virus-expressing canine interferon (IFN)-gamma (vv/cIFN-gamma) was constructed. In rabbit kidney (RK13) and canine A72 cells infected with vv/cIFN-gamma, IFN activity was detected in the culture supernatants of both cell types. Canine IFN-gamma was also detected in both cell extracts by Western blot. The activity of the recombinant canine IFN-gamma in RK13 cells was higher than that in A72 cells. The vv/cIFN-gamma could not grow in A72 cells at a low multiplicity of infection, probably due to the antiviral activity of the canine IFN-gamma produced. Although exogenous IFN-gamma did not inhibit the growth of vaccinia virus, addition of anti-canine IFN-gamma serum recovered the growth of the vv/cIFN-gamma on A72 cells in a dose-dependent manner. These results suggest that the growth of vv/cIFN-gamma was inhibited by IFN-gamma produced in a paracrine and autocrine manner. In addition, the recombinant canine IFN-gamma inhibited the multiplication of canine herpesvirus, pseudorabies virus and canine adenovirus type 1 in Madin-Darby canine kidney cells. The antiviral effect of canine IFN-gamma was more effective than that of canine IFN-beta. From the present studies, we concluded the recombinant virus may be a useful suicide viral vector.     

HTL V-I from Iranian Mashhadi Jews in Israel is phylogenetically related to that of Japan,India, and South America rather than to that of Africa and Melanesia

A new endemic focus of human T-lymphotropic virus type I (HTL V-I) was recently reported among Mashhadi Jews, a group of immigrants from northeastern Iran to Israel. We extracted DNAs from fresh peripheral blood mononuclear cells (PBMCs) and/or gargle mouthwash from 10 HTL V-I carriers, who consisted of members of one family, and HTL V-I-associated myelopathy (HAM) and adult T-cell leukemia (ATL) patients. Long terminal repeat (LTR) regions of proviral DNAs were sequenced and analyzed phylogenetically. In a phylogenetic tree, all the Mashhadi HTL V-I isolates belonged to subtype A, one of the three subtypes of the cosmopolitan type of HTL V-I, and made a tight cluster distinct from the other isolates of subtype A from Japan, India, the Caribbean Basin, and South America. Although a few nucleotide substitutions were observed among the clones sequenced, no characteristic sequence variation was found in different disease manifestations, even in one family or different sources of DNA preparation.