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K Okano

Publications and source records attributed to K Okano.

At least 37 records · Page 2Linked to original sources
PubMed2001

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations.

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.

Amino Acid Sequence↗
PubMed2001

A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori.

The doublesex (dsx) gene is known as the final gene of the sex-determining cascade in Drosophila melanogaster. We have isolated a homologue of dsx in the silkworm, Bombyx mori, which has an epistatic feminizing gene located on the W chromosome. RT-PCR analysis indicated that B. mori dsx (Bmdsx) was transcribed in all the examined tissues, and the size of the amplified products was different between males and females. In Northern blot hybridization of poly(A)(+) RNA, the Bmdsx probe also detected a band with a sex-specific size difference. The male-specific cDNA lacked the sequence between 713 and 961nt of the female-specific cDNA. An RNase protection assay indicated that this sequence was male-specifically removed from the Bmdsx pre-mRNA. Southern blot analysis showed that Bmdsx is present at a single copy in the genome. These results suggested that the primary Bmdsx transcript is alternatively spliced to yield male- and female-specific mRNA isoforms. These sex-specific isoforms encode polypeptides with a common amino-terminal sequence but sex-specific carboxyl termini. DNA binding domain (DM domain) of BmDSX has 80% identity with D. melanogaster DSX proteins. These results suggest the Bmdsx would also regulate sexual differentiation, as does the Drosophila dsx gene.

Alternative Splicing↗
PubMed2001

Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation.

BACKGROUND: In a transcription/translation-based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1-CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized. RESULTS: We identified expression of the Per2, Bmal1, Bmal2 and Clock genes in the chicken pineal gland. Messenger RNA levels of these genes exhibited overt circadian rhythms in the pineal cells, both in vivo and in culture. In vitro functional analyses revealed the formation of cBMAL1-cCLOCK and cBMAL2-cCLOCK heteromers. Both of the cBMAL-cCLOCK heteromers activated E-box element-dependent transcription, which was negatively regulated by cPER2 in luciferase assays. Co-expression of cCLOCK, cBMAL1 and cBMAL2 co-operatively activated E-box element-dependent transcription, and a greater level of expression of cBMAL2 inhibited the activation. In the cultured pineal cells, an over-expression of either cBMAL1 or cBMAL2 disrupted the circadian rhythm of melatonin production. CONCLUSION: The functional characterization of the chicken pineal clock molecules supports the key roles of BMAL1, BMAL2 and CLOCK which contribute to the E-box-dependent transcriptional regulation in the circadian clock system.

ARNTL Transcription Factors↗
PubMed2001

Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin in rats.

The pharmacokinetics and pharmacodynamics of recombinant human erythropoietin (rh-EPO; CAS for EPO: 11096-26-7) after repeated intravenous and subcutaneous administrations in rats were studied. Administration of rh-EPO by both routes caused significant increases in hematocrit. The pharmacokinetics of rh-EPO after intravenous and subcutaneous administration exhibited nonlinearity. The pharmacodynamics of rh-EPO was analyzed using the maximum effect (Emax) and sigmoid maximum effect (sigmoid Emax) models. Both models involved the assumption that rh-EPO in plasma would stimulate the proliferation of erythroid progenitor cells. Akaike's information criterion for the Emax model was lower than that for the sigmoid Emax model, suggesting that the Emax model might be an optimal model. The rh-EPO concentration at which the effect is half of the maximum was 0.383 ng/ml. This pharmacodynamic analysis suggests that the maintenance of effective plasma concentration might be important for the efficacy of rh-EPO.

Animals↗
PubMed2001

DNA hybridization using "bead-array": probe-attached beads arrayed in a capillary in a predetermined order.

We present a DNA analysis device called "Bead-Array", in which DNA-probe-attached beads (100 microns) are arrayed in a capillary in a predetermined order. We developed this device to overcome the problems of using DNA microarray technology, including high cost and a lengthy analysis time. Bead-arrays can easily be mass-produced even for many different probe combinations, and the small reaction volume and the use of sample flows enables faster hybridization. To demonstrate this, we examined DNA hybridization experiments using 18-mer DNA probes and targets and compared them to ones with beads in tubes. The results show that hybridization in bead-arrays progresses more than 100 times faster and reach the plateau in less than three min. These features suggest that the bead-array particularly meets the need for high-speed and disposable devices, for example diagnosis devices.

DNA↗
PubMed2001

Comparative analysis for expressed genes by polymerase chain reaction using module-shuffling primers.

We have developed a method for comparative analysis of gene expression. It is based on competitive PCR amplification with module-shuffling primers, followed by gel electrophoresis in a fluorescent DNA sequencer. In this method, tagged-cDNA restriction fragments derived from different sources were amplified in one tube at the same amplification efficiency. The method can detect different amounts of each expressed gene, up to difference in amounts of 30%. The method was successfully used for comparative analysis of expressed genes in yeast.

DNA Primers↗
PubMed2001

Classified fingerprinting: A method of comprehensive analysis for comparing megabase genomes.

The objective of the work we describe is to establish elementary methods for investigating the functions of genes; specifically, a fingerprinting method for analyzing entire DNA fragments in a mixture. Our goal is to develop a method for comparing genes with a size of several megabases. We improved a method of amplified fragment length polymorphism (AFLP) so that it could be used to analyze all the restriction fragments in a mixture. This method could be used to detect 90% of the DNA fragments produced from 100-kb model genomes by using a four-base cutter enzyme.

Base Sequence↗
PubMed2001

Passive immunization with milk produced from an immunized cow prevents oral recolonization by Streptococcus mutans.

Cell surface protein antigen (PAc) and water-insoluble glucan-synthesizing enzyme (GTF-I) produced by cariogenic Streptococcus mutans are two major factors implicated in the colonization of the human oral cavity by this bacterium. We examined the effect of bovine milk, produced after immunization with a fusion protein of functional domains of these proteins, on the recolonization of S. mutans. To prepare immune milk, a pregnant Holstein cow was immunized with the fusion protein PAcA-GB, a fusion of the saliva-binding alanine-rich region (PAcA) of PAc and the glucan-binding (GB) domain of GTF-I. After eight adult subjects received cetylpyridinium chloride (CPC) treatment, one subgroup (n = 4) rinsed their mouths with immune milk and a control group (n = 4) rinsed with nonimmune milk. S. mutans levels in saliva and dental plaque decreased after CPC treatment in both groups. Mouth rinsing with immune milk significantly inhibited recolonization of S. mutans in saliva and plaque. On the other hand, the numbers of S. mutans cells in saliva and plaque in the control group increased immediately after the CPC treatment and surpassed the baseline level 42 and 28 days, respectively, after the CPC treatment. The ratios of S. mutans to total streptococci in saliva and plaque in the group that received immune milk were lower than those in the control group. These results suggest that milk produced from immunized cow may be useful for controlling S. mutans in the human oral cavity.

Adult↗
PubMed2001

Stiffness index of the calcaneus measured by quantitative ultrasound and menopause among Japanese women: The Hizen-Oshima Study.

Quantitative ultrasound (QUS) is a recently developed technique for evaluating fracture risk that can assess both bone mass and architecture. Although numerous studies have shown that menopause is associated with accelerated loss of bone mineral density, there are only a few studies on the association of QUS parameter (stiffness index) with menopausal status, especially among Japanese population. We examined age-specific changes in stiffness index, and relation with age, body mass index (BMI) and menopausal status among 506 community-dwelling Japanese women aged 40-89 years. Mean age at menopause (standard deviation) among 459 women with natural menopause was 49.4 (4.0) years. Stiffness index significantly decreased with increasing age. Stiffness index among 80-89 years age group was 40% lower, compared with that of 40-49 years age group. The greatest decline among adjacent ten-year age groups in stiffness index (15%) was found between 40-49 and 50-59 years of age. Multiple regression analysis showed that menopause related with decreased stiffness index, independent of age and BMI. Our findings indicate that menopause influences loss of bone mass and induces deterioration of bone trabecular microstructure.

Adult↗
PubMed2001

Analysis of lupus activity in end-stage renal disease treated by hemodialysis.

OBJECTIVE: The activity of systemic lupus erythematosus (SLE) has been reported to decrease in patients who have developed end-stage renal disease (ESRD). However, extrarenal symptoms attributable to the disease activity are noted, especially during the first year of dialysis. We studied the clinical course and evaluate the disease activity of SLE in patients with ESRD on hemodialysis for more than 6 months. SUBJECT AND METHODS: Fourteen patients with SLE who had been initiated on maintenance dialysis at our center between 1982 and 1999 were examined retrospectively. Their clinical details, organ system manifestations, serologic profiles and immunosuppressive treatment regimens were reviewed. Patients with and without postdialysis flaras of SLE were compared statistically. RESULTS: Five patients exhibited 6 SLE flares under treatment with corticosteroids. Two flares occurred within the first year of the initiation of dialysis, and in 1 patient, aggravation of the disease activity was noted 98 months after the initiation of dialysis. Polyarthritis was noted in 5 cases and fever in 4 cases. The serum complement levels decreased in all 6 cases with relapse of SLE activity. Compared with the other 9 patients who did not exhibit SLE relapse, no significant differences were found in 5 patients who did with respect to the demographic and serologic features at the initiation of dialysis. CONCLUSION: We conclude that the disease activity does not always burn out in patients of SLE who show progression to ESRD. SLE flares can sometimes occur even after one year of the initiation of dialysis. SLE patients on dialysis should be carefully followed up by clinical and serological monitoring, and treated by appropriate immunosuppressive therapy.

Adolescent↗
PubMed2001

Evaluation of prognostic factors for myeloperoxidase anti-neutrophil cytoplasmic antibody- (MPO-ANCA) associated glomerulonephritis.

AIMS: To identify prognostic factors for myeloperoxidase anti-neutrophil cytoplasmic antibody- (MPO-ANCA) associated glomerulonephritis. MATERIALS: We analyzed the relations between the clinical and histological features of MPO-ANCA-associated glomerulonephritis and clinical outcome in 14 patients with the disease. The patients were divided into two groups: group 1 consisted of 5 patients with progressive deterioration of renal function leading to end-stage renal disease or chronic dialysis, group 2 consisted of 9 patients in whom the initial deterioration of renal function had improved by the time of the final examination. RESULTS: Creatinine clearance at the time of biopsy was significantly lower in group 1 than in group 2, and urinary protein was higher. The mean interval between onset of symptoms and biopsy in both groups was almost the same. Recovery of renal function was correlated with the percentage of global sclerosis, but patients who had severe crescent formation did not always have a poor response to steroid therapy. There was no statistical difference between the two groups in treatment regimens. Four patients required hemodialysis at the time of biopsy (3 in group 1 and 1 in group 2). Plasmapheresis was performed in 5 patients (1 in group 1 and 4 in group 2). CONCLUSIONS: Degree of proteinuria and renal dysfunction are indicators of a poor prognosis in MPO-ANCA-associated glomerulonephritis. Global sclerosis is a histological feature that is an indicator of a poor prognosis, whereas cellular crescent formation is a predictor of a good response to steroid therapy.

Adult↗
PubMed2001

Tubular osteopontin expression in patients with ANCA-associated glomerulonephritis.

OBJECTIVE: To elucidate the role of osteopontin (OPN) in monocyte recruitment in crescentic glomerulonephritis, we investigated immunohistochemical localization of OPN in the kidney and its correlation with clinical and histopathologic parameters in biopsy specimens of patients with myeloperoxidase antineutrophil cytoplasmic autoantibody- (MPO-ANCA) associated glomerulonephritis. METHODS: Twelve patients with MPO-ANCA-associated glomerulonephritis were enrolled in this study. Clinical parameters such as creatinine clearance and urinary protein excretion of each patient were obtained at the time of biopsy. Paraffin-embedded sections were used for immunohistochemical staining using the LSAB method. Five cortical interstitial fields randomly selected at original magnification x 200 were assessed using a computer-assisted color image analyzer. Tubular OPN expression was assessed as the percentage of positive area in the tubulointerstitium. Double immunofluorescent staining using antibodies against OPN and alpha(v)beta3 was performed. RESULTS: In all of the cases studied, OPN was occasionally localized within the glomeruli, and expressed slightly in proximal tubular epithelium and significantly in distal tubular epithelium. Tubular OPN expression tended to be promoted in the interstitium infiltrating by numerous monocytes/macrophages. The extent of tubular OPN expression was positively correlated with serum ANCA titers and urinary OPN concentrations. Enhanced alpha(v)beta3 expression appeared in the distal tubular epithelium expressing OPN. CONCLUSION: These results suggest that inducible expression of OPN and alpha(v)beta3 in the tubular epithelium seems to be associated with interstitial moncyte infiltration and subsequent tubulointerstitial changes in human MPO-ANCA-associated glomerulonephritis.

Adult↗
PubMed2000

Testis developments and puberty in the male Tokara (Japanese native) goat.

In male Tokara (Japanese native) goats, testis development and the onset of puberty were studied histologically and observing sexual behaviour. Testes weight increased from 36+/-5.4 (S.E.) g (n=5) at 3 months of age to 126+/-6.3 g (n=6) at 12 months. The degree of the testis development differed among kids at 3 months of age and only one of four had produced spermatozoa in the testis. Large number of spermatozoa were always present in seminiferous tubules and epididymal ducts from 4 months of age. The diameter of seminiferous tubules increased from 133+/-9.9 microm (n=4) at 3 months to 198+/-1.0 microm (n=3) at 6 months with little increase thereafter. Mounting and pelvic thrusts onto a teaser doe started at from 9 to 14 weeks of age. Ejaculated semen contained sperm with good motility for the first time from 17 weeks. The male Tokara goat reaches puberty at around 4 months of age but testis development continues to 12 months.

Age Factors↗
PubMed2000

Fibrous pseudocapsule of metastatic liver tumors from colorectal carcinoma. Clinicopathologic study of 152 first resection cases.

BACKGROUND: The presence of pseudocapsules of liver tumors has been recognized in hepatocellular carcinoma and is known to be a favorable prognostic factor. Although several studies have reported pseudocapsules around metastatic liver tumors, to the authors' knowledge there have been few evaluations of the clinical significance of such features in patients with metastatic colorectal carcinoma. METHODS: A clinicopathologic study was conducted in 152 patients who underwent initial hepatic resection for metastatic colorectal carcinoma. RESULTS: The presence of fibrous tissue between the tumor and the surrounding hepatic parenchyma was noted in 93 of the 152 patients (61%) with colorectal liver metastases. These patients were classified as having a thin pseudocapsule (n = 46) or a thick pseudocapsule (n = 47), according to the number of collagen bundles (< 10 or >/= 10) between the tumor and the hepatic parenchyma in histologic sections. Pathologically, the presence and thickness of the fibrous pseudocapsule were related closely to less invasiveness into adjacent vessels (P = 0.004) and the presence of macroscopic intrabile ductal invasion (P = 0.008). The postresection survival was significantly better in patients with thick or thin pseudocapsules than in those without a pseudocapsule (3-year and 5- year cumulative survival rates of patients with no, thin, and thick pseudocapsules were 41% and 31%, respectively, 71% and 64%, respectively, and 88% and 88%, respectively; P < 0.001). In a multivariate analysis using the Cox proportional hazards model, after adjusting for other potential prognostic factors (vascular invasion, bile ductal invasion, number of tumors, tumor size, and curativeness of surgery), the presence of a fibrous pseudocapsule was an independent predictor of a favorable outcome after hepatic resection. CONCLUSIONS: The presence of fibrous tissue between the tumor and the liver parenchyma was a promising indicator of a better prognosis after resection in patients with colorectal liver metastases.

Adult↗
PubMed2000

Pheromone-producing cells in the silkmoth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli.

A method to isolate functional clusters of viable pheromone gland cells of Bombyx mori was developed. The 8th-9th intersegmental invaginated membrane corresponding to the pheromone gland was dissected, trimmed and separated into two distinct layers, the outer and inner layers, by enzymatic digestion with papain. The outer layer mainly consists of cuticle, while the inner layer consists of homogeneous cells with many refractile granules. The solubilized microsome fraction prepared from the inner layer retained the ability to produce bombykol in vitro, whereas the outer layer fraction did not produce bombykol. Moreover, in tissue incubations, the inner layer - but not the outer layer - produced bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. These results indicate that the inner-layer cells are indeed the pheromone-producing cells, which retain their functional integrity after separation with papain. These cells could be cultured successfully in Grace's medium for at least 5days.The presence or absence of pheromonotropic stimuli prior to dissection greatly influenced the size, number and distribution of refractile granules in the cytoplasm of the pheromone-producing cells. Staining with Nile Red proved that these refractile granules were lipid droplets. When pheromone production was studied under normal conditions or stimulated in decapitated females with pheromone-biosynthesis-activating neuorpeptide (PBAN) charge, the size of lipid droplets observed in the pheromone-producing cells reduced prominently and their number increased dramatically with time. By contrast, when pheromone production was suppressed by decapitation, the size and number of the lipid droplets remained constant. Lipid droplets observed in the pheromone-producing cells could be carriers of pheromone precursors and/or the pheromone bombykol. The present results suggest that the isolated cell preparation can be used for quantitative visualization of the cellular dynamics during pheromone production in B. mori.

Journal Article↗
PubMed2000

cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori.

We have isolated two acyl-CoA desaturase clones from a pheromone gland cDNA library by using the EST (expressed sequence tag) database of Bombyx mori. The putative acyl-CoA desaturases encoded by the clones desat 1 (2029bp) and desat 2 (2341bp) have 98% identity, and both proteins show 61% identities to Trichoplusia ni acyl-CoA Delta(11) desaturase. The deduced amino acid sequences conserve well the histidine clusters that are catalytically essential for acyl-CoA desaturase activity. Northern blot and RT-PCR analyses revealed that both transcripts of desat 1 and desat 2 were expressed predominantly in the pheromone gland. Both transcripts detected 3days before adult eclosion dramatically increased a day before adult eclosion, keeping the mRNA levels high even after eclosion. These results, combined with the fact that Delta(11) and Delta(10, 12) desaturation of palmitate is a key step to synthesize pheromone in B. mori, suggest that the desaturases encoded by desat 1 and desat 2 are involved in either or both of the desaturation steps in the pheromone biosynthetic pathway of B. mori. The mRNA levels of desat 1 and desat 2 were not affected by decapitation or injection of the pheromone biosynthesis activating neuropeptide (PBAN) into the adult female moth, suggesting that the transcription of desat 1 and desat 2 is not regulated by PBAN. In addition to the clones in the pheromone gland, eight other clones encoding the same Delta(9) desaturase homolog were found in an embryonic cDNA library by searching from the EST database of B. mori. The deduced amino acid sequence from one of the clones (desat 3) shows 79% identity to T. ni Delta(9) desaturase but only 52% identity to the desaturases in the pheromone gland of B. mori. Northern blot analysis showed that the mRNA corresponding to the desat 3 was detected in the ovary and fat body, but not in the pheromone gland. Abundance of the Delta(9) desaturase clones (eight out of the 762 randomly sequenced clones) in the library prepared from diapause-destined embryos (40h after oviposition) suggests that the Delta(9) desaturase encoded by desat 3 plays an important role in embryonic development in B. mori.

Amino Acid Sequence↗
PubMed2000

Diversity of opsin immunoreactivities in the extraretinal tissues of four anuran amphibians.

The pineal complex, deep brain, and skin have been known to function as extraretinal photoreceptors in non-mammalian vertebrates. To see the diversity of localization of extraretinal photoreceptors in lower vertebrates having different habitats, we analyzed the opsin-like immunoreactivities in anuran amphibians, Xenopus laevis, Rana catesbeiana, Rana nigromaculata, and Bufo japonicus. An antiserum (toad Rh-AS) was raised against rhodopsin purified from the retinas of Japanese toad, B. japonicus. In the retina of all the anurans examined, the outer segments of rods were immunopositive to toad Rh-AS. The outer segments of most pinealocytes were immunopositive in R. catesbeiana, R. nigromaculata, and B. japonicus. The outer segments of photoreceptor-like cells within the frontal organ of R. nigromaculata were immunostained. Interestingly, toad Rh-AS immunostained many secretory cells of mucous glands in the head skin of B. japonicus, implying the presence of a novel photoreceptive molecule. Within the hypothalamus, toad Rh-AS immunostained many cells in the magnocellular preoptic nucleus of R. catesbeiana and B. japonicus. Toad Rh-AS also labeled cerebrospinal fluid (CSF)-contacting cells in the anterior preoptic nucleus of R. nigromaculata and those adjacent to the lateral ventricle within the septum of R. catesbeiana. Thus the distribution patterns of the rhodopsin-like immunoreactivities among the anurans were highly diverged, and there was no relationship between the distribution patterns and their habitats. J. Exp. Zool. 286:136-142, 2000.

Animals↗