Genetic analysis of rabies virus isolates in the Philippines

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.     

Detection and induction of CTLs specific for SYT-SSX-derived peptides in HLA-A24(+) patients with synovial sarcoma

To investigate the immunogenic property of peptides derived from the synovial sarcoma-specific SYT-SSX fusion gene, we synthesized four peptides according to the binding motif for HLA-A24. The peptides, SS391 (PYGYDQIMPK) and SS393 (GYDQIMPKK), were derived from the breakpoint of SYT-SSX, and SS449a (AWTHRLRER) and SS449b (AWTHRLRERK) were from the SSX region. These peptides were tested for their reactivity with CTL precursors (CTLps) in 16 synovial sarcoma patients using HLA-A24/SYT-SSX peptide tetramers and also for induction of specific CTLs from four HLA-A24(+) synovial sarcoma patients. Tetramer analysis indicated that the increased CTLp frequency to the SYT-SSX was associated with pulmonary metastasis in synovial sarcoma patients (p < 0.03). CTLs were induced from PBLs of two synovial sarcoma patients using the peptide mixture of SS391 and SS393, which lysed HLA-A24(+) synovial sarcoma cells expressing SYT-SSX as well as the peptide-pulsed target cells in an HLA class I-restricted manner. These findings suggest that aberrantly expressed SYT-SSX gene products have primed SYT-SSX-specific CTLps in vivo and increased their frequency in synovial sarcoma patients. The identification of SYT-SSX peptides may offer an opportunity to design peptide-based immunotherapeutic approaches for HLA-A24(+) patients with synovial sarcoma.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

A repressor protein,PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.     

A simple method for enrichment of uninucleate conidia of Aspergillus oryzae

Aspergillus oryzae produces multinucleate conidia, which makes the obtaining of homokaryons labor-intensive. Analysis of conidia by flow cytometry clarified the relationship that conidia of lower nuclear number were smaller in size. Based on this, we have developed a simple way to enrich uninucleate conidia with a membrane filter. Our results also suggest that the method is useful for elimination of heterokaryons.     

Simple and sensitive high-performance liquid chromatographic method for the investigation of dynamic changes in the redox state of rat serum albumin

Serum albumin is a mixture of mercaptalbumin (reduced form) and non-mercaptalbumin (oxidized form), i.e. a protein redox couple in serum. To investigate dynamic changes in the redox state of rat serum albumin (RSA), we developed a simple and sensitive high-performance liquid chromatographic (HPLC) system using an ion-exchange column with a linear gradient of ethanol concentration. Furthermore, we applied this HPLC system to examine dynamic changes in the redox state of RSA caused by severe oxidative stress such as exhaustive physical exercise. Using this system, we successfully separated RSA to rat mercaptalbumin (MA(r)) and rat non-mercaptalbumin (NA(r)), and also found the best conditions for the clear separation of RSA. In the experiments with exhaustive exercise, mean values for the MA(r) fraction in control and exercise groups were 76.2+/-1.8 and 69.0+/-3.5%, respectively. The MA(r) in the exercise group was significantly oxidized compared with that of the control group (P<0.01). These results suggested that RSA might act as one of the major scavengers in extracellular fluids under severe oxidative stress.     

Two pathways of iron uptake in bovine spleen apoferritin dependent on iron concentration

Iron incorporation by bovine spleen apoferritin either with ferrous ammonium sulfate in different buffers or with ferrous ammonium sulfate and phosphate was studied. Iron uptake and iron autoxidation were recorded spectrophotomerically. The buffers [4-(2-hydroxyethyl)-1-piperazinyl]ethanesulphonic acid (Hepes) and tris(hydroxymethyl)aminoethane (Tris) exhibited pH-dependent iron autoxidation, with Tris showing less iron autoxidation than Hepes. An Eadie-Scatchard plot (v/[s] versus v) of the iron uptake rate in Hepes was a curved rather than a straight line, suggesting that there are two iron uptake pathways. On the other hand, the Eadie-Scatchard plots of Tris and of Hepes after the addition of phosphate showed a straight line. Phosphate accelerated the iron uptake rate. The iron loading kinetics of apoferritin in Hepes was dependent on apoferritin concentration. The K_m value obtained from iron uptake kinetics was 4.5 M, corresponding to the physiological iron concentration. These results demonstrate that iron loading of apoferritin was accomplished at physiological iron concentrations, which is essential for iron uptake, via two uptake pathways of dependent on iron concentration.     

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.     

Roles of insulin-like growth factors and their binding proteins in the differentiation of mouse tongue myoblasts

To study the roles of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the differentiation of tongue myoblasts, we established a mouse tongue organ culture system and examined the effects of exogenous IGF-I, exogenous IGFBP4, 5, 6, and des(1-3)IGF-I, an IGF-I analogue with reduced affinity for IGFBPs, on the differentiation of tongue myoblasts. The exogenous IGF-I stimulated differentiation of tongue myoblasts and induced the expressions of endogenous IGFBP4, 5, and 6, suggesting that these IGFBPs were involved in the regulation of tongue myoblast differentiation by the IGF-I. Exogenous IGFBP4 and 5 slightly stimulated early tongue myoblast differentiation in which myogenin was involved. The stimulation seems to be due to the protection of endogenous IGFs from proteolytic degradation by the binding of these IGFBPs to endogenous IGFs. A low concentration of des(1-3)IGF-I stimulated tongue myoblast differentiation, whereas high concentrations of des(1-3)IGF-I inhibited it. The abnormal shape of the tongue, low cell density and low staining intensity with hematoxylin and eosin in tongues treated with high concentrations of des(1-3)IGF-I, suggest that the inhibition is due to abnormal reactions of tongue tissues to the toxicity caused by high concentrations of des(1-3)IGF-I. From these results, we suggest that IGFBPs may function to regulate the differentiation of mouse tongue myoblasts by controlling the concentration of free IGFs within a range suitable for the progress of tongue myoblast differentiation.     

Elucidation of correspondence between swine chromosome 4 and human chromosome 1 by assigning 27 genes to the ImpRH map, and development of microsatellites in the proximity of 14 genes

Loci affecting swine intramuscular fat content, backfat thickness, carcass weight, and daily weight gain were assigned to regions of swine chromosome (SSC) 4, which were shown to correspond to human chromosome (HSA) 1p22--> q25 by ZOO-FISH, bidirectional chromosome painting, as well as by the linkage map of genes. In order to select candidate genes responsible for the above traits from the human genome database, precise correspondence between SSC4 and HSA1 is a prerequisite. In the present study, 27 genes, PTGFR, GBP1, GBP2, GFI1, GCLM, ABCD3, EXTL2, KCNA3, ADORA3, KCND3, WNT2B, NRAS, SYCP1, PTGFRN, IGSF2, NOTCH2, S100A10, SHC1, SSR2, LMNA, CCT3, CD5L, PEA15, FCER1G, EAT2, DDR2, and LAMB3, located in the HSA1 region corresponding to SSC4 or possibly SSC4, were assigned to the IMpRH map. The alignment of genes from centromere to telomere in the SSC4 q arm is basically conserved in HSA1p22-->q25 with the direction from the q arm to the p arm, which is in good agreement with results from linkage mapping. In addition, the present study first demonstrated that WNT2B residing in the middle of the HSA1 region was assigned to SSC18 with a high lod score (> 5), and that at least three intrachromosomal rearrangements occurred in the region in the process of swine and human evolution. PTGFR, and LAMB3 localized at both ends of the HSA1 region were assigned to SSC6 and SSC9, respectively, which is consistent with regional correspondence reported earlier. In the course of the above analysis, microsatellite markers were developed in the proximity of eleven genes localized on SSC4, and three genes on other swine chromosomes.