Sequential steps of macroautophagy and chaperone-mediated autophagy are involved in the irreversible process of posterior silk gland histolysis during metamorphosis of Bombyx mori.

To elucidate the degradation process of the posterior silk gland during metamorphosis of the silkworm ITALIC! Bombyx mori, tissues collected on the 6th day after entering the 5th instar (V6), prior to spinning (PS), during spinning (SP) and after cocoon formation (CO) were used to analyze macroautophagy, chaperone-mediated autophagy (CMA) and the adenosine triphosphate (ATP)-dependent ubiquitin proteasome. Immediately after entering metamorphosis stage PS, the levels of ATP and phosphorylated p70S6 kinase protein decreased spontaneously and continued to decline at SP, followed by a notable restoration at CO. In contrast, phosphorylated AMP-activated protein kinase α (AMPKα) showed increases at SP and CO. Most of the Atg8 protein was converted to form II at all stages. The levels of ubiquitinated proteins were high at SP and CO, and low at PS. The proteasome activity was high at V6 and PS but low at SP and CO. In the isolated lysosome fractions, levels of Hsc70/Hsp70 protein began to increase at PS and continued to rise at SP and CO. The lysosomal cathepsin B/L activity showed a dramatic increase at CO. Our results clearly demonstrate that macroautophagy occurs before entering the metamorphosis stage and strongly suggest that the CMA pathway may play an important role in the histolysis of the posterior silk gland during metamorphosis.

Japanese encephalitis vaccine-facilitated dengue virus infection-enhancement antibody in adults.

BACKGROUND: Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus, and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination. METHODS: Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells. RESULTS: A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %). CONCLUSION: In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.

Identification and characterization of the short variable region of the Japanese encephalitis virus 3' NTR.

Since the 1980s, the Japanese encephalitis virus (JEV) variants with slightly short variable regions (VR) of the 3' non-translated region (NTR) have been found; however, the implications of these short VR remain unclear. We recently identified two novel types of short VR (5 and 9 nt shorter than that of major group of genotype I JEV strains) of genotype I JEV isolates. To elucidate the impact of these short VR on the replication and virulence of JEV, we generated five recombinant JEV viruses: M41-d5 and M41-d9 have deletions in the VR that correspond to those observed in some recent JEV isolates, M41-d5d9 has both the 5- and 9-nt deletions in the VR, M41-d27 has a large deletion that encompasses both the 5- and 9-nt deletion regions, and M41-a13 has a 13-nt sequence insertion of the genotype III JEV strain Beijing-1 into the parent genotype I JEV strain Mie/41/2002 genome. The recombinant viruses and the parent virus, except for the M41-d27 mutant, showed similar growth properties in mammalian and mosquito cell lines. Mouse challenge experiments indicated that no significant differences among the recombinant viruses M41-d5d9, M41-d27, M41-a13, and the parent virus. Our results suggest that the short VR in JEV 3' NTR do not affect its growth in vitro or its pathogenicity in mice.

Detecting Dengue Virus Nonstructural Protein 1 (NS1) in Urine Samples Using ELISA for the Diagnosis of Dengue Virus Infection.

Dengue virus (DENV) infection is a serious global health threat. For the surveillance and control of dengue, there is a need for robust diagnostic tools that are relatively easy to use and reliable in various clinical settings. We investigated the applicability of NS1 antigen detection in urine samples for the diagnosis of DENV. About 118 urine samples, obtained from 96 dengue patients at various phases of disease, were used for this study. NS1 antigen was detected by ELISA in the urine samples obtained from patients after 2-17 days of disease onset. Positive detection rates of NS1 antigen ranged between 13-43%. Based on real-time RT-PCR, positive detection rates of viral genome in the urine samples ranged between 20-33% on days 0 to ≥15. On days 11 to ≥15 after the disease onset, NS1 antigen was detected at similar rates in serum and urine samples. Additionally, NS1 antigen was detected in 2 urine samples, but not in the serum samples, on days 7 and 16 after the onset of the disease. The results confirm the applicability of NS1 antigen detection in urine samples using ELISA to diagnose acute DENV infection and suggests that the assay is potentially useful when only limited amounts of serum samples are available and in limited resource settings.

Involvement of sigma(H) and related sigma factors in glucose-dependent initiation of morphological and physiological development of Streptomyces griseus.

We cloned and characterized sigH encoding a stress-response sigma factor, sigma(H), of Streptomyces griseus. Nucleotide sequencing of the sigH gene cluster revealed an identical gene organization as in the orthologous region of Streptomyces coelicolor A3(2). Transcriptional analysis by S1 nuclease mapping showed the presence of three tandem promoters (P1-P3) that direct transcription of sigH. The activity of P1 was markedly reduced in a sigH-depleted mutant, suggesting its dependence on sigma(H). P1 was induced by addition of 0.7 M NaCl, and P2 was induced by heat shock at 45 degrees C or addition of 4% ethanol. The sigH mutant of S. griseus showed conditional defect in aerial mycelium formation and streptomycin production depending on high concentration of glucose (>2%). Meanwhile, the wild-type strain of S. griseus introduced with rshA encoding a probable anti-sigma factor for sigma(H) on a high-copy-number plasmid was unable to perform development on media containing 1% glucose while it showed wild-type phenotype on media containing 1% maltose. RshA inhibited the sigma(H)-dependent in vitro run-off transcription at P1, which confirmed its role as a negative regulator for sigma(H). Analysis of RshA-sigma interaction by an Escherichia coli two-hybrid system showed specific interaction of RshA with sigma(H) and two related sigma factors, sigma(L) and sigma(F). We speculate that the high copy number of rshA represses morphological and physiological development of S. griseus through simultaneous inactivation of sigma(H) and related stress-response sigma factors, which play an essential role in the onset of cellular differentiation and antibiotic production on glucose media.

Cloning of the conserved regulatory operon by its aerial mycelium-inducing activity in an amfR mutant of Streptomyces griseus.

We report cloning and characterization of a 2.8 kb DNA fragment that suppressed the aerial mycelium-deficient phenotype of an amfR mutant of Streptomyces griseus when it was introduced on a high-copy-number plasmid. Nucleotide sequencing revealed that the cloned DNA fragment contained a part of a regulatory operon homologous to one of the conserved operons identified in the genome of Streptomyces coelicolor A3(2). The operon appeared to consist of 5 CDSs (rarA-E; restoration of aerial mycelium formation in an amfR mutant): rarA encoded a membrane protein with weak similarity to the histidine kinase of the two-component regulatory system; rarB and rarC products did not show marked similarity to other proteins with known function; rarD encoded a G-protein carrying two GTP-binding consensus sequences conserved in the eukaryotic Ras-like proteins; rarE product showed end-to-end homology to cytochrome P450. The 2.8 kb fragment contained a 5'-end incomplete rarA and complete rarB-D in the downstream from the promoter region of mel operon of the vector plasmid. Subcloning showed that the region containing rarA only is sufficient for the aerial mycelium-inducing activity. The truncation of rarA at its 5' terminus was essential for the restoration activity, which implied that the mutated rarA product causes unusual signaling that directs the onset of morphogenesis without amfR function. Inactivation of both rarA in Streptomyces griseus and cvnD9, a rarD ortholog in S. coelicolor resulted in precocious and glucose-resistant formation of aerial mycelium and secondary metabolites, which suggested that the operon negatively regulates the onset of differentiation. S1 nuclease protection analysis showed that the transcriptional activity of the promoter preceding rarA is developmentally regulated in an amfR- and glucose-dependent manner.

Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus.

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

LL-Z1272alpha epoxide, a precursor of ascochlorin produced by a mutant of Ascochyta viciae.

A novel metabolite, LL-Z1272alpha epoxide, structurally related to ascochlorin, was isolated from the cultured mycelium of Ascochyta viciae J-29, a mutant derived from A. viciae Libert. The structure was elucidated on the basis of spectroscopic data. The epoxide is proposed to be enzymatically formed from LL-Z1272alpha and is a precursor of ascochlorin, an antiviral and antitumor antibiotic. The conversion of the epoxide to ascochlorin by cyclization of its farnesyl chain to a cyclohexanone ring is similar to that of squalene 2, 3-oxide to sterols. Unlike ascochlorin, the new metabolite had no growth inhibitory activity against Candida albicans in the paper-disc agar diffusion assay.

Isolation and characterization of a new Clostridium sp. that performs effective cellulosic waste digestion in a thermophilic methanogenic bioreactor.

A methanogenic bioreactor that utilized wastepaper was developed and operated at 55 degrees C. Microbial community structure analysis showed the presence of a group of clostridia that specifically occurred during the period of high fermentation efficiency. To isolate the effective cellulose digester, the sludge that exhibited high fermentation efficiency was inoculated into a synthetic medium that contained cellulose powder as the sole carbon source and was successively cultivated. A comprehensive 16S rRNA gene sequencing study revealed that the enriched culture contained various clostridia that had diverse phylogenetic positions. The microorganisms were further enriched by successive cultivation with filter paper as the substrate, as well as the bait carrier. A resultant isolate, strain EBR45 (= Clostridium sp. strain NBRC101661), was a new member of the order Clostridiales phylogenetically and physiologically related to Clostridium thermocellum and Clostridium straminisolvens. Specific PCR-based monitoring demonstrated that strain EBR45 specifically occurred during the high fermentation efficiency period in the original methanogenic sludge. Strain EBR45 effectively digested office paper in its pure cultivation system with a synthetic medium.

Desferrioxamine E produced by Streptomyces griseus stimulates growth and development of Streptomyces tanashiensis.

The authors previously reported that interspecific stimulatory events between Streptomyces species for antibiotic production and/or morphological differentiation mediated by putative diffusible metabolites take place at a high frequency. This paper reports the isolation and characterization of a substance produced by Streptomyces griseus that stimulates the growth and development of Streptomyces tanashiensis. The substance was purified from the culture supernatant of S. griseus by using anion-exchange chromatography, gel filtration chromatography and reverse-phase HPLC. FAB-MS and NMR analyses of the purified preparation indicated the substance to be desferrioxamine E (synonym: nocardamine), a siderophore that is widely produced by Streptomyces species and related organisms. Similar stimulatory effects on the growth and development of S. tanashiensis were exerted by desferrioxamine E produced by another actinomycete strain, but not by other siderophores tested, including ferrichrome and nocobactin and free ferric ion. An exogenous supply of desferrioxamine E stimulated secondary metabolite formation and/or morphological differentiation in various actinomycete strains. Disruption of the desferrioxamine biosynthesis gene cluster in Streptomyces coelicolor A3(2) abolished the production of desferrioxamine E and the activity to stimulate the growth and differentiation of S. tanashiensis. The S. coelicolor mutant showed impaired growth and development on Bennett's/glucose agar medium, but it was rescued by the exogenous supply of desferrioxamine E. These results indicate that desferrioxamines play an important role in streptomycete physiology. Similar to several pathogenic bacteria and fungi, S. tanashiensis may be defective in the production of siderophores; however, it can utilize the siderophores excreted by other organisms.

A novel extracytoplasmic phenol oxidase of Streptomyces: its possible involvement in the onset of morphogenesis.

Exogenous addition of copper stimulates cellular differentiation in Streptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants forA-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.

AmfS, an extracellular peptidic morphogen in Streptomyces griseus.

The amf gene cluster was previously identified as a regulator for the onset of aerial-mycelium formation in Streptomyces griseus. The nucleotide sequences of amf and its counterparts in other species revealed a conserved gene organization consisting of five open reading frames. A nonsense mutation in amfS, encoding a 43-amino-acid peptide, caused significant blocking of aerial-mycelium formation and streptomycin production, suggesting its role as a regulatory molecule. Extracellular-complementation tests for the aerial-mycelium-deficient phenotype of the amfS mutant demonstrated that AmfS was secreted by the wild-type strain. A null mutation in amfBA, encoding HlyB-like membrane translocators, abolished the extracellular AmfS activity without affecting the wild-type morphology, which suggests that AmfBA is involved not in production but in export of AmfS. A synthetic C-terminal octapeptide partially induced aerial-mycelium formation in the amfS mutant, which suggests that an AmfS derivative, but not AmfS itself, serves as an extracellular morphogen.