Antiviral activity of hypothiocyanite produced by lactoperoxidase against influenza A and B viruses and mode of its antiviral action.

Hypothiocyanite (OSCN-) is a natural component of human saliva and is produced by the lactoperoxidase (LPO)/thiocyanate/hydrogen peroxide (H2O2) system. OSCN- has been previously shown to exhibit antiviral activity against influenza viruses (IFV) A/H1N1/2009 and A/H1N2/2009 in vitro as well as antimicrobial and antifungal activities. We elucidated the antiviral activity of OSCN- against both IFV types A and B and the mode of its antiviral action. OSCN- was produced constantly at 900 ± 200 µmol/l in Na3PO4 buffer solution containing NaSCN and LPO in the presence of H2O2 as an original OSCN- solution. In a plaque reduction assay, IFV A/PR/8/34 (H1N1), A/Fukushima/13/43 (H3N2), B/Singapore/222/97, and B/Fukushima/15/93 were exposed to various concentrations of OSCN- for 0 to 30 min before adsorption to MDCK cells, and plaque formation was examined. OSCN- exhibited significant similar antiviral activities against all four viruses without cytotoxicity, and the EC50 values for them were from 57 ± 16 to 148 ± 27 µmol/l regardless of the exposure times. The exposure of MDCK cells to OSCN- before viral adsorption did not affect its anti-IFV activity (EC50: more than 450 µmol/l), but the exposure after viral adsorption affected it moderately (EC50: 380 ± 40 µmol/l). Moreover, the exposure of virus particles to OSCN- at 450 µmol/l did not affect the hemagglutinin activity of IFV in hemagglutination inhibition assay. These results suggest that the attachment of OSCN- to the viral envelope critically contributes to the mode of antiviral action of OSCN- without interfering with viral adsorption. Keywords: hypothiocyanite; influenza virus type A; influenza virus type B; lactoperoxidase; antiviral activity.

Human C-reactive protein enhances thrombus formation after neointimal balloon injury in transgenic rabbits.

BACKGROUND: High plasma levels of C-reactive protein (CRP) constitute a powerful predictive marker of cardiovascular events. Several lines of evidence suggest that CRP has prothrombogenic effects. However, whether CRP directly participates in the pathogenesis of thrombosis in vivo has not been fully clarified. OBJECTIVE: To test whether human CRP (hCRP) affects arterial thrombus formation after balloon injury of smooth muscle cell (SMC)-rich or macrophage-rich neointima. METHODS: We compared the susceptibility of transgenic (Tg) rabbits expressing hCRP (46.21 ± 13.85 mg L(-1), n = 22) and non-Tg rabbits to arterial thrombus formation after balloon injury of SMC-rich or macrophage-rich neointima. RESULTS: Thrombus size on SMC-rich or macrophage-rich neointima was significantly increased, and was accompanied by an increase in fibrin content in hCRP-Tg rabbits, as compared with non-Tg rabbits. Thrombus size did not significantly differ between SMC-rich and macrophage-rich neointima in hCRP-Tg rabbits. Tissue factor (TF) mRNA expression and activity in these neointimal lesions were significantly increased in hCRP-Tg rabbits as compared with non-Tg rabbits. The degree of CRP deposition correlated with the elevated TF expression and thrombus size on injured neointima. In addition, hCRP isolated from hCRP-Tg rabbit plasma induced TF mRNA expression and activity in rabbit cultured vascular SMCs. CONCLUSIONS: These results suggest that elevated plasma hCRP levels promote thrombus formation on injured SMC-rich neointima by enhancing TF expression, but have no additive effects in macrophage-rich neointima.

RNA editing in the anticodon of tRNA Leu (CAA) occurs before group I intron splicing in plastids of a moss Takakia lepidozioides S. Hatt. & Inoue.

RNA editing of cytidine (C) to uridine (U) transitions occurs in plastids and mitochondria of most land plants. In this study, we amplified and sequenced the group I intron-containing tRNA Leu gene, trnL-CAA, from Takakia lepidozioides, a moss. DNA sequence analysis revealed that the T. lepidozioides tRNA Leu gene consisted of a 35-bp 5' exon, a 469-bp group I intron and a 50-bp 3' exon. The intron was inserted between the first and second position of the tRNA Leu anticodon. In general, plastid tRNA Leu genes with a group I intron code for a TAA anticodon in most land plants. This strongly suggests that the first nucleotide of the CAA anticodon could be edited in T. lepidozioides plastids. To investigate this possibility, we analysed cDNAs derived from the trnL-CAA transcripts. We demonstrated that the first nucleotide C of the anticodon was edited to create a canonical UAA anticodon in T. lepidozioides plastids. cDNA sequencing analyses of the spliced or unspliced tRNA Leu transcripts revealed that, while the spliced tRNA was completely edited, editing in the unspliced tRNAs were only partial. This is the first experimental evidence that the anticodon editing of tRNA occurs before RNA splicing in plastids. This suggests that this editing is a prerequisite to splicing of pre-tRNA Leu.

A case of pulmonary aspergilloma molecular biological identification and typing of the isolates from antemortem sputa and autopsy fungus ball.

Filamentous fungi were isolated from antemortem sputum and an autopsy fungus ball of the lung in a case of aspergilloma. Both of the isolates were analyzed for the sequences of species or strain-specific nuclear ribosomal DNA (partial 28S and ITS1 regions), and were identified as Aspergillus fumigatus. The molecular biological technique saved time and is thought to be a powerful tool in the accurate diagnosis of pulmonary fungal infection to assure effective treatment.

Potential use of Amplicor PCR kit in diagnosing pulmonary tuberculosis from gastric aspirate.

Polymerase chain reaction (PCR) detection of mycobacteria from gastric aspirate for the diagnosis of tuberculosis is not fully evaluated up to now. A total of 116 gastric aspirate specimens were collected from patients with suspected pulmonary tuberculosis. The breakdown of diagnosis was 67 pulmonary tuberculosis, 16 nontuberculous mycobacterial infection, 5 extra pulmonary tuberculosis, and 28 other lung diseases. The conventional methods were shown to have a sensitivity of 47.8% and a specificity of 79.6%; on the other hand, Amplicor had 34.9% and 97.0%, respectively. The Amplicor provided a more rapid and specific method for diagnosing tuberculosis and was more useful than the conventional.

Blood lysate staining, a new microscopic method for diagnosis of fungemia using peripheral blood.

We developed a microscopy method for the detection of fungal cells in peripheral blood, termed blood lysate staining, using an approximately 5x5 mm dotted blood lysate. This method was able to detect the emerging fungal pathogen Trichosporon asahii in murine models of systemic fungal infection and fungemia in patients quickly and at minimal cost. Pathogenic yeasts were successfully detected in 6 of 8 blood samples which were taken from feverish immunocompromised patients who were clinically suspected of having fungal infections. Fungal cells were observed as ovoid to elongated, 3x3 to 7x10 microm, and occurred singly, budding, and in short chains and clusters in a periodic acid-Schiff-stained blood smear. The yeast cells were easily distinguished from blood-cell debris by their size, shape and smooth yet rigid outline.

A novel nucleic acid-binding protein in the cyanobacterium Synechococcus sp. PCC6301: a soluble 33-kDa polypeptide with high sequence similarity to ribosomal protein S1.

Cyanobacteria are prokaryotes that carry out plant-type photosynthesis and contain several eukaryotic-type RNA-binding proteins. Using a single-stranded DNA column, a 33-kDa protein was isolated and characterized from Synechococcus sp. PCC6301. This protein of 293 amino acids is similar in overall structure to the ribosomal protein S1 found in the same species, and contains three repeated units that are highly similar to the S1 motif originally found in the ribosomal protein S1 of Escherichia coli. However, the 33-kDa protein was found not to be associated with ribosomes and its nucleic acid binding specificity is distinct from that of the ribosomal protein S1. As this protein has high affinity for both single- and double-stranded DNA, as well as for poly(G) and poly(A), we tentatively named it nucleic acid-binding protein 1 (Nbp1).

Organization of a large gene cluster encoding ribosomal proteins in the cyanobacterium Synechococcus sp. strain PCC 6301: comparison of gene clusters among cyanobacteria, eubacteria and chloroplast genomes.

The structure of a large gene cluster containing 22 ribosomal protein (r-protein) genes of the cyanobacterium Synechococcus sp. strain PCC6301 is presented. Based on DNA and protein sequence analyses, genes encoding r-proteins L3, L4, L23, L2, S19, L22, S3, L16, L29, S17, L14, L24, L5, S8, L6, L18, S5, L15, L36, S13, S11, L17, SecY, adenylate kinase (AK) and the alpha subunit of RNA polymerase were identified. The gene order is similar to that of the E. coli S10, spc and alpha operons. Unlike the corresponding E. coli operons, the genes for r-proteins S4, S10, S14 and L30 are not present in this cluster. The organization of Synechococcus r-protein genes also resembles that of chloroplast (cp) r-protein genes of red and brown algal species. This strongly supports the endosymbiotic theory that the cp genome evolved from an ancient photosynthetic bacterium.

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1.

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem II P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.