Intracellular localization of tyrosine kinase substrates beneath crosslinked surface immunoglobulins in B cells

Crosslinking of surface immunoglobulins (sIg) in B cells led to the accumulation of submembranal phosphotyrosine, which was followed morphologically with the PY20 antiphosphotyrosine monoclonal antibody. Phosphotyrosine was not detected before sIg crosslinking. After sIg crosslinking, phosphotyrosine-containing proteins were redistributed from scattered small clusters near the plasma membrane to a juxtanuclear region, where immunofluorescent staining decreased with time. Double immunofluorescent staining of individual cells showed accumulation of phosphotyrosine beneath crosslinked sIg molecules at the cell surface. The sIg molecules were subsequently internalized more rapidly than the phosphotyrosine-containing molecules were redistributed. Genistein, a protein tyrosine kinase (PTK) inhibitor, blocked intracellular tyrosine phosphorylations but not cell surface patching of crosslinked sIg. When polyacrylamide beads coated with anti-Ig antibodies were added to the cells, intracellular tyrosine phosphorylation occurred beneath the regions of contact with the beads. This study provides an independent line of evidence confirming recent biochemical experiments that show that crosslinking of the antigen receptor induces PTK activity in B cells, and that components of the newly described sIg complex are among the PTK substrates. The surprising finding that the bulk of the induced phosphotyrosine remains associated with crosslinked sIg for many minutes suggests a role for complex local protein interactions in phosphotyrosine-mediated signal transduction through the antigen receptor of B cells

Improved Adsorption of an Enterococcus faecalis Bacteriophage ΦEF24C with a Spontaneous Point Mutation

Some bacterial strains of the multidrug-resistant Gram-positive bacteria Enterococcus faecalis can significantly reduce the efficacy of conventional antimicrobial chemotherapy. Thus, the introduction of bacteriophage (phage) therapy is expected, where a phage is used as a bioagent to destroy bacteria. E. faecalis phage ΦEF24C is known to be a good candidate for a therapeutic phage against E. faecalis. However, this therapeutic phage still produces nonuniform antimicrobial effects with different bacterial strains of the same species and this might prove detrimental to its therapeutic effects. One solution to this problem is the preparation of mutant phages with higher activity, based on a scientific rationale. This study isolated and analyzed a spontaneous mutant phage, ΦEF24C-P2, which exhibited higher infectivity against various bacterial strains when compared with phage ΦEF24C. First, the improved bactericidal effects of phage ΦEF24C-P2 were attributable to its increased adsorption rate. Moreover, genomic sequence scanning revealed that phage ΦEF24C-P2 had a point mutation in orf31. Proteomic analysis showed that ORF31 (mw, 203 kDa) was present in structural components, and immunological analysis using rabbit-derived antibodies showed that it was a component of a long, flexible fine tail fiber extending from the tail end. Finally, phage ΦEF24C-P2 also showed higher bactericidal activity in human blood compared with phage ΦEF24C using the in vitro assay system. In conclusion, the therapeutic effects of phage ΦEF24C-P2 were improved by a point mutation in gene orf31, which encoded a tail fiber component

Cutavirus on the skin in an Asian cohort: identification of a novel geographically related genotype

Abstract Background Cutavirus (CuV) is the newest human parvovirus and is currently receiving increasing attention because of its possible association with cutaneous T-cell lymphoma. Despite the pathogenetic potential of CuV, it has been detected in normal skin; however, little is known about the prevalence, infection levels, and genetic variations of this virus in the skin of the general population. Methods We investigated the CuV DNA prevalence and viral loads concerning age, sampling location, and gender using 678 skin swabs collected from the normal-appearing skins of 339 Japanese participants aged 2–99 years. Phylogenetic analyses were also conducted based on the near-full-length CuV sequences identified in this study. Results Both the CuV DNA prevalence and viral loads were significantly higher in the skin of elderly persons aged ≥60 years compared with those of persons aged < 60 years. CuV DNA tended to persist in the skin of elderly individuals. No significant difference in viral loads was observed between the skin of the upper arm and the skin of the forehead in CuV DNA-positive specimens. Significantly higher viral loads were evident in men vs. women, although no gender-associated differences in viral prevalence were noted. Phylogenetic analyses demonstrated the existence of Japanese-specific viruses that were genetically distinct from viruses prevalent in other areas, especially Europe. Conclusions This large study suggests that high levels of CuV DNA are prevalent on the skin of elderly adults. Our findings also indicated the prevalence of geographically related CuV genotypes. A follow-up study of this cohort should provide helpful information on whether CuV may become pathogenic

Additional file 3 of Cutavirus on the skin in an Asian cohort: identification of a novel geographically related genotype

Additional file 3: table S2 Nucleotide identities between the near-full-length cutavirus sequences (4455 bp) identified in the current study

Additional file 1 of Cutavirus on the skin in an Asian cohort: identification of a novel geographically related genotype

Additional file 1: table S1 Sequences of primers used for PCR analysis

Additional file 2 of Cutavirus on the skin in an Asian cohort: identification of a novel geographically related genotype

Additional file 2: figure S1 Phylogenetic analysis of cutavirus based on complete VP2 sequences