Biomedical subjects
Publications and source records attributed to M Nishijima.
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Lipid A 3-ether analogues were synthesized from allyl 2-deoxy-4,6-O-isopropylidene-2-trifluoroacetamido-alpha-D-glucopyr anoside and 3,4,6-tri-O-acetyl-2-trifluoroacetamido-alpha-D-glucopyranosyl bromide. The compound lost completely the endotoxic activity.
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A phosphatidylserine-auxotrophic mutant of cultured Chinese hamster ovary (CHO) cells, PSA-3, is defective in phosphatidylserine synthase I activity. Transfection of PSA-3 cells with a cDNA expression library of CHO-K1 (the parent of PSA-3) yielded a phosphatidylserine-prototrophic transformant with normal phosphatidylserine synthase I activity. Using a cDNA segment retrieved from the transformant as a probe, a cDNA clone (pssA) responsible for phosphatidylserine prototrophy was isolated from the original cDNA library by colony filter hybridization. Introduction of the pssA cDNA into PSA-3 cells restored the phosphatidylserine content, and the resultant transformant exhibited 15-fold higher specific phosphatidylserine synthase I activity than CHO-K1 cells. The nucleotide sequence of the pssA cDNA contained a single long open reading frame capable of encoding a protein of 471 amino acid residues with several putative membrane-spanning domains. Our results indicated that the pssA cDNA encodes an integral membrane protein essential for phosphatidylserine synthase I activity.
A phosphatidylserine-auxotrophic mutant of cultured Chinese hamster ovary cells, PSA-3, manifests a defect in phosphatidylserine synthase I activity (Kuge, O., Nishijima, M., and Akamatsu, Y. (1986) J. Biol. Chem. 261, 5790-5794). We cloned a Chinese hamster gene, designated pssC, which was able to transform the PSA-3 cell line to a phosphatidylserine prototroph. The resultant transformant contained phosphatidylserine in normal amounts but remained defective in phosphatidylserine synthase I activity, indicating that pssC is a suppressor gene. Using the genomic fragment of pssC as a probe, a cDNA clone of pssC was isolated, and its nucleotide sequence was determined. A computer search through a protein data bank revealed that pssC had homology with the Escherichia coli psd gene encoding the proenzyme of phosphatidylserine decarboxylase at the amino acid level. Introduction of the cloned pssC gene into PSA-3 resulted in a 2-fold increase in phosphatidylserine decarboxylase activity. When the pssC cDNA was placed downstream of the yeast GAL1 promoter and introduced into yeast Saccharomyces cerevisiae cells, the phosphatidylserine decarboxylase activity increased in a galactose-dependent manner. These results indicate that pssC encodes phosphatidylserine decarboxylase. The mechanism by which pssC complements the defect of PSA-3 in phosphatidylserine biosynthesis is discussed.
Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM). Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment. Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.
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UNLABELLED: In the present study, in order to clarify the diurnal changes in pregnant women, fetuses and neonates, pineal hormone (melatonin) was analyzed with a RIA Kit. RESULTS: 1. The venous melatonin concentration at night was higher than during the day in healthy women and full term pregnant women without labor. 2. Melatonin concentrations in maternal venous blood (MV) (67.44 pg/ml) were significantly higher than in umbilical venous blood (UV) (37.78 pg/ml). 3. A distinct midnight peak of melatonin was observed in MV, and UV at delivery. 4. Significant correlations were observed between MV, UV or umbilical arterial blood (UA), but those were non linear. 5. In neonates on the first to fourth day after birth, no midnight peak was able to be clearly observed, but fluctuations were observed. These result indicate that there is a diurnal change in melatonin in full term pregnant women without labor and women in labor. And we suggest that the pineal gland of early neonates secretes melatonin, but no diurnal change in melatonin is established up to 4th day after birth.
Two cases of a disproportionately large communicating fourth ventricle (DLCFV) which developed after suboccipital craniectomy are reported. The first case was that of a 43-year-old female who sustained subarachnoid hemorrhage, and the second case was that of a 12-year-old male with medulloblastoma. Patency of the aqeductal canal and occlusion of the fourth ventricle outlets were demonstrated in both cases by metrizamide ventriculography, with intraventricular injection of contrast material. Clinical history was analyzed with repeated CT scan, and pathogenesis of DLCFV was discussed with review of the literature.
We devised an in situ assay method for the activity of serine palmitoyltransferase (SPT) that catalyzes the first step in sphingolipid biosynthesis and isolated a temperature-sensitive mutant of Chinese hamster ovary cells with thermolabile SPT. This mutant stopped growing at 40 degrees C after several generations, although the cells grew at 33 and 37 degrees C at rates similar to those of the parent. The SPT activity in cell homogenates of the mutant grown at low temperatures was 4-8% of that in the parent homogenates. When the cells were cultured for several generations at 40 degrees C, the activity in the mutant homogenate became negligible. When cell homogenates were incubated at 45 degrees C before enzyme assay, mutant SPT was more markedly inactivated than parental SPT, indicating that mutant SPT had become thermolabile. The rates of de novo synthesis of sphingolipids in the mutant were much slower at 40 degrees C than at lower temperatures, in contrast to those in the parent. The sphingomyelin content in the mutant cultivated at 40 degrees C for several generations was also less than that at low temperatures. These results indicate that SPT functions in the main pathway for sphingolipid biosynthesis. The temperature-sensitive growth of the mutant defective in sphingolipid synthesis suggests that sphingolipid(s) plays an essential role in cell growth.
By means of an in situ colony autoradiographic assay for the incorporation of [14C]inositol into the trichloroacetic acid-insoluble fraction, we have isolated a mutant of cultured Chinese hamster ovary cells defective in inositol transport, named mutant 648. Through comparison of the inositol uptake activity of 648 cells with that of the parental cells with various concentrations of inositol and sodium, it has been demonstrated that Chinese hamster ovary cells possess a sodium-dependent transport system for inositol, and that 648 cells lack this system. The sodium-dependent uptake is inhibited by 2,4-dinitrophenol and ouabain, and the intracellular concentration of inositol exceeds the extracellular concentration during the uptake period, indicating that it is active transport, at least partially driven by the sodium gradient generated by Na+,K(+)-ATPase. The apparent Km for inositol has been estimated to be 12.0 microM. It is inhibited by hyperglycemic concentration of D-glucose in a competitive fashion.
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Lipopolysaccharide (LPS)-resistant mutants which did not respond to LPS were isolated from a macrophage-like mouse cell line, J774.1. Unlike the parental J774.1 cells, these mutants grew even in LPS added medium as well as in normal growth medium without any morphological changes. Assay of 125I-LPS binding to the cell monolayers revealed that one of these LPS-resistant mutants (LR-9) was strikingly defective in LPS-binding activity. Scatchard plot showed that LR-9 cells lacked the high affinity binding sites which were present in J774.1. The high affinity binding was inhibited by addition of excess unlabeled LPS, lipid A, lipid IVA (tetraacyl-beta(1'-6)-linked D-glucosamine disaccharide-1,4'-bisphosphate), and lipid X (2,3-diacylglucosamine 1-phosphate) and sensitive to proteinase K. LPS enhanced O2- generation and the release of arachidonic acid in J774.1 cells but not in LR-9 cells. Other stimulants such as zymosan and 12-O-tetradecanoylphorbol 13-acetate, however, induced the release of arachidonic acid in LR-9 cells as well as in J774.1 cells. LPS-photocross-linked assay allowed the identification of 65- and 55-kDa LPS-binding proteins in the membrane fraction of J774.1 cells. Both of the bands were not detectable in that of LR-9 cells and disappeared by competing with unlabeled LPS or lipid X. These results show that one or both of the two LPS-binding proteins might relate to the specific membrane receptor for LPS.
Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.
A modified indicator gas washout method was developed to measure functional residual capacity (FRC) during high-frequency oscillatory ventilation (HFOV) without interruption of HFOV. A hot-wire flowmeter and medical gas analyzer measured the flow rate and argon concentration, respectively, at the expiratory end of the respiratory circuit. Upstream of the hot-wire flowmeter, two heat-and-moisture exchangers for resistance and a rubber balloon for capacitance were placed to convert the oscillating expiratory flow to an almost continuous flow. This made it possible to measure FRC during HFOV without interrupting HFOV. To measure the volume of the entire respiratory circuit, a 10 percent argon in 90 percent oxygen gas mixture was initially used as a bias flow, and after equilibration, the test gas was switched to 100 percent oxygen. By electrical integration of the product of the expiratory flow rate and argon concentration, the total amount of argon equilibrated in the entire respiratory circuit was calculated. The volume of the circuit was calculated by dividing the total amount of argon by the initial argon concentration. Functional residual capacity plus the volume of the respiratory circuit was similarly calculated and the difference was estimated as FRC. The accuracy and reproducibility of our method were evaluated by using a one-compartment lung model. There was a high correlation between the volume setting of the model lung and the estimated FRC. This method can be used to estimate FRC in a one-compartment lung model during HFOV, and it is potentially useful in clinical situations.
Two cases of vertigo with subclavian artery stenosis are presented. In both cases, the angiograms revealed left subclavian artery stenosis at the middle portion of the clavicle as well as kinked origin of the vertebral artery. After resection of the anterior scalene muscle, the stenosis and the vertigo resolved.
The phosphatidylethanolamine (PE) N-methyltransferase (MT) system is known to convert PE to phosphatidylcholine by three successive N-methylations. Phosphatidyl-N-monomethylethanolamine (PME) MT was purified 1,400-fold from mouse liver microsomes and separated from the PE-MT activity for the first time. This enzyme catalyzes N-methylations of PME and phosphatidyl-N,N-dimethylethanolamine, the intermediates of PE-MT system, but not PE, the initial substrate of the PE-MT system. In addition, a preparation with a different affinity to S-adenosyl-L-homocysteine catalyzing all the three methylations was obtained. These results suggest that at least two enzymes are involved in the PE-MT system.