Biomedical subjects
Publications and source records attributed to G Tamura.
The concomitant injection of isoproterenol with house dust allergen inhibited the immediate skin allergic reaction in asthmatic patients. Pretreatment with dibutyryl cyclic AMP showed an inhibitory effect on the whealing formation to the allergen. Those results showed the inhibitory effect of isoproterenol on the skin reaction through increasing intracellular cyclic AMP levels.
The effectiveness of acupuncture therapy for asthmatic patients was studied by continuous monitoring of the respiratory function. Using the forced 5 Hz oscillating technique, the investigators could estimate the change of total respiratory resistance (Rrs) of asthmatic patients throughout their reception of acupuncture therapy. They performed a single-blind study consisting of a placebo acupuncture, a placebo stimulation and real acupuncture therapy For further control of this study, they selected an easily locatable point for needle placement among the many traditional meridian points. In 10 out of 26 acupuncture therapies. Rrs significantly decreased while in only one of 17 placebo treatments did Rrs decrease.
The promoter and the amino-terminal region of phoA, the structural gene for alkaline phosphatase of Escherichia coli K12, was cloned by using a promoter cloning vector pMC1403. The nucleotide sequence of the cloned fragment has been determined. A sequence encoding the amino-terminal portion of mature alkaline phosphatase is found and it is preceded by a sequence encoding the signal peptide. The signal peptide consists of 21 amino acids; Met-Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr-Lys-Ala. The translation initiation codon is GUG, which is preceded by the Shine-Dalgarno sequence GGAG. Upstream to these sequences, there is a typical procaryotic promoter. TATAGTC for the Pribnow box. Around the Pribnow box, there are several dyad symmetrical sequences, which may probably be concerned with the regulation of this gene.
When deoxycorticosterone acetate (DOCA)-loaded uninephrectomized rats were fed on standard laboratory pellet diet and 1% saline for 5 weeks, caloric homeostasis became abnormal resulting in (a) hyperlipidemia, (b) cholesterol deposit in the heart, (c) significant reduction of triglycerides in the aorta, heart and liver and (d) a 60% increase in the cardiac free fatty acids (FFA) on one hand and a 50% reduction of the hepatic FFA on the other. These facts suggest that the hypertension severely reduces hepatic lipogenesis, whereas the cardiovascular system depends much more on FFA as a metabolic fuel than on glucose. This idea is supported by the deficiency in total body potassium (K) and decrease in serum immunoreactive insulin (IRI) which occur in the hypertension. These alterations were attenuated by the fungal prenylphenols, 4-0-methylascochlorin (MAC) and ascofuranone (AF). The protective effect seems to be partly attributable to the counteraction to DOCA. In addition, the agents caused a specific increase of renal water reabsorption. MAC treatment resulted in a particularly marked reduction of saline intake and excretion of unusually thick urine with 2.8 times higher sodium (Na) concentration than in the DOCA/saline control rats.
A mutant, CTM422, resistant to low dose of tunicamycin (TM) was isolated from Chinese hamster ovary (CHO) cells, and it showed 7- to 10-fold higher resistance to TM than CHO. We further mutagenized CTM422, to isolate TM-high-resistant mutants which were resistant to about 100-fold higher dose of TM than CHO. The TM-high-resistant mutants (N101 and N102) acquired about 4-fold higher cross-resistance to 2-deoxy-d-glucose than CHO or CTM422, while both CHO and CTM422 showed similar sensitivity to 2-deoxy-d-glucose. TM-low-resistance appeared to be codominant, and TM-high-resistance was partially codominant against TM sensitivity, respectively. The transfer activity of N-acetylglucosamine from UDP-N-acetylglucosamine into the lipid fraction with CHO and CTM422 cell extracts was inhibited by TM to a similar extent, while the extract of N102 cells showed about 10-fold higher resistance to TM than CHO or CTM422.
The effect of 4-O-methylascochlorin (MAC), an experimental hypocholesterolemic agent, on cholesterol metabolism was investigated in rats in two separate experiments. The administration of MAC for 2 and 6 consecutive weeks at daily doses of 100-135 mg/kg resulted in reduction in serum cholesterol levels of 16% after 2 weeks of treatment in the first experiment, and 13% after 6 weeks in the second experiment in comparison to the corresponding controls. MAC administered at a daily dose of 100 mg/kg for 2 weeks showed a significant increase in the biliary excretion of bile acids and cholesterol in bile-duct cannulated rats with or without the administration of taurocholate. In the second experiment, MAC treatment for 6 weeks produced a marked increase in the fecal output of acidic sterols during a 2 to 6-week period. MAC treatment also further enhanced hepatic cholesterol 7 alpha-hydroxylase in the rats. Therefore, it appears that the mechanism of serum cholesterol lowering due to MAC is related to the enhancement of hepatic bile acid synthesis and the increase in biliary and fecal excretion of bile acids.
L . P3 cells grown in serum-free synthetic medium produced a colony-stimulating factor (CSF: a sialoglycoprotein stimulating proliferation of granulocyte-macrophage progenitor cells). Addition of tunicamycin (2 microgram/ml) or 2-deoxy-D-glucose (10 mM) to the culture neither decreased the yield of CSF relative to the cell number grown nor induced heterogeneity of the produced CSF. However, CSF produced in the presence of tunicamycin (CSF-tm) was about 23 percent smaller in its molecular weight than normally produced CSF (CSF-normal). Similarly, the addition of 2-deoxy-D-glucose resulted in the formation of a CSF (CSF-dGlc) which was about 16 percent smaller than CSF-normal. However, both CSF-tm and CSF-dGlc seemed to have retained sialic acid residues, because they were focused respectively at pH 4.2 and pH 3.7 upon isoelectric focusing, and both were converted to a pH 5.2 species by treatment with neuraminidase. In addition, CSF-tm was significantly less heat-stable than CSF-normal, whereas CSF-dGlc was only slightly less stable. These results suggest that complete glycosylation of the factor is not necessary for its production nor for its stimulatory action on the proliferation of myeloid stem cells, but is necessary for its maximal stabilization.
A new antibiotic, reductiomycin was isolated from the culture broth of Streptomyces griseorubiginosus. The antibiotic has the molecular formula C14H15O6N(M.W. 293). Reductiomycin is active against Gram-positive bacteria, fungi, and newcastle disease virus.
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Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor), which is suggested to be a glycoprotein. On the other hand, growth and differentiation of normal precursor cells of macrophages and granulocytes can be stimulated by a glycoprotein termed colony-stimulating factor (CSF). Mouse fibroblast L929 cells were found to produce both the D-factor and CSF. The properties of the D-factor and CSF and the roles of carbohydrates in the molecules of these factors were examined using tunicamycin, a specific inhibitor of asparaginase-linked glycosylation. Although both the D-factor and CSF were produced by L-cells in usual medium containing fetal calf serum, production of D-factor, but not CSF, was reduced by omission of serum from the medium. The activity of the D-factor was slightly decreased by treating the L-cells with tunicamycin (0.5 microgram/ml) in the presence of 2% fetal calf serum, without any decrease in CSF activity. Conditioned medium of L-cells incubated with or without tunicamycin was fractionated by gel filtration on a Sephadex G-200 column. Normal D-factor appeared as a single peak with an apparent molecular weight of 67,000. D-factor produced in the presence of tunicamycin had an apparent molecular weight of 25,000. On the other hand, most of the CSF was eluted in the void volume, even when it was produced in the presence of tunicamycin. The D-factor produced in the presence of tunicamycin was more sensitive than normal D-factor was to trypsin or heat treatment at 70 degrees. The CSF produced in the presence of tunicamycin was resistant to these treatments. These results indicate that the D-factor is distinct from CSF. Furthermore, the results suggest that the D-factor produced by L-cells is also a glycoprotein and that, although carbohydrate is not essential for production or activity of the D-factor, it contributes to stabilizing the protein portion of D-factor.
A newly synthesized mycophenolic acid (MPA) derivative, ethyl O-[N-(p-carboxyphenyl)-carbamoyl]-mycophenolate (CAM, NSC-297879D) was tested for antitumor activity, when given orally, against transplantable murine tumors. The compound was markedly effective against transplantable murine tumors. The compound was markedly effective against leukemia P388 and L1210, lymphoma L5178Y, mastocytoma P815 and sarcoma Meth-A, moderately effective against sarcoma-180, C3MC2 and BAMC1, Ehrlich carcinoma, Lewis lung carcinoma and melanoma B16 and marginally effective against hepatoma MH134. The antitumor effects were manifested not only in growth inhibitory effects on subcutaneously transplanted tumors but also in the prolongation of life span of mice int which the tumors had been inoculated intraperitoneally or subcutaneously. The growth of primary transplants of a mammary tumor which developed spontaneously in a C3H/He mouse was inhibited by consecutive administration of CAM frm the 34th day after the transplantation. Oral CAM was more potent than its mother compound, MPA, in the tumor models examined. These results indicate that orally administered CAM has a wide antitumor spectrum.
Whole-embryo culture techniques have advanced to the point where the study of normal and abnormal primary palate development in vitro is possible. The purpose of this study was to ascertain whether localized administration of tunicamycin (TM) an inhibitor of protein glycosylation, into the region of the developing primary palate would induce cleft lip in culture. Rat embryos were explanted on late day 11 of gestation and cultured with open yolk sacs for 40 h. TM was administered by implanting a sterile eyelash soaked in TM into the nasal placode region. The contralateral placode was used as the control by implanting an eyelash lacking TM. Under these conditions, TM-treated placodes were found to develop cleft lip in 14 out of 15 embryos compared to 0 for the controls. These experiments demonstrate that localized administration of TM results in cleft lip formation in whole embryo culture. The technique of localized administration of drugs and teratogens in whole embryo culture should prove useful for similar studies on embryonic development.
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Tunicamycin, an antibiotic that specifically blocks the synthesis of N-acetylglucosamine-lipid intermediates and thereby prevents glycosylation of glycoproteins, induced differentiation of both human (HL-60) and murine (M1) myeloid leukemia cell lines in culture. At 0.1-1.0 microgram/ml, it induced differentiation of both HL-60 and M1 cells, characterized by increase in phagocytic cells and changes to resemble mature myeloid cells. Fc receptors were also induced in M1 but not in HL-60 cells; induction of intracellular lysozyme activity was not detected in either HL-60 or M1 cells. With this concentration of tunicamycin, there was marked decrease in rate of incorporation of radioactive glucosamine into macromolecules and a decrease in the rate of DNA synthesis. These data show that glycosylation of cellular proteins has an important role in maintaining these myeloid leukemia cells in an undifferentiated state in culture. The results also indicate that induction of phagocytosis in both HL-60 and M1 myeloid leukemia cells and of Fc receptors in M1 cells does not require continued synthesis of the oligosaccharide portions of cellular proteins by the lipid-linked pathway.
Many substances in the plant kingdom and in man's diet occur as glycosides. Recent studies have indicated that many glycosides that are not mutagenic in tests such as the Salmonella test become mutagenic upon hydrolysis of the glycosidic linkages. The Salmonella test utilizes a liver homogenate to approximate mammalian metabolism but does not provide a source of the enzymes present in intestinal bacterial flora that hydrolyze the wide variety of glycosides present in nature. We describe a stable cell-free extract of human feces, fecalase, which is shown to contain various glycosidases that allow the in vitro activation of many natural glycosides to mutagens in the Salmonella/liver homogenate test. Many beverages, such as red wine (but apparently not white wine) and tea, contain glycosides of the mutagne quercetin. Red wine, red grape juice, and tea were mutagenic in the test when fecalase was added, and red wine contained considerable direct mutagenic activity in the absence of fecalase. The implications of quercetin mutagenicity and carcinogenicity are discussed.
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