Evidence of three HIV-1 subtypes in subgroups of individuals in Thailand.
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HIV Infections↗
Biomedical subjects
K Okuda
Publications and source records attributed to K Okuda.
Presence of cholesterol 7 alpha-hydroxylase enzyme protein in COS-cells leads to increased HMG CoA reductase activity.
Transfection of COS-cells with a cDNA coding for human cholesterol 7 alpha-hydroxylase resulted in significant production of cholesterol 7 alpha-hydroxylase enzyme and intracellular accumulation of the product, 7 alpha-hydroxycholesterol. Presence of this enzyme activity was always associated with increased HMG CoA reductase activity. In five different independent transfection experiments resulting in a cholesterol 7 alpha-hydroxylase activity of 0.26 +/- 0.05 pmol/min/mg in the transfected cells, the HMG CoA reductase activity increased to 158 +/- 14% of that of the control cells (p < 0.01). This change was not associated with significant changes in the cholesterol content or LDL-receptor expression of the COS-cells. It is evident that the two key enzymes in cholesterol synthesis and degradation interact with each other also in extra-hepatic cells that are unable to degrade cholesterol into bile acids. Possible mechanisms for the finding is discussed.
Animals↗
Ligand-binding properties and N-glycosylation of alpha 1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate(AMPA)-selective glutamate receptor channel expressed in a baculovirus system.
The alpha 1 subunit of the mouse alpha-amino-3-hydroxy-5-methyl-4-isoxazole- propionate(AMPA)-selective glutamate receptor channel has been expressed in insect Spodoptera frugiperda cells using a baculovirus system. The recombinant receptor proteins were identified by immunocytochemical detection, Western-blot analysis, and [35S]methionine/[35S]cysteine metabolic labeling experiments. The effect of tunicamycin on the metabolic labeling and immunoblots suggested that the two products, a major protein species of approximately 104 kDa and a minor species of approximately 100 kDa, correspond to glycosylated and non-N-glycosylated forms, respectively, which was also supported by the enzymic deglycosylation experiments. The lack of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-binding activity of non-N-glycosylated glutamate receptor expressed in the presence of tunicamycin suggested that N-glycosylation is required, directly or indirectly, for functional expression in insect cells for ligand binding. Scatchard analysis of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding showed a single binding site with Kd 30 nM and a Bmax value of 2.6 x 10(5) binding sites/cell or 1.5 pmol/mg protein in the total particulate fraction. Among the compounds tested in the competition studies, beta-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl)-L-alanine (quisqualate) was the most potent inhibitor of the 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate binding (IC50 = 30 nM), followed in decreasing order by alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionate, L-glutamate, 6,7-dinitroquinoxaline-2,3-dione, 6-cyano-7-nitroquinoxaline-2,3-dione, and 2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetate (kainate). Thus, in this study we present detailed analysis of alpha-amino-3-hydroxy-5-methyl-4- isoxazole-propionate-binding activity of the homomeric (single subunit) glutamate receptor channel of mouse alpha 1 subunit and discuss possible roles of N-glycosylation of the glutamate receptor channel alpha 1 subunit.
Amino Acid Sequence↗
Leukemic meningitis in a patient with splenic lymphoma with villous lymphocytes (SLVL). Meningitis as a possible initial manifestation of SLVL.
BACKGROUND: Splenic lymphoma with villous lymphocytes (SLVL) is a low grade, non-Hodgkin's lymphoma with a stable or slowly progressive clinical course. To the authors' knowledge, central nervous system involvement has not been described previously in patients with SLVL. METHODS: Morphologic, immunocytochemical, and immunohistochemical analyses were conducted to determine the nature of villous lymphocytes in the peripheral blood, spleen, and cerebrospinal fluid (CSF) of a patient with massive splenomegaly. RESULTS: A diagnosis of SLVL was made, based on tartrate-resistant acid phosphatase-negative peripheral villous lymphocytosis with CD19+, CD20+, HLA-DR+ phenotypes, and the involvement of spleen white pulp with these cells. Mononuclear cells in the CSF showed the same morphologic and immunocytochemical features seen in the villous lymphocytes in the peripheral blood and spleen. Splenectomy and intrathecal chemotherapy were successful in clearing leukemic cells from the CSF. CONCLUSION: In this patient with SLVL in whom leukemic meningitis developed, meningitis was found to be a possible initial manifestation of SLVL.
B-Lymphocytes↗
Measurements of urinary adipic acid and suberic acid using high-performance liquid chromatography.
A sensitive and specific method was developed for measuring medium-chain dicarboxylic acids (adipic and suberic acid) in urine. These acids were extracted from urine with diethyl ether and converted into fluorescent derivatives with 9-anthryldiazomethane, which can be separated by high-performance liquid chromatography. The reproducibility was high and the recovery from urine was above 90%. Urinary concentrations of adipic acid in streptozotocin-induced diabetic rats were significantly higher than those in control rats. In diabetic patients, both adipic acid and suberic acid tended to be high, but not significantly. This method should be useful for measuring dicarboxylic acids in urine.
Adipates↗
Antigenic alteration of an anomalous human luteinizing hormone caused by two chorionic gonadotropin-type amino-acid substitutions.
We analyzed the nucleotide sequence of the luteinizing hormone beta subunit (LH beta) in a patient with an anomalous LH. This anomalous LH showed abnormal immunogenicity, but normal bioactivity, suggesting that this variance of antigenicity was caused by amino acid substitution(s). In the anomalous LH, two single amino acid substitutions, Trp(TGG) to Arg(CGG) and Ile(ATC) to Thr(ACC), were found at the codon for the 8th and 15th residue of LH beta. These two substituted amino acid residues of the anomalous LH are identical to those of chorionic gonadotropin, but not to those of LH, although the rest of the region showed the normal sequence of human LH beta. Pedigree analysis by direct DNA sequencing revealed that the parents of the patient and the healthy sister were heterozygotes for the mutation and the patient and the healthy brother were homozygotes.
Abortion, Habitual↗
Human monocyte chemoattractant protein-1 expressed in a baculovirus system.
Human monocyte chemoattractant protein-1 (hMCP-1) was produced using a baculovirus system. The hMCP-1 cDNA was inserted into the genomic DNA of Autographa californica nuclear polyhedrosis virus (AcNPV) using a transfer vector, pJVP10Z. Spodoptera frugiperda insect cells, which were infected with this recombinant virus, secreted recombinant hMCP-1 (re-hMCP-1) at the level of 10-20 micrograms/ml of culture medium. This product was shown to chemoattract monocytes. Three distinct bands of 11, 11.5 and 12 kDa were revealed by immunoblotting analysis, and this heterogeneity was assigned to differences in carbohydrate processing. N-terminal amino-acid sequence analysis of the purified product revealed identity with hMCP-1. Thus, in this system, re-hMCP-1 was produced in large quantities and modified in a manner similar to native hMCP-1.
Amino Acid Sequence↗
Elastase gene expression in non-elastase-producing Pseudomonas aeruginosa strains using novel shuttle vector systems.
In order to determine whether non-elastase-producing strains of Pseudomonas aeruginosa such as N-10, PA103 and IFO3080 can express foreign elastase genes, we introduced elastase genes from P. aeruginosa IFO3455 (elastase-producing) as well as from PA103 and N-10 into non-elastase-producing P. aeruginosa strains. Results suggested that gene expression, secretion, and precursor processing systems of elastase were essentially normal in P. aeruginosa N-10 and IFO3080. Our studies using various elastase genes showed that both the elastase structural gene and 5'-upstream regions of P. aeruginosa PA103 were also normal. This was confirmed by the finding that P. aeruginosa N-10 and IFO3080 which carry the PA103 elastase gene produced elastase. Several deleted or chimeric genes were constructed using the 5'-upstream regions of elastase genes from P. aeruginosa N-10 or PA103 and studies of expression revealed that two individual DNA bases seem to be important in suppressing P. aeruginosa N-10 elastase gene expression. Possible reasons for the lack of elastase expression in these non-elastase-producing strains are discussed.
Base Sequence↗
rel Is rapidly tyrosine-phosphorylated following granulocyte-colony stimulating factor treatment of human neutrophils.
Stimulation of neutrophils with granulocyte-colony stimulating factor (G-CSF) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of G-CSF are mediated by binding to specific, high affinity receptors. G-CSF receptors lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the G-CSF rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be p80c-rel, a proto-oncogene belonging to a family of transcriptional regulators which include NF-kB. The induction of tyrosine phosphorylation of p80c-rel was unique to G-CSF in that granulocyte-macrophage colony stimulating factor which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of p80c-rel. The consequences of p80c-rel tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated p80c-rel is capable of binding to DNA, and G-CSF stimulation results in an increase in the amount of p80c-rel which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following G-CSF stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of p80c-rel. Thus, the tyrosine kinase activated by G-CSF appears to directly transduce a signal to a protein which functions as a transcriptional regulator.
Adult↗
Magnetic resonance imaging of the olfactory apparatus.
The magnetic resonance imaging visualization of the olfactory apparatus is reported in this article. We used a superconductor-type magnetic resonance device (Siemens Magnetom H15 (1.5 T) with a surface coil. The subjects were patients with posttraumatic anosmia. The olfactory bulbs were clearly visualized in the frontal plane magnetic resonance images, the olfactory bulbs and the foramina of the cribriform plate were visualized in the sagittal section, and the olfactory bulbs, tracts, and other structures were visualized in the coronal sections. The olfactory nerves could not be seen.
Brain Diseases↗
Effect of intrahepatic portal-systemic shunting on hepatic ammonia extraction in patients with cirrhosis.
Increased plasma ammonia levels in patients with advanced cirrhosis have been attributed to reduced conversion of enteric ammonia to urea by the diseased liver and to entry of enteric ammonia into systemic circulation by way of portal-systemic shunts. Because single-pass extraction is high for portal venous ammonia, reduction of portal blood supply to hepatocytes may have detrimental effects on the hepatic extraction of ammonia. To assess how the development of intrahepatic portal-systemic shunts alters hepatic ammonia metabolism, we determined portal and hepatic venous ammonia levels along with measurements of intrahepatic portal-systemic shunts using 99mTc-macroaggregated albumin in 46 patients with portal hypertension. Hepatic venous ammonia levels in the groups of patients with idiopathic portal hypertension, Child class A cirrhosis and Child class B or C cirrhosis were 36 +/- 17, 75 +/- 26 and 93 +/- 52 micrograms/dl, respectively, in increasing order, and portal venous ammonia extraction rates as calculated with the equation (portal venous ammonia-hepatic venous ammonia)/portal venous ammonia x 100% were decreased in the same order (77% +/- 14%, 50% +/- 21%, 40% +/- 25%, respectively). Furthermore, we noted a significant negative correlation between the intrahepatic shunt indexes as calculated by counts per minute in lungs/counts per minute in lungs and liver x 100% and the ammonia extraction rates. It was noteworthy that among Child class C patients, the ammonia extraction rates were significantly lower in patients with high intrahepatic shunt indexes than in those with low shunt indexes. These results demonstrate a significant direct relationship between hepatic ammonia extraction rates and intrahepatic shunting in cirrhosis.
Adult↗
Quantitative analysis of Lewis antigens on erythrocytes by flow cytometry.
We have developed a method for the quantitative analysis of Lewis antigens on human red blood cells (RBC) using immunofluorescence labeling and flow cytometry. Initially, Lewis a and Lewis b (Le(a) and Le(b)) antigens were labeled with monoclonal anti-Le(a) or anti-Le(b) antibodies followed by labeling with the fluorescein isothiocyanate (FITC)-conjugated second antibody. This method was not sensitive enough to identify the Lewis antigens on RBC, although the FITC method is very commonly used for antigens on white blood cells. Next, we selected the enhanced labeling technique using the avidin-biotin procedure. Biotinylated anti-mouse IgM was used for the second label and the reaction with R-phycoerythrin (RPE)-conjugated streptavidin followed to produce the fluorescence. The method was found to be effective for our objectives. From the results analyzed by the enhanced labeling technique, differences were not found in either the levels of the antigen-positive percentage and the peak mean channel of Le(a) antigens on RBC in the groups of blood type O and A (in ABO system). On the other hand, both the levels of Le(b) antigens on RBC were higher in the groups of blood type O than in those of blood type A. We found both Le(a) and Le(b) antigens on RBC from a few blood type O subjects. We conclude that enhanced labeling and flow cytometry constitute a useful technique for the determination of Lewis antigens on RBC and that this method enables the precise quantification of such antigens.
Bacterial Proteins↗
The importance of NaCl concentration in a chemically defined medium for the development of bovine oocytes matured and fertilized in vitro.
Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined medium (modified Tyrode's solution) without glucose. When different concentrations of NaCl were added to the medium, the proportions of embryos developed to the >or=8-cell, morula and blastocyst stages 96, 144 and 192 h post insemination, respectively, were significantly higher at 89 to 114 mM than 64 to 76 and 126 to 139 mM NaCl. A high proportion (28%) of blastocyst-stage embryos 192 h post insemination was obtained at 89 mM NaCl. When calculated osmolarity in the medium with 64 mM NaCl was varied by adding D-sorbitol, significantly higher proportions of morula-stage embryos were obtained at 265 to 315 mOsm (27 to 38%) than 215 (9%) and 365 (2%) mOsm, but the development to the blastocyst stage was difficult at any osmolarities (215 to 365 mOsm) tested. In the medium with a fixed osmolarity (315 mOsm) but with different concentrations (64 to 114 mM) of NaCl, there were no differences in the proportions (29 to 33%) of morula-stage embryos among different NaCl concentrations. However, significantly higher proportions of embryos developed to the blastocyst stage at 89 to 101 mM (22 to 23%) than 64 to 76 (0 to 9%) and 114 (11%) mM NaCl. When Cl- concentration in the medium with 64 mM NaCl was adjusted by adding choline chloride, significantly higher proportions of embryos developed to the morula stage at 97 to 122 mM (32 to 40%) than 72 (6%) and 147 (2%) mM Cl-, but few embryos developed to the blastocyst stage at any Cl- concentrations (72 to 147 mM) tested. In the medium with 64 or 114 mM NaCl and each with 2 different Na+/K+ ratios, there were no differences in the proportions of morula- and blastocyst-stage embryos between different Na+/K+ ratios (31 and 39 at 64 mM NaCl, and 39 and 47 at 114 mM NaCl) at each NaCl concentration. When glucose was added to the medium with 89 mM NaCl 120 h post insemination, there were no significant differences in the proportions (40 to 48%) of morula-stage embryos 144 h post insemination among different concentrations (0 to 6.95 mM) of glucose. The proportion (33%) of blastocysts 192 h post insemination at 2.78 mM glucose was significantly higher than the values at 0 (22%), 5.56 (19%) and 6.95 (15%) mM but not different compared with the values at 1.39 (23%) and 4.17 (28%) mM. In conclusion, NaCl concentration in a defined medium is one of the most important factors for the development of bovine embryo to the blastocyst stage, but the development of embryos up to the morula stage is also regulated by osmolarity and/or Cl-concentration.
Journal Article↗
Effects of fetal calf serum in culture medium on development of bovine oocytes matured and fertilized in vitro.
Bovine oocytes matured and fertilized in vitro were cultured in a chemically defined bovine embryo culture medium (BECM) or polyvinylalcohol (PVA)-free BECM supplemented with 10% heat-treated or unheated fetal calf serum (FCS). When PVA in BECM was replaced with FCS 8 h postinsemination, the proportion of >/=-cell-stage embryos 48 h postinsemination was significantly lower in heat-treated FCS (74%) than in PVA (91%) medium, but the value (84%) in unheated FCS was not different from the values in PVA and heat-treated FCS. However, the addition of either heat-treated FCS or unheated FCS to PVA-free BECM did not depress or increase the proportions (63 to 70%) of >/=-cell-stage embryos 96 h postinsemination and stimulated blastocyst formation 144 (21 to 28%) and 192 (47 to 54%) h postinsemination. Blastocyst formation was also stimulated when heattreated FCS or unheated FCS was added to PVA-free BECM 120 h postinsemination. The mean cell number of blastocysts developed in PVA-free BECM with heat-treated FCS (150 cells) or unheated FCS (152 cells) significantly increased compared with that (119 cells) developed without FCS. These results indicate that, although BECM can support bovine embryo development, a high proportion (about 50%) of morphologically normal blastocysts can be produced from oocytes matured and fertilized in vitro when they are cultured in PVA-free BECM containing FCS.
Journal Article↗
Hepatitis C infection unrelated to blood transfusion in hemodialysis patients.
Hepatitis C virus antibodies were studied using both the 1st and 2nd generation tests in 485 patients who were on maintenance hemodialysis. One hundred and eighty-seven tested positive for antibodies (38.6%); 139 of them had a history of past blood transfusion. There was a crude correlation between the amount of blood given and the antibody positivity rate among those who had a history of blood transfusion. Of 152 patients who had no blood transfusion history, 48 or 31.2% were positive for the antibodies. The length of the period during which these patients had undergone dialysis was closely correlated with the positivity rate; 50% of those who had been on dialysis for more than 10 years were positive for anti-HCV. The positivity rate among the new dialysis patients with chronic renal failure as the control was 4.6%. The difference may be accounted for by nosocomial hepatitis C virus infection. It appears that with two new needle holes made along the anastomosed blood vessels two to three times a week, the chances of patient exposure to hepatitis C virus may increase with time.
Adolescent↗
Activation of bovine oocytes penetrated after germinal vesicle breakdown.
The present study was designed to examine the ability of bovine oocytes, after germinal vesicle breakdown (GVBD), to be activated by sperm penetration and the sequence of sperm nuclear transformation. Bovine oocytes cultured for 8 h in maturation medium (tissue culture medium TCM-199 containing 10% fetal calf serum) were inseminated in Brackett and Oliphant's medium supplemented with bovine serum albumin (10 mg/ml), caffeine (5 mM) and heparin (10 micrograms/ml). When oocytes were transferred to the maturation medium 8 h after insemination and additionally cultured for 5-40 h at 39 degrees C in 5% CO2 in air, 71-76% of oocytes were penetrated and polyspermy (67-75%) was common. The proportions of penetrated oocytes that were activated significantly increased with the lapse of the additional culture time, reaching 88% and 87% by 25 and 40 h after additional culture, respectively. When compared with unpenetrated oocytes, significantly higher proportions of penetrated oocytes reached metaphase II or beyond 15 and 25 h after additional culture. After penetration, sperm nuclei were transformed into metaphase chromosomes and then to telophase chromosomes before the formation of male pronuclei. These results provide evidence that bovine oocytes acquire the ability to respond to sperm-mediated activation soon after GVBD.
Animals↗
Clinical features of hepatocellular carcinoma in the elderly: a study of 91 patients older than 70 years.
In order to determine the clinical features of hepatocellular carcinoma in the elderly, a total of 622 patients with hepatocellular carcinoma, including 91 patients 70 years or older, were retrospectively analysed with reference to their ages at the time of diagnosis. The proportion of females increased and that of hepatitis B surface antigen-positive cases decreased as age increased. Tumour sizes at the time of diagnosis were somewhat smaller in the elderly than in younger patients, whereas clinical stage taking liver function into consideration was similar in the two age groups. The prognosis in the elderly patients was similar to that in the younger ones in a clinical stage-matched comparison. Furthermore, by a multivariate analysis using the Cox proportional hazards model with inclusion of age and other clinical parameters, age was not selected in the final model as an independent predictor for survival. These results indicate that elderly patients with hepatocellular carcinoma have certain clinical features different from those in younger patients and that their prognosis is not necessarily poorer than in the latter.
Adolescent↗
Direct determination of NotI cleavage sites in the genomic DNA of adult mouse kidney and human trophoblast using whole-range restriction landmark genomic scanning.
Restriction landmark genomic scanning (RLGS) is a method for visualizing restriction landmarks, employing direct labeling of restriction sites of genomic DNA and high-resolution two-dimensional electrophoresis. We determined the conditions for both the first and second dimensions of RLGS that define all of the restriction fragments which carry the NotI landmark. Using this system, we determined the number of cleavable NotI sites of genomic DNA from the mouse kidney (C57BL/6) and from the human placenta. The mouse and human genomes were cleaved at 2,380 +/- 80 sites (4,760 +/- 160 spots) and 3,240 +/- 110 sites (6,480 +/- 220 spots), respectively with NotI.
Animals↗