Molecular cloning of the cyanobacterial adenylate cyclase gene from the filamentous cyanobacterium Anabaena cylindrica.
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Adenylyl Cyclases↗
Biomedical subjects
M Ohmori
Publications and source records attributed to M Ohmori.
Characterization of an up-stream thyroid transcription factor-1-binding site in the thyrotropin receptor promoter.
A thyroid transcription factor-1 (TTF-1)-binding element in the rat TSH receptor (TSHR) promoter, between -189 and -175 basepairs (bp), is important for both thyroid-specific expression and thyroid-specific TSH/cAMP autoregulation of the TSHR. The identification of an up-stream TTF-1-binding site and its relationship to the function of the down-stream TTF-1 element are the subjects of this report. Sequence analysis identifies a potential TTF-1 site at -878 bp; deoxyribonuclease-I footprinting shows that the -881 to -866 bp region is protected by recombinant TTF-1 protein and by nuclear extracts from FRTL-5 thyroid cells that contain TTF-1, but not by extracts from nonfunctioning FRT thyroid or Buffalo rat liver (BRL) cells, which have no TTF-1, or by Pax-8. FRTL-5, but not FRT or BRL cell nuclear extracts, form a specific protein-DNA complex with this region in gel mobility shift analyses; its formation is prevented by TTF-1-binding elements from the thyroglobulin promoter. The upstream TTF-1 site acts as an enhancer when coupled to a heterologous simian virus-40 promoter-chloramphenicol acetyltransferase (CAT) chimera and transfected into FRTL-5 thyroid cells. There is a greater increase, 3-vs. 2-fold (P < 0.05), when TSHR promoter-CAT chimeras, which contain the up-stream TTF-1 element, pTRCAT5'-907 or pTRCAT5'-886, as opposed to those in which it is deleted, pTRCAT5'-907 delta USTTF-1, are transfected into FRTL-5 cells or cotransfected with a TTF-1 expression vector into BRL cells, which have no endogenous TTF-1. The TTF-1-dependent activity of pTRCAT5'-907 delta USTTF-1 is the same as that of the minimal promoter, -220 to -39 bp, containing only the down-stream TTF-1 site in both cells. Transfection of chimeric TSHR-CAT plasmids with the down- and/or up-stream TTF-1 site deleted reveals that the down-stream TTF-1 element functions in the absence of the up-stream element, but function of the up-stream site requires the down-stream TTF-1 element. Like the down-stream TSHR TTF-1-binding site, the up-stream TTF-1 site is different from TTF-1 sites in the thyroglobulin and thyroid peroxidase promoter, in that it does not interact with Pax-8.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Single strand DNA-binding proteins and thyroid transcription factor-1 conjointly regulate thyrotropin receptor gene expression.
An element, -186 to -176 base pairs (bp), in the minimal TSH receptor (TSHR) promoter binds thyroid transcription factor-1 (TTF-1) and is important for both constitutive expression and TSH/cAMP-induced negative autoregulation of the TSHR in thyroid cells. An element on the noncoding strand of the TSHR, contiguous with the 5'-end of the TTF-1 element, has single strand binding activity. It is distinct from the TTF-1 site, as evidenced by competition experiments using gel shift assays; but the association of the two elements is not random. Thus, the single strand binding protein (SSBP) element also exists contiguous to the 5'-end of an upstream TTF-1 site, -881 to -866 bp; mutation of two conserved nucleotides in each SSBP element results in the loss of SSBP binding and cross-competition. Transfection experiments indicate that full, constitutive TSHR gene expression in FRTL-5 thyroid cells requires the binding of both SSBPs and TTF-1, since mutation of either element halves thyroid-specific promoter activity, whereas mutation of both decreases promoter activity to values near those of a control vector. Transfection experiments with rat liver cells support their independent activities and show that the SSBP site contributes to TSHR gene expression in non-thyroid tissue. The SSBPs function conjointly with TTF-1 in thyroid-specific, TSH/cAMP-induced negative autoregulation of the TSHR. Thus, TSH or forskolin-treated FRTL-5 cells coordinately decrease TSHR RNA levels and TSHR DNA binding to both the SSBPs and TTF-1; also the maximal TSH/cAMP-induced decrease in gene expression requires both elements. The TSH-induced effect in each case is inhibited by cycloheximide; the TSH-induced decrease in SSBP/DNA complex formation requires the presence of insulin or calf serum, exactly as does TSH-induced down-regulation of TSHR RNA levels. In sum, full, constitutive expression of the TSHR in thyroid cells requires TTF-1 and the SSBPs to bind separate, contiguous elements on the TSHR promoter. TSH/cAMP decreases the binding of each factor to its respective site, thereby decreasing TSHR gene expression. The role of the SSBP and TTF-1 sites in constitutive TSHR expression and in TSH/cAMP-induced negative regulation of the TSHR is, therefore, additive and independent.
Animals↗
[Noninvasive recording of pre-P wave related to sinus node electrical activity using P wave-triggered signal-averaging technique].
To determine whether the sinus node electrogram (SNE) could be detected noninvasively, a specially modified P wave-triggered signal-averaged electrocardiogram was recorded through the standard V1, V2 and V3 precordial leads in 39 patients. The data between 500 ms before and 300 ms after the trigger point were processed using a digital bandpass filter of 0.5 Hz (12 dB/oct)-20 Hz (18 dB/oct). Pre-P signals were observed just before the P wave resembling SNE recorded directly. Pre-P potential duration closely correlated with direct SACT (r = 0.856, P < 0.0005). Thus, it is believed that signals obtained by this new method would be related to sinus electrical activity and that it could be useful for noninvasive estimation of SACT.
Body Surface Potential Mapping↗
[The estimates of the future trend of tuberculosis incidence and the control programs for its elimination].
In most European and other industrialized countries, it is estimated that tuberculosis would be eliminated around 2030, and in the U.S. and Western European countries, new tuberculosis strategies were developed to eliminate tuberculosis. The Ministry of Health and Welfare (MOHW) in Japan too, has also introduced some new tuberculosis control plans and goals. In Japan, it is estimated that the elimination of tuberculosis would be achieved around 2060, and MOHW has set the final goal for the elimination of tuberculosis by the year 2030. MOHW has also set two interim goals by the year 2000, i.e., (1) to achieve the tuberculosis incidence of below 20 per 100,000 population, (2) to achieve the tuberculosis incidence in children to the level of elimination, that is below 0.1 per 100,000. Wishing the success of these plans, the author tried to examine the possibility of achieving the goals by 2000. The decline of tuberculosis incidence rate in Japan has been gradually slower since 1977. However, the declining trend has become almost constant since around 1983, although it is still very slowly decreasing. Therefore, assuming that the future incidence rates will continue at the same decreasing speed as that of average annual decline rate during the last 10-year period from 1983 to 1992, the incidence rate in Japan in 2000 would be estimated to become 30.9 per 100,000 (95% C.I.; 29.8-31.7), and the incidence rate in children would be 0.87 per 100,000 (95% C.I.; 0.71-1.05). Examining the possibility of acceleration of the declining speed of tuberculosis incidence, it was discussed that there could be a possibility of removing such cases as the preventable cases, non-tuberculosis and inactive tuberculosis cases from newly registered cases in advance.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Identification of a novel insulin-responsive element in the rat thyrotropin receptor promoter.
By transfecting TSH receptor (TSHR)-chloramphenicol acetyltransferase (CAT) chimeras into FRTL-5 thyroid cells in the presence or absence of insulin, we identify an insulin-responsive element (IRE) between -220 and -190 bp of the TSHR 5'-flanking region. The region between -220 and -192 bp is footprinted by nuclear extracts from FRTL-5 cells and, coupled to a heterologous SV40-CAT chimera, an oligonucleotide containing the protected region induces insulin responsiveness in FRTL-5 cells. FRTL-5 cell nuclear extracts form two groups of protein-DNA complexes, A and B, in gel shift assays using an oligonucleotide having the protected sequence; mutation data indicate only the A complexes are increased by exposure of FRTL-5 cells to insulin; TSH can also increase A complex formation, but the TSH action is insulin-dependent. The nuclear factor(s) in FRTL-5 cells that interact with the TSHR IRE are distinct from thyroid transcription factor-2 (TTF-2), the insulin regulatory factor of the thyroglobulin promoter, as evidenced by the absence of competition in gel shift assays; there is no apparent sequence similarity of this region with other known IREs. The IRE is immediately upstream of a thyroid transcription factor-1 (TTF-1) binding site, -189 to -175 bp; mutation of the TTF-1 site causing a loss of TTF-1 activity also causes a loss of insulin responsiveness when the TSHR-CAT chimera at -220 bp is transfected into FRTL-5 cells and an altered IRE footprint by nuclear extracts. The TSHR appears, therefore, to contain a novel IRE whose activity depends at least in part on TTF-1, a thyroid-specific, homeodomain-containing transcription factor important both for thyroid-specific TSHR gene expression and TSH/cAMP autoregulation of the TSHR.
Animals↗
delta 9 Acyl-lipid desaturases of cyanobacteria. Molecular cloning and substrate specificities in terms of fatty acids, sn-positions, and polar head groups.
In cyanobacteria, the biosynthesis of unsaturated fatty acids is initiated by delta 9 acyl-lipid desaturase which introduces the first double bond at the delta 9 position of a saturated fatty acid that has been esterified to a glycerolipid. We have cloned genes, designated desC, for delta 9 acyl-lipid desaturases from two cyanobacteria, namely Anabaena variabilis and Synechocystis sp. PCC 6803. These desaturases, when expressed in Escherichia coli, desaturated stearic acid to yield oleic acid at the C-1 positions of phosphatidylethanolamine and phosphatidylglycerol, but did not desaturate palmitic acid, palmitoleic acid, and cis-vaccenic acid. These results indicate that the delta 9 acyl-lipid desaturases are specific to stearic acid esterified at the C-1 position of a glycerolipid and are nonspecific with respect to the polar head group of the glycerolipid. The deduced amino acid sequences of the delta 9 acyl-lipid desaturases are similar in part to those of stearoyl-CoA desaturases of the rat, the mouse, and Saccharomyces cerevisiae, but not to those of acyl-(acyl-carrier-protein) desaturases of higher plants.
Amino Acid Sequence↗
Identification of conserved domains in the delta 12 desaturases of cyanobacteria.
Cyanobacterial genes for enzymes that desaturate fatty acids at the delta 12 position, designated desA, were isolated from Synechocystis PCC6714, Synechococcus PCC7002 and Anabaena variabilis by cross-hybridization with a DNA probe derived from the desA gene of Synechocystis PCC6803. The genes of Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis encode proteins of 349, 347 and 350 amino acid residues, respectively. The transformation of Synechococcus PCC7942 with the desA genes from Synechocystis PCC6714, Synechococcus PCC7002 and A. variabilis was associated with the ability to introduce a second double bond at the delta 12 position of fatty acids. The amino acid sequence of the products of the desA genes revealed the presence of four conserved domains. Since one of the conserved domains was also found in the amino acid sequences of omega 3 desaturases of Brassica napus and mung bean, this domain may play an essential role in the introduction of a double bond into fatty acids bound to membrane lipids.
Amino Acid Sequence↗
Cloning of omega 3 desaturase from cyanobacteria and its use in altering the degree of membrane-lipid unsaturation.
Cyanobacteria respond to a decrease in temperature by desaturating fatty acids of membrane lipids to compensate for the decrease in membrane fluidity. Among various desaturation reactions in cyanobacteria, the desaturation of the omega 3 position of fatty acids is the most sensitive to the change in temperature. In the present study, we isolated a gene, designated desB, for the omega 3 desaturase from the cyanobacterium, Synechocystis sp. PCC 6803. The desB gene encodes a protein a 359 amino-acid residues with molecular mass of 41.9 kDa. The desB gene is transcribed as a monocistronic operon that produced a single transcript of 1.4 kb. The level of the desB transcript in cells grown at 22 degrees C was 10 times higher than that in cells grown at 34 degrees C. In order to manipulate the fatty-acid unsaturation of membrane lipids, the desB gene in Synechocystis sp. PCC 6803 was mutated by insertion of a kanamycin-resistance gene cartridge. The resultant mutant was unable to desaturate fatty acids at the omega 3 position. The desA gene, which encodes the delta 12 desaturase of Synechocystis sp. PCC 6803, and the desB gene were introduced into Synechococcus sp. PCC 7942. Whilst the parent cyanobacterium can only desaturate membrane lipids at the delta 9 position of fatty acids, the resultant transformant was able to desaturate fatty acids of membrane lipids at the delta 9, delta 12 and omega 3 positions. These results confirm the function of the desB gene and demonstrate that it is possible to genetically manipulate the fatty-acid unsaturation of membrane lipids in cyanobacteria.
Amino Acid Sequence↗
Comparison of the binding of beta-cyclodextrin and alpha- and gamma-cyclodextrins with pullulanase from Klebsiella pneumoniae as studied by equilibrium and kinetic fluorometry.
The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was investigated at 25 degrees C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied. The dissociation constant, Kd, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, Ki, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The Kd values of beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin were approximately two orders of magnitude smaller than those of alpha- and gamma-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among alpha-, beta-, and gamma-cyclodextrins for binding with the enzyme, which indicates that the binding region of beta-cyclodextrin overlaps those of alpha- and gamma-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for alpha- and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin, whereas beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of beta-cyclodextrin is slightly different from those of alpha- and gamma-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacterial Proteins↗
Three cases of extrapulmonary small cell carcinoma occurring in the prostate, stomach, and pancreas.
Small cell carcinomas are fairly common neoplasms in the lung, but tumors featuring similar histological profiles may occur in extrapulmonary organs. Three cases of small cell carcinomas occurring in the prostate (case 1), stomach (case 2), and pancreas (case 3) are presented. Production of hormones was demonstrated immunohistochemically in all cases. In case 2 alpha-fetoprotein (AFP) was elevated in the serum and observed immunohistochemically in tumor cells. Production of AFP is a distinctive feature, which has not been reported in the pulmonary and extrapulmonary cases of small cell carcinoma. Amplification and/or expression of myc gene family have been suggested to be related to the prognosis of pulmonary small cell carcinoma. Amplification of myc genes was not detected in any of our cases, but c-myc protein was demonstrated immunohistochemically in tumor cells of case 1.
Adult↗
[Thyrotropin receptor peptide antibody with thyroid stimulating activity induces an experimental hyperthyroidism in rabbits].
It has been presumed that N-terminal region of the TSH-R is important for TSH or TSH-R autoantibody binding to the receptor. We have synthesized human TSH-R peptide corresponding to the N-terminus of the extracellular domain, and immunized it into 8 rabbits. All these rabbits produced antibody against the peptide. Seven out of 8 antibodies showed thyroid stimulating antibody (TSAb) activity. We found that T3 and T4 concentrations in the sera of the immunized rabbits were significantly higher than those of the pre-immune sera, and increased time-dependently. These results suggest that anti-TSH-R peptide antibody with TSAb activity induced an increase in thyroid hormones in rabbits.
Animals↗
[Commemorative lecture of receiving Imamura Memorial Prize. III. Estimating the year of eradication of tuberculosis in Japan].
The year by which tuberculosis could be eradicated, has been discussed for several foreign countries, and based on those results, new strategic plans and goals have been elaborated. Therefore, in Japan too, it was desired to estimate the point at which eradication of tuberculosis would be achieved. The author estimated the year of eradication of tuberculosis, according to the criterion proposed by Dr. Styblo that "tuberculosis is eradicated when the prevalence of tuberculosis infection in the general population has fallen below 1% and continues to decrease". If the risk of infection is changing at a regular rate, it is possible to estimate the risk of infection at any particular time. Once the risk of infection is determined, it is also possible to calculate the age-specific prevalence of infection and the proportion of the population infected with tubercle bacilli at any given time. In Japan, the risk of infection before World War II was assumed to be around 4%; since then, it has declined on average, by 10 to 11% annually. The incidence rate in Japan also has declined, on average, by 10 to 11% annually. However, since late 1970s, the annual speed of decline of the incidence rate has slowed down. Therefore, I assumed that the recent trend of the infection risk is the same as the trend of the recent incidence rate among the 0-29 year age-group. The size of the effect of age on the risk of infection has been discussed. The author also considered age-effects in the model.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
Marked apoptosis of human myelomonocytic leukemia cell line P39: significance of cellular differentiation.
Myelodysplastic syndrome (MDS)-derived leukemia cell line P39/Tsugane could be induced to apoptosis by a variety of agents including metabolic inhibitors, a calcium ionophore and differentiation-inducing agents. As evaluated by characteristic morphological changes and oligonucleosomal lengths DNA ladder, the levels of apoptosis in P39 cells induced by actinomycin D, or A23187, were far greater than in other myeloid lines examined in this study. When 22-oxa-1 alpha, 25(OH)2D3 (D3), dimethyl sulfoxide (DMSO) and all-trans retinoic acid (RA) were used as differentiation-inducers, varying degrees of apoptosis were seen. D3 induced monocytoid differentiation, but not apoptosis above the control level. On the other hand, RA induced profound apoptosis concomitant with the progressive expression of differentiation markers. Studies on morphology, functions and phenotypes of P39 cells exposed to differentiation inducers suggest that the incidence of apoptosis was not affected by the process of differentiation, but cells in the process of varying degrees of differentiation may die via apoptosis. Moreover, RA-treated P39 cells are unique in the simultaneous occurrence of profound apoptosis and differentiation. We propose that RA-treated P39 differentiation model is ideally suited for the study of MDS.
Apoptosis↗
Combined analysis of flow cytometry and morphometry of ovarian granulosa cell tumor.
BACKGROUND: It is difficult to determine the prognosis of granulosa cell tumors (GCT) at the time of diagnosis. METHODS: The nuclear DNA content of 17 patients with ovarian GCT was investigated by flow cytometry using paraffin-embedded tissue. Nuclear area (NA), nuclear perimeter (NP), and nuclear shape factor (NSF) were measured by an image analyzer using hematoxylin- and-eosin-stained sections. RESULTS: The follow-up period of the patients ranged from 2 months to 11 years. Thirteen tumors were diploid or near diploid, whereas one was tetraploid, and three were aneuploid. Two tumors had varying degrees of DNA content heterogeneity. Crude survival of the patients with an euploid tumor (13 diploid, 1 tetraploid) was more favorable than that of the patients with an aneuploid tumor. Patients with S-phase fraction (SPF) greater than 10% or DNA content heterogeneity experienced disease recurrence or metastasis. A significant difference was observed in NA and NP between those with and without metastasis. CONCLUSIONS: Our results indicate that DNA aneuploidy, large SPF, DNA content heterogeneity, and large NA and NP are adverse prognostic factors in GCT. Thus, flow cytometric and morphometric measurement may provide a rapid and valuable method to predict the biologic behavior of GCT.
Adult↗
Which subgroup of patients with dilated cardiomyopathy would benefit from long-term beta-blocker therapy? A histologic viewpoint.
OBJECTIVES: The purpose of this study was to elucidate whether the effectiveness of long-term beta-blocker therapy could be predicted before this therapy is started. BACKGROUND: Long-term beta-blocker therapy has recently been reported to provide a favorable effect in treatment of congestive heart failure due to dilated cardiomyopathy. METHODS: Several measurements including histologic variables before administration of metoprolol were retrospectively compared among 18 good responders (showing improvement of at least one New York Heart Association functional class or an increase in ejection fraction > or = 0.10 12 months after drug administration) and 12 poor responders without such improvement. RESULTS: Although there were no significant differences between the two groups in age, gender, functional class, heart rate, blood pressure, pulmonary capillary wedge pressure, cardiac index, left ventricular end-diastolic dimension and ejection fraction, percent fibrosis estimated by the point-counting method in endomyocardial biopsy specimens was significantly lower in good than in poor responders (7.6 +/- 5.7 vs. 14.2 +/- 9.7%, p < 0.05). Moreover, when the types of fibrosis were classified as interfascicular and intercellular by the dominance of counted points, there were 13 cases of interfascicular fibrosis and 5 cases of intercellular fibrosis in good responders and 1 case of interfascicular fibrosis and 11 cases of intercellular fibrosis in poor responders (p < 0.001, sensitivity 72%, specificity 91%, predictive accuracy 80%). These results suggest that improvement with long-term beta-blocker therapy may be more likely to occur in patients with less myocardial fibrosis, with interfascicular fibrosis the dominant type. CONCLUSIONS: The extent and type of fibrosis may be important factors in the prediction of the effectiveness of long-term beta-blocker therapy for dilated cardiomyopathy.
Adrenergic beta-Antagonists↗
Detection of c-Ki-ras point mutation from pancreatic juice. A useful diagnostic approach for pancreatic carcinoma.
Cytological diagnosis of pancreatic carcinoma sometimes poses difficulties in distinguishing malignant from benign cells. Recent molecular study of pancreatic carcinoma has revealed a very high incidence of a point mutation of the c-Ki-ras oncogene at codon 12 in this neoplasm. To take advantage of this technique for the diagnosis of pancreatic carcinoma, we attempted to amplify the c-Ki-ras gene from endoscopically obtained pancreatic juice by isolation of DNA and polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). PCR was possible in approx 70% of the cases. A point mutation was nonradioisotopically detected in 4 of 6 pancreatic carcinomas and in one intraductal papillary neoplasm, whereas no mutation was detected in other cases. Thus, this method was thought to be useful for the diagnosis of pancreatic carcinoma.
Codon↗
New approach to the estimation of the extent of myocardial fibrosis in patients with dilated cardiomyopathy: use of signal-averaged electrocardiography.
To determine whether the extent of myocardial fibrosis in dilated cardiomyopathy could be estimated noninvasively, signal-averaged electrocardiograms were recorded in 32 patients with dilated cardiomyopathy, followed by left ventricular endomyocardial biopsy. The root mean square voltage for the last 40 msec (V40), the duration of the filtered QRS complex (fQRSd) and the duration of low amplitude signals < 40 microV (LAS) were obtained by signal-averaged electrocardiography. The extent of fibrosis in all biopsy samples was measured by the point-counting method. The extent of myocardial fibrosis closely correlated with fQRSd (r = 0.623, p < 0.001), LAS (r = 0.570, p < 0.001), and V40 (r = -0.355, p < 0.05). When fibrosis was classified into intercellular and interfascicular types, the extent of intercellular fibrosis more closely correlated with fQRSd (r = 0.695, p < 0.0001), LAS (r = 0.640, p < 0.0001), and V40 (r = -0.533, p < 0.005). These results suggest that signal-averaged electrocardiograms might be useful for estimation of the extent of myocardial fibrosis, especially intercellular fibrosis in patients with dilated cardiomyopathy.
Adult↗