Assay of vitamin D derivatives and purification of vitamin D hydroxylases.
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Animals↗
Biomedical subjects
K Okuda
Publications and source records attributed to K Okuda.
Molecular cloning of vitamin D3 hydroxylases.
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Alcohol Dehydrogenase↗
Arginine-481 mutation abolishes ligand-binding of the AMPA-selective glutamate receptor channel alpha1-subunit.
Arginine-481 is located in the putative agonist-binding region preceding the putative transmembrane segment M1 of the alpha1-subunit of the AMPA-selective glutamate receptor (GluR) channel. This amino acid is completely conserved among GluR proteins. A site-directed mutagenesis study using a baculovirus expression system showed that substitution of glutamate, glutamine and lysine for arginine-481 of the recombinant alpha1-subunit protein abolishes binding to [3H]AMPA completely. The present study provides the first direct experimental evidence that the conserved charged arginine-481 residue is essential, directly or indirectly, for the acquisition of ligand-binding activity by the receptor protein.
Animals↗
Synthetic peptide from the V3 loop consensus motif with a potent anti-HIV activity inhibits ristocetin-mediated vWF-GPIb interaction.
The V3 loop consensus motif. Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB), inhibits an interaction of HIV with CD4-positive lymphocytes. Recently, both proline-rich peptides and peptides containing proline-glycine loops (beta-turns) form a complex with ristocetin dimers. These peptides interact with ristocetin-loaded platelet membrane glycoprotein (GP) Ib and act as inhibitors of von Willebrand factor (vWF)-GPIb interaction by preventing the subsequent formation of ristocetin dimer bridges. The Pro-Gly sequence is also present in the V3 loop consensus motif, Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile (HIV-1 IIIB). In this report, we have evaluated the effect of the HIV-1 IIIB peptide on vWF binding to GPIb. This peptide only inhibited vWF binding to GPIb as well as platelet aggregation in the presence of ristocetin while it had no effect on botrocetin-mediated vWF interaction with platelets. The peptide inhibited a binding of anti-vWF monoclonal antibody (RG-46) to immobilized vWF. Furthermore, ristocetin inhibited the binding of HIV-1 IIIB peptide to immobilized CXC-chemokine receptor-4 (CXCR-4) peptide. These results indicate that ristocetin may prevent HIV infection and would be useful a tool to understand the mechanism of HIV tissue tropism and infection.
Amino Acid Sequence↗
DNA vaccination followed by macromolecular multicomponent peptide vaccination against HIV-1 induces strong antigen-specific immunity.
The induction of a strong and long-lasting immunity characterized by both a humoral and cell-mediated immune (CMI) response is one of the most important considerations in developing an effective HIV vaccine. In previous studies, we have independently developed both DNA vaccine and macromolecular multicomponent peptide vaccine (VC1) candidates. In the present study, we attempted to optimize the vaccination protocol using mice, guinea pigs, rabbits and Macaca fuscata monkeys. Repeated vaccination with VC1 induced a substantial level of multivalent antibodies which neutralized various HIV-1 strains, as determined using a p24 inhibition assay. On the other hand, repeated immunization with DNA vaccine induced and sustained high levels of cytotoxic T lymphocytes (CTLs). In addition, when DNA vaccination was followed by multicomponent peptide vaccination, levels of both humoral immunity and CMI increased, and this effect continued for at least 10 months. These data clearly demonstrate that for inducing HIV-1 specific immunity, immunization with DNA vaccine followed by VC1 boosting produces better results than immunizing with either vaccine alone.
AIDS Vaccines↗
HIV-1-specific cell-mediated immunity is enhanced by co-inoculation of TCA3 expression plasmid with DNA vaccine.
We developed a candidate DNA vaccine designated pCMV160IIIB with pcREV (pCMV160IIIB/REV) that encodes gp160 of human immunodeficiency virus (HIV)-1IIIB and Rev driven by the cytomegalovirus (CMV) promotor. This vaccine induced both HIV-1-specific antibodies and cytotoxic T lymphocyte (CTL) activity. In the present study, we inoculated the TCA3 expression plasmid into mouse skeletal muscle with pCMV160IIIB/REV to determine whether this cytokine expression plasmid was able to modify the immune response. Results of a delayed-type hypersensitivity (DTH) assay using footpad swelling as well as those of a CTL assay clearly demonstrated that cell-mediated immunity (CMI) elicited by co-inoculation of pCMV160IIIB/REV with the TCA3 expression plasmid was markedly enhanced compared with that obtained using pCMV160IIIB/REV alone. When TCA3 expression plasmid was inoculated with anti-TCA3 antibody, enhancement of the DTH response was suppressed below the level of that obtained with pCMV160IIIB/REV alone. The titre of HIV-1-specific IgG2a was slightly high when pCMV160IIIB/REV was co-inoculated with this plasmid, suggesting that T-helper 1 (Th1) response was predominant in TCA3-inoculated mice. Infiltration of mononuclear cells was seen in the muscles at sites where TCA3 expression plasmid had been inoculated. Our present data suggest that TCA3 expression plasmid has potent adjuvant activity that results in an augmented CMI response.
AIDS Vaccines↗
HIV-1-specific cell-mediated immune responses induced by DNA vaccination were enhanced by mannan-coated liposomes and inhibited by anti-interferon-gamma antibody.
The adjuvant effect of mannan-coated liposomes on human immunodeficiency virus type-1 (HIV-1) DNA vaccine and the mechanism of this enhancement were studied. Coating of cationic liposomes with mannan significantly enhanced the ability of this vaccine to induce an HIV-specific delayed-type hypersensitivity (DTH) response. HIV-specific cytotoxic T-cell (CTL) activity elicited by DNA vaccination was also significantly enhanced with the mannan-liposome cocktail. This mannan-liposome-mediated activity was greatly inhibited by in vivo injection of anti-interferon (IFN)-gamma antibody, which suggests that IFN-gamma plays an important role in this HIV-specific immune response. The results of both isotype-specific antibody and cytokine analysis revealed that mannan-liposome-mediated DNA vaccination enhances Th1-mediated immunity.
AIDS Vaccines↗
Induction of HIV-1 specific mucosal immune responses by DNA vaccination.
DNA vaccination has been shown to induce immunity against several different pathogens including HIV-1. The authors demonstrate here that administration of DNA vaccines via the intranasal route is sufficient to induce immune responses both at distal mucosal sites and systemically. Since transmission of HIV-1 occurs largely across mucosal surfaces, the intranasal route provides a further means of application for DNA immunization.
Administration, Intranasal↗
Functional oxytocin receptors in bovine granulosa cells.
The aim of this study was to identify the presence of functional oxytocin (OT) receptors on bovine granulosa cells. Freshly prepared bovine granulosa cells from small (3-5 mm in diameter) or preovulatory (mature) follicles were examined for OT receptors by a radioreceptor assay. Scatchard analysis revealed that both binding capacity and affinity in granulosa cells from small follicles were significantly higher than those in granulosa cells from mature follicles (p < 0.01). With use of a reverse transcriptase polymerase chain reaction analysis, expression of OT receptor mRNA was detected in granulosa cells obtained from both small and mature follicles. When the granulosa cells obtained from small follicles were cultured in Dulbecco's Modified Eagle's Medium and Ham's F-12 medium (1:1 [v:v]) with 10% calf serum up to 72 h, as the period of culture was prolonged, the concentration of OT receptor decreased with increases of progesterone and OT release in the medium. However, the binding affinity was not changed during culture for 72 h. When bovine follicular oocytes with cumulus oophorus were cultured for 24 h in tissue culture Medium-199 with 10% fetal calf serum and OT (0-10 nM), the percentages of oocytes reaching maximum cumulus expansion were significantly increased at 0.5, 1, and 10 nM OT, although nuclear maturation in oocytes surrounded by compact cumulus cells was not affected by the addition of OT. Coexposure with OT antagonist blocked the stimulatory effect of OT on cumulus expansion, confirming the specificity of the effect. Furthermore, anti-OT rabbit serum inhibited the percentages of oocytes with expanded cumulus compared to those supplemented with normal rabbit serum (p <0.05). The overall results indicate the presence of functional OT receptors in bovine granulosa cells and support the hypothesis that OT plays a role (or roles) in regulating the function of granulosa cells as an autocrine factor during follicular growth.
Animals↗
Regulation of oxytocin receptors in bovine granulosa cells.
It is reported that oxytocin (OT) receptors in bovine granulosa cells decrease in concentration during follicular development. However, the factor or factors that regulate OT receptors are not known. In the present study, we evaluated hormonal control of OT receptors in bovine granulosa cells obtained from small antral follicles (3-5 mm in diameter). Granulosa cells were cultured for 48 h and exposed to FSH, LH, progesterone, and/or estradiol-17beta (estradiol) in the final 15 h of culture. The relative binding of OT decreased to 63% of the control value following treatment with FSH (100 ng/ml). The inhibitory effect of FSH was mimicked by an adenylate cyclase activator, forskolin. In contrast, estradiol (10(-7) M) increased the number of OT receptors by 77% compared with that in untreated controls, without changing binding affinity. The effects of estradiol were dose dependent and were diminished by an estradiol antagonist, tamoxifen (10(-6) to 10(-5) M). Although tamoxifen (10(-5) M) alone did not change OT binding, the stimulatory effects of 10(-9) M and 10(-8) M estradiol were inhibited by treatment with tamoxifen (10(-5) M). Furthermore, when the granulosa cells were exposed to FSH (10 ng/ml) and estradiol (10(-10) to 10(-7) M) in various combinations, estradiol inhibited the reduction of OT receptors by FSH. On the other hand, LH and progesterone did not affect OT binding in the cultured granulosa cells. Additionally, OT secretion from cultured granulosa cells was not changed by any treatment used in the present study. These findings suggest that both FSH and estradiol are significant regulators of OT receptors in granulosa cells during follicular development. FSH might down-regulate OT receptors in this phase, and the inhibitory effects of FSH are mediated by the adenylate cyclase-cAMP-protein kinase A system. Furthermore, estradiol seems to play a role in neutralizing the effects of FSH.
Adenylyl Cyclases↗
Up-regulation of oxytocin receptors in porcine endometrium by adenosine 3',5'-monophosphate.
Porcine endometrial cells (a mixture of epithelial, stromal, and glandular cells) were examined for the presence of oxytocin (OT) receptors using a cell culture system and a 125I-labeled OT antagonist. Binding specificity was tested in displacement studies with various related peptides. Scatchard analyses revealed the presence of a binding site with a dissociation constant (Kd) = 0.9 nM and a capacity of 1.9 fmol/10(5) cells. These cells, which were obtained from prepubertal gilts and thus had not been exposed to endogenous ovarian steroids, were used as a model to evaluate the possible action of ovarian steroids and intracellular cAMP on OT receptors. Although ovarian steroids showed no effect on OT receptors, forskolin (an adenylate cyclase activator) and dibutyryl cAMP caused 1.5- to 1.6-fold increases in specific binding of OT without changing the binding affinity. When the endometrial cells were exposed to OT (0.1-1000 nM) in combination with arachidonic acid (10 microM), OT stimulated prostaglandin F2 alpha secretion in a dose-dependent manner. These results indicate the presence of functional OT receptors in prepubertal porcine endometrial cells and suggest that the concentration of OT receptors may be regulated by one or more substances that raise intracellular cAMP levels.
Animals↗
Genetic control of immune responses to HIV-1 env DNA vaccine.
We investigated the genetic control of immunoglobulin production and the delayed-type hypersensitivity (DTH) response produced by an HIV-specific DNA vaccine using several strains of mice. Murine antigen-specific immunoglobulin production was determined by ELISA. The DTH response was assessed in terms of the footpad swelling reaction. All strains of mice, except for B10.RIII and B10.T(6R), exhibited strong immunoglobulin production and footpad swelling in response to the DNA vaccine. In vitro treatment of lymphoid cells with monoclonal antibodies showed that the footpad swelling response was mediated by CD4+8- and Ia- T cells. However, CD8+ T cells did not suppress footpad swelling. There was no difference in the induction of HIV-specific immunoglobulin production or DTH response induced by the DNA vaccine among the strains, suggesting that HIV-specific DNA vaccine is useful for immunizing various populations against HIV-1.
AIDS Vaccines↗
Genetic control of immune response to a synthetic fimbrial antigen of Actinobacillus actinomycetemcomitans.
The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10.A (5R) and B10.S (9R). These results indicate that mice with E(beta s):E(alpha k), E(beta r):E(alpha r) and E(beta b):E(alpha k) respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.
Aggregatibacter actinomycetemcomitans↗
GB virus-C infection among chronic haemodialysis patients: clinical implications.
It is known that patients on chronic haemodialysis are frequently infected with hepatitis C virus (HCV). It has recently been found that GB virus-C (GBV-C) and hepatitis G virus frequently coinfect patients with HCV. This study aimed at elucidating the clinical implications of GBV-C infection among haemodialysis patients who have and do not have HCV infection. GBV-C RNA was detected in sera of randomly selected 98 anti-HCV-positive and 85 -negative patients on dialysis by reverse transcription-polymerase chain reaction using two sets of amplification primers made from the reported sequences of the non-structural protein 3 and 5' untranslated regions. In these patients, liver function tests were carried out at regular intervals. There were six patients who were coinfected with HCV and GBV-C and three who had only GBV-C RNA. All had a history of past blood transfusion. The onset of mild hepatitis was identified in three HCV-negative patients; elevation of alanine aminotransferase (ALT) following blood transfusion was very mild but recognizable, and aspartate aminotransferase (AST) was higher than ALT. In two of six coinfected patients, the onset of liver disease was recognized with a peak ALT of 72 and 90 IU/L, respectively. Two of these six were Amplicore (HCV-RNA) negative and asymptomatic, two had low-grade HCV viraemia and two moderate-grade HCV viraemia. Of the 98 anti-HCV-positive cases, 41 were thought to have had nosocomial infection of HCV or non-A, non-B virus; none of them had GBV-C. GBV-C RNA was negative in nine patients who had chronic non-A-E hepatitis. GBV-C infection was detected in 6.1% of anti-HCV-positive and in 3.5% of -negative dialysis patients. All had blood transfusion in the past, and there was no evidence of patient-to-patient spread of GBV-C in hospital. The liver disease was very mild and self-limited in GBV-C infection alone. The natural history of coinfected patients may be similar to that of those with chronic HCV infection, but the liver disease appears to be milder.
Aged↗
Case report: right-sided periadrenal metastasis supplied by the hepatic artery. Clue to the genesis of pedunculated hepatocellular carcinoma.
The adrenal is the second most common site of haematogenous metastasis of hepatocellular carcinoma (HCC). The right adrenal is much more frequently affected than the left, but no reason has been offered for this difference. An aetiological connection has never been suggested between adrenal metastasis and pedunculated HCC. Hepatocellular carcinoma was resected in two patients who subsequently developed right-sided adrenal metastasis diagnosed by imaging. The adrenal mass was enhanced by hepatic arteriography and took up lipiodol injected into the hepatic artery. Reoperation was performed to remove the adrenal mass, which was abutting on the liver but was readily separable. Histopathologically, the adrenal gland was compressed by a metastatic HCC that developed in the immediate periadrenal tissue or adrenal capsule. By conventional imaging, the adrenal gland could not be recognized and the mass was thought to have arisen within the adrenal gland. In conclusion, periadrenal growth of HCC is a hitherto unrecognized type of metastasis and must have been mistaken either for an adrenal metastasis or a pedunculated HCC in the past. If left unresected, it would have fused with the liver and grown into a pedunculated HCC. Cancer cell invasion through an adrenohepatic fusion is the most likely mode of periadrenal metastasis; it explains the arterial communication between the mass and the liver.
Adrenal Gland Neoplasms↗
Hepatocellular carcinoma: clinicopathological aspects.
The histopathology and clinical picture of hepatocellular carcinoma (HCC) varies between individual patients and regions. These variations are perhaps due to differences in the genetic alterations that precede hepatocarcinogenesis. In this study, the clinicopathological features of HCC were compared between southern African blacks and Japanese, indicating large differences in the frequency of underlying cirrhosis, grade of cancer cell differentiation and clinical course. Intra-abdominal bleeding and febrile, rapidly progressive HCC are more common among blacks. Such a difference is accounted for, in part, by frequent encapsulation of the tumour which is well differentiated, and grows slowly in an expanding fashion in Japan. Encapsulated HCC was not seen among the black patients studied. Other distinct clinicopathological types discussed in this paper include diffuse-type HCC which is usually caused by multiple portal spread occurring almost simultaneously; the clinical course is fulminant. Sclerosing carcinoma is frequently associated with hypercalcaemia in the United States, but not in Japan. Fibrolamellar carcinoma is nearly non-existent in Asia, whereas it is common among young adults in the West. Its prognosis is generally better than ordinary HCC. Hepatocellular carcinoma has a strong propensity to invade vessel and duct systems. Portal invasion does not produce distinct clinical signs although it may aggravate portal hypertension. Patients with tumour occlusion in the major portal vein may give rise to ischaemic hepatitis when blood pressure drops suddenly in the preterminal stage. Liver parenchyma develops submassive necrosis and clinically there is an acute rise in alanine aminotransferase (ALT). Invasion into a major hepatic vein and the inferior vena cava also occurs, but less frequently compared with portal invasion. The patient can live even with a tumour thrombus in the atrium crossing the tricuspid valves. Intraductal invasion causes acute jaundice as well as an occasional haemobilia with pain. We recently found that a distinct pathological type called 'extrahepatic growth' or 'pedunculated HCC' develops as a result of fusion of right-sided adrenal metastasis of HCC and the liver, perhaps through the 'adreno-hepatic fusion' which is rather common in cirrhotic livers.
Bile Ducts↗
Adsorption of hepatitis C virus particles onto the dialyzer membrane.
It was recently found that the blood level of hepatitis C virus (HCV) RNA is significantly reduced after each dialysis procedure in patients on chronic hemodialysis. This study was designed to elucidate the mechanism for this phenomenon. In two patients with high serum levels of HCV RNA, the filtrate through the dialyzer (TF-alpha, Teijin Co., Tokyo, Japan) was analyzed for viral RNA using the polymerase chain reaction. At the end of dialysis, the filter was washed with saline, and during the saline washing, aliquots were taken for quantification of RNA by the branched DNA method. The HCV core antigen was quantified as a measure of viral particles, and hemoglobin was also measured for correcting for blood contamination. After the clearance of the blood, the filter was washed with guanidinium isothiocyanate, and the recovery of RNA was measured. The filtrate did not contain detectable RNA. The saline washing of the filter after dialysis contained a significant amount of RNA. Washing with guanidinium isothiocyanate of the thoroughly saline washed filter also recovered a significant amount of RNA. During saline washing, the recovery of RNA in the washing was much delayed behind that of hemoglobin, suggesting the adsorption of the former onto the filter membrane. There was a discordant recovery of RNA and HCV core antigen in the washing, the recovery of the former being delayed behind that of the latter. These results indicate that viral particles are adsorbed onto the inner surface of the filter membrane during dialysis. Some of these adsorbed viral particles are perhaps destroyed by hydraulic pressure applied to blood for dialysis.
Adsorption↗
Role for the S-layer of Campylobacter rectus ATCC33238 in complement mediated killing and phagocytic killing by leukocytes from guinea pig and human peripheral blood.
OBJECTIVE: The role of the S-layer of Campylobacter rectus ATCC 33238 in complement-mediated killing and interaction with leukocytes of the intraperitoneal cavity from guinea pigs and human peripheral blood was studied in vitro. MATERIALS AND METHODS: Rabbit polyclonal anti-serum to whole C. rectus cells, a monoclonal antibody which recognizes 150 KDa S-layer protein antigen and a monoclonal antibody against lipopolysaccharide (LPS) were prepared. Sensitivities of C. rectus cells against complement mediated killing and phagocytic killing by peritoneal leukocytes of guinea pig and human peripheral leukocytes were examined in the presence or absence of a specific antibody. RESULTS: C. rectus ATCC 33238 cells were moderately sensitive to complement mediated killing in the presence of rabbit polyclonal antibody against whole cells, and slightly sensitive in the presence of monoclonal antibody against S-layer. Ingestion and phagocytic killing of C. rectus cells by leukocytes were enhanced by the rabbit anti-serum and monoclonal antibody against S-layer, but not by the monoclonal antibody against LPS, pre-immune rabbit serum or control ascites. Viability of leukocytes was dependent on the number of ingested C. rectus cells. CONCLUSIONS: The present study demonstrates that S-layer possessing C. rectus cells are resistant to complement mediated killing and phagocytic killing by leukocytes in the absence of specific antibody.
Animals↗