Biomedical subjects
Publications and source records attributed to K Okubo.
A 3'-directed partial cDNA clone that matches exactly a genomic sequence in GenBank was isolated while collecting transcribed sequences from adult lung by a random approach. This is the first report of active gene identification on genomic sequence without the aid of Northern hybridization.
The band 3 protein of the red blood cell membrane catalyzes anion exchange that is inhibited by the stilbenedisulfonate derivative H2DIDS (4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid). There is one H2DIDS binding site per 95,000-Da band 3 polypeptide. The single bound H2DIDS molecule can react covalently with 2 different lysine residues. The 2 lysines that react covalently with H2DIDS have been localized directly by sequencing fragments of human band 3 from cells labeled with [3H]H2DIDS. The most rapid covalent reaction is with Lys-539, in agreement with site-directed mutagenesis studies. The slower reaction is with Lys-851, which is known to be the primary site of binding of another anion transport inhibitor, pyridoxal phosphate (Kawano et al., 1988). These results indicate that the protein is folded to bring these 2 residues into close enough proximity to react covalently with the same H2DIDS molecule. In addition to defining the residues that react with H2DIDS, these studies have also defined new in situ proteolytic cleavage sites in band 3.
From the hypocotyl of scarlet runner bean (Phaseolus coccineus), a new 2,3-dihydro-2,5,-dihydroxy-6-methyl-4H-pyran-4-one conjugated saponin, named soyasaponin alpha a, was isolated together with two known saponins, soyasaponin alpha g and beta g. Their structures were elucidated by 1H NMR and 13C NMR spectroscopy and chemical techniques.
An expression profile of genes active in the human colonic mucosa was obtained by collecting 959 partial sequences from a 3'-directed cDNA library. Seven genes were found to produce mRNA each of which comprised more than 1% of total mRNA. Four of these genes are novel, and are likely to be uniquely expressed in the colonic mucosa, and the other three have been identified as genes for fatty acid binding protein, immunoglobulin lambda chain, and carcinoma-associated antigen GA733-2. In the remaining 952 clones, 310 were composed of 118 species occurred recurrently but less than 1%, and 533 clones appeared only once. Because the 3'-directed cDNA library faithfully represents the mRNA population in the source tissue, these numbers represent the relative activities of the gene expression. Altogether 156 gene species were identified in GenBank, and a significant portion of these genes encode proteins found in Golgi apparatus and lysosomes, chromosome-encoded mitochondrial proteins, cell surface proteins, and components in the protein synthesis machinery. The types and proportions of genes identified is consistent with the known major activities of the colonic mucosa such as mucous protein production, energy-dependent water absorption, and rapid cell proliferation and turnover.
By collecting 3'-directed cDNA sequences called gene signatures (GSs) on a large scale, it is possible to make an expression profile of genes in a particular tissue, as well as discovering a number of novel genes. A total of 305 novel GSs collected from granulocytoid cells derived from HL60, a human promyelocytic leukemia cell line, by exposure to dimethyl sulfoxide were radiolabeled and used for Southern blot analyses to determine the copy number of the corresponding genes. Of these, 198 GSs identified as representing single-copy genes were then used as probes for hybridization analyses using a monochromosomal hybrid cell DNA panel. Sixty-nine of them were assigned to individual chromosomes. These results demonstrate that the chromosomal distribution of the GSs seems not to be proportional to the cytogenetic length of each chromosome.
Overlapping primers and tailed short primers are effective agents for mapping very short cDNA sequences. By using such primers, human cDNAs as short as 32 nucleotides in length can produce PCR bands. Using these and other primers of ordinary size, 44 cDNAs were assigned to chromosomes, of which 24 were assigned to single chromosomes, and 2 were assigned to two chromosomes and two were assigned to three chromosomes, respectively. Among the 24 cDNAs, all of which matched GenBank entries, 6 cDNAs were observed to map to the same chromosomes as reported previously.
An expression profile of genes active in the human lung was obtained by collecting 797 partial sequences from a 3'-directed cDNA library. Three genes were found to produce mRNA each of which comprised more than 1% of total mRNA. These three have been identified as genes for pulmonary surfactant apoprotein (PSP-A), Clara cells 10-kDa secretory protein, and HLA-E heavy chain. In the remaining 745 clones, 221 were composed of 89 species that occurred recurrently, and 524 clones appeared only once. Because the 3'-directed cDNA library faithfully represents the mRNA population in the source tissue, these numbers represent the relative activities of the gene expression. Altogether 437 gene species were novel, and 179 gene species were identified in GenBank. A significant portion of these genes encode proteins found in secretory proteins, cell surface proteins, and components in the protein synthesis machinery, representing the function of the lung.
The mechanisms of the stimulatory effect of the bombesin-like peptide neuromedin C on pancreatic exocrine secretion were examined in conscious rats. Rats were prepared with cannulae draining bile and pancreatic juice separately. Intravenous infusion of 0.35 nmol/kg/h of neuromedin C significantly increased the secretions of pancreatic bicarbonate and protein, and transiently increased the plasma cholecystokinin (CCK) concentration. The increase in pancreatic secretion persisted for 90 min, whereas the increase in plasma CCK was observed only after 15 and 30 min from the beginning of neuromedin C infusion. Intravenous infusion of CR-1409, a specific CCK-receptor antagonist, inhibited, but did not abolish, the protein secretion stimulated by neuromedin C. Intraduodenal infusion of a potent proton pump inhibitor, omeprazole, suppressed, but did not abolish, protein secretion induced by neuromedin C. Omeprazole abolished the increase in bicarbonate secretion produced by neuromedin C. These results indicate that neuromedin C induces release of CCK and that its induction of pancreatic hypersecretion is due to both its direct effect and CCK. The results also suggest that gastric hypersecretion may have a role in the bicarbonate hypersecretion induced by neuromedin C.
A study was conducted to ascertain the presence of peroxidized low-density lipoprotein (LDL) in plasma and to determine the chemical structure of the peroxidized LDL so that the mechanism by which vascular complications develop may be elucidated in patients with familial hypercholesterolemia (FH). Family trees showing hypercholesterolemia and moderate grade thickness of the Achilles' tendon were confirmed in all patients. Cholesteryl ester, triglycerides (TG), free fatty acid (FFA), free cholesterol (FC), and phospholipids in normal LDL were stained on a thin-layer chromatography (TLC) plate, but from peroxidized LDL, spot X1 between TG and FFA, and spot X2 between FFA and FC were identified clearly except for the lipids recognized in normal LDL on the TLC plates. From intermediate-density lipoprotein (IDL), LDL1, and LDL2 of FH patients, spot X1 was clearly recognized. Hydroperoxidized cholesteryl linoleate (HPO-CL) was estimated and identified from Cu(2+)-treated standard CL, Cu(2+)-treated LDL and LDL obtained from FH patients by means of high-pressure liquid chromatography, mass spectrometry, nuclear magnetic resonance, positive reaction of p-methoxydiphenylpyrenyl-phosphine and various chemical reactions. The percentage of HPO-CL to total lipids in LDL was in the order of IDL < LDL1 < LDL2 in FH patients, and it was markedly higher in FH patients when compared to healthy persons. Spot X1 obtained from Cu(2+)-treated CL, from Cu(2+)-treated normal LDL and from LDL of FH patients was chemically identical, consisting of at least 4 kinds of HPO-CL isomer. LDL2-cholesterol levels were higher in FH patients than those of LDL1-cholesterol, whereas in healthy persons LDL1 cholesterol levels were higher compared to those of LDL2 cholesterol. The capacity of LDL2 for peroxidization by Cu2+ was greater than that of LDL1 in healthy persons. Biological functions, such as acceleration of platelet aggregation, great internalization into macrophages and injuries of the arterial endothelia, were reported for LDL with HPO-CL in our previous experiments. Thus, the existence of peroxidized LDL with HPO-CL may be one of the important risks for vascular complications in FH patients.
A 69-year-old Japanese man with recurrent annular erythema localized on palms and flexor surface of fingers with anti-SSA/Ro and anti-SSB/La antibodies, is reported. The present case indicates that even in an elderly man with atypical localization, annular erythema with autoimmunity may be considered and serological analysis should be performed. Systematic studies will be required to understand the relationship between 'recurrent annular erythema associated with anti-SSB/La antibodies' in Japanese, subacute cutaneous lupus erythematosus (SCLE) and 'annular erythema associated with Sjogren's syndrome'.
Pancreatic exocrine secretion in conscious rats is regulated by bile and pancreatic juice in the proximal intestine (luminal feedback regulation), and bile-pancreatic juice diversion from the intestine results in cholecystokinin (CCK) release and pancreatic hypersecretion. Pancreatic protein secretion increases to a maximum 60-90 min after bile-pancreatic juice diversion, and then decreases slightly to a steady level of two times the basal level. Change in plasma CCK concentration parallels protein secretion. In this study, the mechanism of the decreases of protein secretion and CCK concentration was examined by stimulation with various species of peptides having different stimulatory mechanisms. Cannulae for draining bile and pancreatic juice separately and a duodenal cannula and extrajugular vein cannula were inserted into male Wistar rats. Four days later, basal levels in a 90-min period were determined, bile and pancreatic juice were diverted for 90 min, and then either secretin (1.2 nmol/kg/h), CCK-8 (25 and 100 pmol/kg/h), neuromedin C (350 pmol/kg/h and 3.5 nmol/kg/h), or CCK-JMV-180 (200 nmol/kg/h) was infused intravenously for 60 min. Infusion of secretin significantly increased protein secretion and prevented its decrease after its maximum induced by bile-pancreatic juice diversion. The plasma CCK concentrations were not increased further by neuromedin C. In conclusion, pancreatic exocrine secretion and CCK release in conscious rats are maximally stimulated by luminal feedback regulation that the decrease after maximal protein output may be due to limitation of secretory capacity and/or desensitization of acinar cells.
We previously found that an increased number of mast cells and eosinophils accumulated in nasal inverted papilloma and in the nasal mucosa of allergic subjects. Two subtypes of mast cells, i.e., mucosal mast cells and connective tissue mast cells are known to be present in the allergic nasal mucosa. Eosinophils in the allergic nasal mucosa are also heterogeneous. In addition, we demonstrated accumulation of formalin-sensitive mast cells at the tumor site of nasal inverted papilloma. The morphological characteristics and function of mast cells and eosinophils, however, have not yet been identified. The purpose of this study was to determine the ultrastructural characteristics of mast cells and eosinophils in relation to their function in tumor tissue. The results revealed two subtypes of mast cells in nasal inverted papilloma, one distributed mainly in the tumor site, the other mainly in the stromal site. These two subtypes of mast cells had different ultrastructural characteristics. In contrast to stromal mast cells, mast cells in the tumor site were characterized by a smaller cell diameter, fewer specific granules and a higher rate of degranulation. This suggested that they may have played some role in the pathogenesis of the tumor, however, their precise function is still unknown. In comparison with the mast cells in the allergic nasal mucosa, previously reported by Okuda et al, the mast cells in the tumor site were similar to those in the epithelial layer of the allergic nasal mucosa (MMCs), while mast cells in the stromal site resembled those in the lamina propria (CTMCs). There were no marked morphological differences between eosinophils in the tumor site and the stromal site.(ABSTRACT TRUNCATED AT 250 WORDS)
On the basis of the literature the results of experimental investigations on hypocholesterolemic action of saponins are demonstrated. Also the theories explaining the mechanism of this action are discussed.
As far as we know, IgA and IgG antibodies to purified house dust mite allergens Der fI and Der fII in nasal secretions have never been documented. Therefore, we determined specific IgA, SIgA and IgG antibodies (abs) to crude extract of D. farinae and its purified allergens Der fI and der f II in nasal secretions collected by aspiration from 34 normal subjects, 25 untreated nasal allergic patients and 28 treated nasal allergic patients on parenteral immunotherapy by means of an avidin-biotin ELISA. The following results were obtained. (1) The specific IgA, SIgA and IgG abs to each of the three kinds of allergens correlated with each other. The groups of patients with nasal allergy (both treated and untreated) showed higher levels of specific IgA, SIgA and IgG abs to the allergens than the normal group. (2) In the group of treated patients, the levels of specific abs were not correlated with the clinical improvement of symptoms or the degree of response to nasal challenge. (3) The treated patients failed to show significantly higher levels of abs in nasal secretions than the untreated patients. (4) The specific IgA and SIgA abs in nasal secretions seemed to be predominantly produced locally, and IgG abs might be transudated from the circulation.
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The ultimate goal of the human genome project to decode all the genetic information carried in the genome. Towards this goal, the physical structure of the genome, as well as the functional aspects of the genome, must be understood. We initiated a cDNA project to collect the "expression profiles" of all human genes, a database with which to describe which genes are expressed, and to what extent, in any given human cell at a particular time. Single-cycle sequencing of randomly selected members from a 3'-directed cDNA library is most appropriate for this purpose: the sequence data serve as a "gene signature" to identify the expressing gene, and the frequency of appearance of the gene signature reflects the activity of the gene. The compiled data, which usually covers some 1000 sequencing results per sample, is referred to as an "expression profile". We applied this analysis to HepG2 (a cell line derived from a hepatocellular carcinoma), liver cells and lung cells. The expression profiles shed some light upon the unique features of gene expression in the cell or tissue tested. A comparison of the expression profiles among different cells has allowed active genes to be classified as housekeepers or those with cell-specific functions. A significant fraction of the abundantly expressed genes include those that are unique to the cell. In addition, the resulting collection of thousands of gene signatures is a useful source of probes for mapping and for isolating full size cDNAs.
Results of 65 cases of small cell lung cancer (SCLC) treated from Jan. 1981 to May 1991 were reviewed. There were 58 male and 7 female patients and their age was ranging from 37 to 86 (mean 65). There were 41 limited disease (LD) and 24 extensive disease (ED) cases. According to TNM (UICC 1987) staging system, there were 2 cases of stage I, 4 of stage II, 9 of stage III A, 28 of stage III B and 22 of stage IV. Among 65 cases, 60 cases received radiotherapy and 55 cases of them received radiotherapy for primary site. There were 29 cases received radiotherapy combined with BAI (bronchial artery infusion) and 20 cases received systemic chemotherapy. On survival, the 2-year survival rate was 26% and MST was 13 months in LD patients (n = 41). No 2-year survivors were seen in ED patients and MST was 10 months. Tumor response of primary site was as follows. In systemic chemotherapy group, CR 35%, PR 59%, NC 6% and PD 0% before radiotherapy and CR 59% after radiotherapy were obtained. In BAI group (including BAI + systemic chemotherapy), CR 6% and PR 88% before radiotherapy in BAI group. BAI did not seem to improve response rate compared to systemic chemotherapy. On survival, BAI group did not show significant better survival compared to BAI (-) group in LD cases (n = 31). In responders (evaluable LD cases, n = 24), the MSTs were 25 months in CR cases and 13 months in PR cases. No 2-year survivors were seen in PR cases.(ABSTRACT TRUNCATED AT 250 WORDS)
The ultimate goal of the human genome project is to decode all the genetic information carried in the genome. Towards this goal, the physical structure of the genome, as well as the functional aspects of the genome, must be understood. We initiated a cDNA project to collect the 'expression profiles' of all human genes, a database with which to describe which genes are expressed, and to what extent, in any given human cell at a particular time. Single-cycle sequencing of randomly selected members from a 3'-directed cDNA library is most appropriate for this purpose: the sequence data serve as a 'gene signature' to identify the expressing gene, and the frequency of appearance of the gene signature reflects the activity of the gene. The compiled data, which usually cover some 1000 sequencing results per sample, are referred to as an 'expression profile.' We applied this analysis to HepG2 (a cell line derived from a hepatocellular carcinoma), liver cells and lung cells. The expression profiles shed some light upon the unique features of gene expression in the cell or tissue tested. A comparison of the expression profiles among different cells has allowed active genes to be classified as housekeepers or those with cell-specific functions. A significant fraction of the abundantly expressed genes include those that are unique to the cell. In addition, the resulting collection of thousands of gene signatures is a useful source of probes for mapping and for isolating full-size cDNAs.