[Hydropericardium].
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Diagnosis, Differential↗
Biomedical subjects
M Shimizu
Publications and source records attributed to M Shimizu.
[Chylopericardium].
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Diagnosis, Differential↗
Genetic regulation of telomerase in a multiple pathways model to cellular senescence.
Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the program of senescence in normal cells. Genes involved in the senescence program have been mapped to over 10 different genetic loci by introduction of normal human chromosomes via microcell fusion. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. The loss of indefinite growth potential was either with or without the loss of telomerase activity activity and shortening of telomeres in the microcell hybrids containing the introduced chromosome. The findings suggest that telomerase regulation is one of multiple pathways to cellular senescence.
Cellular Senescence↗
Hydrophilic surroundings requisite for the solubilization of proteins related with their hydrophobicity in the AOT reversed micellar extraction.
The reversed micellar extraction (AOT/isooctane system) using the phase transfer method was investigated in relation to the AOT concentration and the water solubilization for ribonuclease A, lysozyme and cytochrome c. The minimal AOT concentration required for 100% forward extraction was obtained for these proteins. At the minimal AOT concentration, the hydrophilic surroundings, i.e. the molar ratio of water to extracted protein in the organic phase, were independent of the protein concentration for each protein. The hydrophilic surroundings of these proteins were linearly related with Fisher's polarity ratio, p, as an index of the hydrophobicity of the protein. Using this linear relation, a procedure to estimate the sufficient AOT concentration for the protein extraction was proposed. In the cases of cytochrome c and lysozyme, the water concentration was larger than that in the protein-free system in spite of the same AOT condition. On the contrary, in the case of ribonuclease A, this large water uptake in the organic phase was not observed. These differences of water uptake were discussed in relation to the location of the protein in the AOT reversed micelles.
Animals↗
A new approach to determine the effect of mismatches on kinetic parameters in DNA hybridization using an optical biosensor.
We have demonstrated a simple yet direct method for determining the kinetic parameters in DNA-DNA interactions using biosensor technology based on the surface plasmon resonance phenomenon; a technique that does not require complex DNA labeling. To determine the effect of mismatches on the kinetics involved in DNA-DNA interactions, DNA hybridization kinetics were monitored in real time using synthetic oligonucleotides less than 20 bases in length which contained either a complementary sequence or mismatched bases. Upon analysis of the kinetic parameters obtained in oligonucleotide hybridization, we found that they were significantly affected by the presence of mismatches as well as by their number and location in a DNA duplex. In addition, the presented biosensor method is sensitive enough to detect kinetic effects caused by the presence of a single-mismatched base pair. Our findings strongly suggest that analysis of kinetic parameters involved in DNA-DNA interactions is advantageous for detecting the presence of mismatch base pairs in a DNA duplex.
Base Sequence↗
Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA.
The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.
Bacterial Proteins↗
Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases.
Long CTG triplet repeats which are associated with several human hereditary neuromuscular disease genes are stabilized in ColE1-derived plasmids in Escherichia coli containing mutations in the methyl-directed mismatch repair genes (mutS, mutL, or mutH). When plasmids containing (CTG)180 were grown for about 100 generations in mutS, mutL, or mutH strains, 60-85% of the plasmids contained a full-length repeat, whereas in the parent strain only about 20% of the plasmids contained the full-length repeat. The deletions occur only in the (CTG)180 insert, not in DNA flanking the repeat. While many products of the deletions are heterogeneous in length, preferential deletion products of about 140, 100, 60, and 20 repeats were observed. We propose that the E. coli mismatch repair proteins recognize three-base loops formed during replication and then generate long single-stranded gaps where stable hairpin structures may form which can be bypassed by DNA polymerase during the resynthesis of duplex DNA. Similar studies were conducted with plasmids containing CGG repeats; no stabilization of these triplets was found in the mismatch repair mutants. Since prokaryotic and human mismatch repair proteins are similar, and since several carcinoma cell lines which are defective in mismatch repair show instability of simple DNA microsatellites, these mechanistic investigations in a bacterial cell may provide insights into the molecular basis for some human genetic diseases.
Adenosine Triphosphatases↗
Pausing of DNA synthesis in vitro at specific loci in CTG and CGG triplet repeats from human hereditary disease genes.
Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase beta paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70 degrees C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausing increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.
Base Sequence↗
High avidity state of leukocyte function-associated antigen-1 on rheumatoid synovial fluid T lymphocytes.
The integrin LFA-1 (CD11a/CD18) is a cell surface adhesion molecule required for leukocyte extravasation and subsequent immune and inflammatory responses. Rapid transition between nonadherent and adherent states of LFA-1 is of key importance to Ag-specific recognition of T lymphocytes. In this paper, LFA-1-mediated adhesiveness of peripheral blood (PB) and synovial fluid (SF) T lymphocytes to affinity-purified ICAM-1-coated plates was studied in patients with rheumatoid arthritis (RA) and in patients with non-RA panels, including osteoarthritis, ankylosing spondylitis, erythema nodosum, pseudogout, and pustulosis. LFA-1-mediated adhesiveness of SF T lymphocytes was not observed in any of the 10 non-RA patients studied, although cross-linking of the TCR on lymphocytes from these patients rapidly converted LFA-1 to an adhesive state. In contrast, SF T lymphocytes from 10 of 12 RA patients exhibited LFA-1-mediated adhesiveness without a requirement for cross-linking of the TCR. No difference was seen in the cell surface density of LFA-1 between non-RA and RA T lymphocytes, suggesting that the difference in adhesiveness was due to a high avidity state of LFA-1 on SF T lymphocytes in RA. Furthermore, exposure of PB T lymphocytes, which showed a low avidity state of LFA-1, to whole SF from RA patients that was depleted of T lymphocytes could induce a high avidity state of LFA-1 in vitro. Cellfree SF from RA patients also could stimulate adhesiveness, although to a lesser extent. These data suggest the existence of a LFA-1-activating environment that is selectively found in SF from RA patients.
Adult↗
Determination of 25-hydroxyvitamin D3 in human plasma using a non-radioactive tetranorvitamin D analogue as an internal standard.
A convenient non-radioactive method for assaying plasma 25-hydroxyvitamin D3 is described. The method uses 22-hydroxytetranorvitamin D3 as an internal standard and includes two-step liquid- and solid-phase extractions and quantification by normal-phase HPLC. The intra- and inter-assay coefficients of variation were 2.2% and 2.4%, respectively, and the analytical recovery of 25-hydroxyvitamin D3 added to plasma was quantitative. Assay linearity was obtained in the range 0.5-4.0 ml of plasma. When compared with the method employing a radioactive 25-hydroxyvitamin D3 tracer, the correlation coefficient was 0.990 (slope 0.999 and intercept 1.19 ng/ml).
Adult↗
Effects of long-term omeprazole treatment on adult rat gastric mucosa--enhancement of the epithelial cell proliferation and suppression of its differentiation.
Effects of long-term omeprazole treatment on the process of epithelial cell proliferation and differentiation in the adult rat gastric mucosa were investigated. Animals were treated with omeprazole (25 mg/kg body weight/day) for 28 days to induce anacidity in the stomach. The treatment induced a marked decrease in the number of chief cells in the gastric mucosa and at the same time an increase in that of immature pepsinogen-producing cells expressing class III mucin. This was accompanied by a decrease to 60% and 10% of the control values in the mucosal levels of pepsinogen and its mRNA, respectively. Moreover, the expression of cathepsin E in surface mucous cells was reduced. Cell proliferation studies revealed that the rate of bromodeoxyuridine-labeled cells was increased by omeprazole. The above-described changes were reversed by cessation of the treatment and they were not caused by the omeprazole-treatment at a dose which does not induce anacidity in the stomach. These results suggest that long-term omeprazole treatment reversibly increases the epithelial cell proliferation and suppresses its differentiation in the adult rate gastric mucosa probably by altering the acidic environment specific for the stomach.
Animals↗
Diversity of T cell receptor delta-chain cDNA in the thymus of a one-month-old pig.
Pig TCR delta-chain cDNA sequences were obtained from the anchored PCR products of the reverse-transcribed RNA from the thymus of a 1-mo-old germfree pig. Among 43 sequences analyzed, 4 different J delta sequences were found, and in the cDNA sequences of rearranged V-D-J junctional regions, three putative D delta sequences were assigned. Also, the V delta region sequences were grouped into five families. One family, V delta 1, consisted of a large number of members and the other families consisted of limited members as determined by cDNA sequences as well as genomic Southern blots. Using PCR, V delta 1 gene segments were also obtained from liver genomic DNA of the same individual pig. Thirty-one distinct sequences were found for the V delta 1 family including the sequences obtained from both the thymic RNA and the PCR product of liver DNA. All the V delta 1 genes were found to be associated with almost identical leader gene sequences. The amino acid sequences of the complimentarity-determining region 3 coded by the V-D-J joints gave long and variable lengths with a high content of tryptophan and charged amino acids. The length of the complimentarity-determining region 3 in the porcine TCR delta-chain supports the idea that TCR delta-chain is designed to see Ags directly similar to the Ig heavy chain. Data presented from this study shows that the porcine TCR delta-chain has extensive V region diversity as well as junctional diversity. The combination of these two creates a level of diversity that is the highest of any lymphocyte Ag receptor studied so far.
Amino Acid Sequence↗
Sarin poisoning in Matsumoto, Japan.
A presumed terrorist attack with sarin occurred in a residential area of the city of Matsumoto, Japan, on June 27, 1994. About 600 residents and rescue staff were poisoned; 58 were admitted to hospitals, and 7 died. We examined clinical and laboratory findings of 264 people who sought treatment and the results of health examinations on 155 residents done 3 weeks after the poisoning. Findings for severely poisoned people were decreases in serum cholinesterase, acetylcholinesterase in erythrocytes, serum triglyceride, serum potassium and chloride; and increases in serum creatine kinase, leucocytes, and ketones in urine. Slight fever and epileptiform abnormalities on electroencephalogram were present for up to 30 days. Examination revealed no persisting abnormal physical findings in any individual. Acetylcholinesterase returned to normal within 3 months in all people examined. Although subclinical miosis and neuropathy were present 30 days after exposure, almost all symptoms of sarin exposure disappeared rapidly and left no sequelae in most people.
Acute Disease↗
Study of stimulation-secretion coupling in a flow culture system: periodic secretion of hepatocyte growth factor by interleukin-1 alpha-stimulated human embryonic lung fibroblasts.
In order to closely examine stimulation-secretion coupling, the authors developed a 'flow culture system' in which it is possible to continuously replace the culture medium. This lessens the effects of cell-derived products observed in the conventional culture system. We compared our flow culture system and the conventional culture system based on the secretion patterns and concentrations of human hepatocyte growth factor (HGF) produced by interleukin 1 alpha (IL-1 alpha)-stimulated human embryonic lung fibroblasts (MRC-5). While the cells in the conventional culture system secreted HGF at a nearly constant rate, the cells in the flow culture system secreted HGF periodically. Even short-term stimulation with IL-1 alpha for 4 h resulted in significant HGF secretion continuing for at least 46 h. Thus the inflammatory cytokine IL-1 alpha was shown to modulate fibroblast secretion of HGF. The periodic secretion of HGF may play an important role in tissue repair and regeneration. Based on the results of actually applying it, we conclude that our flow culture system is an efficient and accurate model for the detailed examination of stimulation-secretion coupling.
Cell Line↗
Effect of CCK-B/gastrin receptor antagonist on pepsinogen-producing cells during omeprazole treatment.
The present study investigated the effect of long-term treatment with omeprazole on pepsinogen-producing cells and examined whether the selective CCK-B/gastrin receptor antagonist was able to prevent the omeprazole-induced changes, if occurred, in rat stomach. Rats were treated with omeprazole and/or the CCK-B/gastrin receptor antagonist for 28 days. As a result, omeprazole markedly reduced mucosal pepsinogen activity and its mRNA concentration in rat stomach. Morphologically, in fundic glands omeprazole drastically decreased the proportion of mature chief cells and reciprocally increased that of immature chief cells which were positive for class III mucin. These effects of omeprazole were attenuated by an addition of the CCK-B/gastrin receptor antagonist. Our results suggest that omeprazole retards the differentiation of chief cells in fundic mucosa probably through hypergastrinemia in adult rat.
Animals↗
Arterial-injection chemotherapy for hepatocellular carcinoma using monodispersed poppy-seed oil microdroplets containing fine aqueous vesicles of epirubicin. Initial medical application of a membrane-emulsification technique.
BACKGROUND: Iodized poppy-seed oil (IPSO) has a property of depositing itself selectively in the cells of hepatocellular carcinoma (HCC). A mixture of anticancer agents and IPSO has been used widely because IPSO accumulates in tumors, but its usefulness appears limited because the anticancer agents become separated easily from IPSO and do not remain in the tumor. The authors prepared a long term inseparable, water-in-oil-in-water emulsion (W/O/W) for use in arterial-injection therapy for patients with HCC and evaluated its clinical usefulness. METHODS: The W/O/W was prepared by a membrane-emulsification technique using a controlled pore glass with 10.6-microns pores. From December 1992 to January 1994, the W/O/W containing 8-60 mg of epirubicin was applied to the hepatic arterial-injection therapy for 21 patients with HCC to determine its antitumor and side effects. RESULTS: After arterial infusions with W/O/W, an evident antitumor effect was observed in all 13 patients treated with W/O/W containing 40 mg or more of epirubicin with or without gelatin-sponge particles used simultaneously. In the group treated with the W/O/W containing a high dose (40 mg or more) of epirubicin, even though the gelatin-sponge particles were not used, tumor size was reduced in six of seven patients, and a 50% or greater decrease of initial alpha-fetoprotein (AFP) levels within 14 days was observed in all four patients who showed abnormal levels of serum AFP before treatment. One partial necrosis and two complete necroses of three resected tumors were confirmed histopathologically. Fever (in all patients), nausea (in two), pain in the right upper quadrant of the abdomen (in two), and slight cough (in one) were noted as minor side effects. CONCLUSIONS: To the authors' knowledge, this is the first clinical trial using this emulsion prepared by the membrane-emulsification technique. Emulsification using a fine-pore glass membrane of equal pore size (i.e., controlled-pore glass membrane) is a new technique for preparing lipid microdroplets of equal size (monodispersed) containing aqueous fine microdroplets to form W/O/W: This technique of chemoembolization can be used to treat patients with HCC.
Aged↗
Identity elements of Saccharomyces cerevisiae tRNA(His).
Recognition of tRNA(His) by Saccharomyces cerevisiae histidyl-tRNA synthetase was studied using in vitro transcripts. Histidine tRNA is unique in possessing an extra nucleotide, G-1, at the 5' end. Mutation studies indicate that this irregular secondary structure at the end of the acceptor stem is important for aminoacylation with histidine, while the requirement of either base of this extra base pair is smaller than that in Escherichia coli. The anticodon was also found to be required for histidylation. The regions involved in histidylation are essentially the same as those in E.coli, whereas the proportion of the contributions of the two portions distant from each other, the anticodon and the end of the acceptor stem, makes a substantial difference between the two systems.
Acylation↗
Determination of beta 3-adrenoceptor mediated lipolysis in human fat cells.
The existence and relative importance of beta 3-adrenoceptors in man is still controversial. The aim of the present study was 1) to find further evidence for the existence of functional beta 3-adrenoceptors in human fat, and 2) to investigate factors that may influence this beta 3-adrenoceptor function. Fifty individuals were examined. Lipolysis mediated by the selective beta 3-adrenoceptor agonist CGP 12177 in omental fat cells correlated with the response in subcutaneous fat cells. However, lipolysis was more pronounced in omental as compared to subcutaneous adipocytes, the intrinsic activity for CGP 12177 was 41% and 33%, respectively, while dobutamine, terbutaline and norepinephrine were full agonists. Both the lipolytic response and the sensitivity to CGP 12177 correlated with the effects of norepinephrine in the omental fat cells (r2 = 0.68 and 0.50, respectively, p = 0.0001). The beta 3-adrenoceptor mediated lipolytic response did also correlate with the responses induced by beta 1- and beta 2-agonists and by postreceptor acting agents. The antagonistic properties (pA2) of the beta-adrenoceptor subtypes were also investigated. The pA2 for the selective beta 1- and beta 2-adrenoceptor antagonists versus CGP 12177-induced lipolysis were 2 to 3 log units lower than those for the beta 1-antagonist versus dobutamine or for the beta 2-antagonist versus terbutaline. Furthermore, bupranolol had a significantly better antagonistic effect (pA2 7.17, p < 0.001) on the CGP 12177-induced lipolysis than had the beta 1- and beta 2-adrenoceptor antagonists (pA2 6.26 and 6.05, respectively). These data clearly support the existence of a third human beta-adrenoceptor. Several factors may contribute to the contradictory beta 3-adrenoceptor results in man. The sensitivity of the different lipolytic systems vary considerably. Omental fat cells are preferable to subcutaneous cells for beta 3-adrenoceptor studies in man. The beta 3-responses are more attenuated in isolated fat cell preparations than in tissue fragments. Furthermore, as the beta 3-adrenoceptor activity correlates to the norepinephrine activity, more pronounced effects will be expected in catecholamine sensitive subjects. At present, the number of tools available for beta 3-adrenoceptor studies are limited, and the receptor is hard to study, why it is essential to perform beta 3-adrenoceptor studies under optimal conditions in order to obtain conclusive effects.
Adipocytes↗