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At least 19 recordsLinked to original sources
PubMed1991

Two possibly distinct prostaglandin E1 receptors in N1E-115 clone: one mediating inositol trisphosphate formation, cyclic GMP formation, and intracellular calcium mobilization and the other mediating cyclic AMP formation.

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.

Animals↗
PubMed1992

Dye-linked dehydrogenase activities for formate and formate esters in Amycolatopsis methanolica. Characterization of a molybdoprotein enzyme active with formate esters and aldehydes.

Cell-free extracts of methanol-grown Amycolatopsis methanolica contain dye-linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate-ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were found for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. (1987) Eur. J. Biochem. 166, 575-579] which was at that time considered to be an aldehyde dehydrogenase. Formate-ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and CO2 or a factor-formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.

Aldehydes↗
PubMed1991

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds.

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

8,11,14-Eicosatrienoic Acid↗
PubMed1983

Drug interactions. III. Formation of nitrosamines from therapeutic drugs. Formation, mutagenic properties and safety assessment of propranolol hydrochloride with respect to the intragastric formation of N-nitrosopropranolol under conditions found in patients.

In the preceding report, the kinetics of formation of N-nitrosopropranolol (NNP) from propranolol and inorganic nitrite were determined in solutions of hydrochloric acid over the range of pH similar to that found in the human stomach. In this communication, NNP formation was examined in human gastric juice and in the presence of organic nitrate ester vasodilator drugs. In comparison to HCl solutions, equivalent concentrations of propranolol and nitrite produced similar amounts of NNP in gastric juice; however, the yield increased as the pH was lowered and the kinetics of nitrosamine formation were different. Endogenous nitrite concentrations in 22 samples of human gastric juice were below the minimum concentration (10(-5) M) required for production of detectable levels of NNP. Maximal therapeutic dosages of propranolol (10(-2) M) incubated with isosorbide dinitrate (3,4-6.8 X 10(-3) M) or nitroglycerin (8.6 X 10(-4) M) also failed to produce NNP. However, NNP formed adventitiously during the concentration of aqueous and methylene chloride solutions that contained propranolol and organic nitrates, underscoring the importance of avoiding artifactual formation of nitrosamines. Furthermore, synthetic NNP was not mutagenic in either the Ames tester strains (TA92, TA98, TA100, TA1535, TA1537 and TA1538) or the hepatocyte-mediated mammalian cell mutagenesis assay. We conclude that NNP is unlikely to form in the stomach under conditions normally present in patients. Moreover, even if NNP formed under exceptional circumstances, this compound is unlikely to be a carcinogen. With respect to the potential formation of nitrosamines during drug dissolution in the stomach, long-term therapy with propranolol hydrochloride appears to be safe.

Biotransformation↗
PubMed1973

Formation of hydrogen and formate by Ruminococcus albus.

Radioisotopic growth studies with specifically labeled (14)C-glucose confirmed that Ruminococcus albus, strain 7, ferments glucose mainly by the Embden-Myerhof-Parnas pathway to acetate, ethanol, formate, CO(2), H(2), and an unidentified product. Cell suspensions and extracts converted pyruvate to acetate, H(2), CO(2), and a small amount of ethanol. Formate was not produced from pyruvate and was not degraded to H(2) and CO(2), indicating that formate was not an intermediate in the production of H(2) and CO(2) from pyruvate. Cell extract and (14)C-glucose growth studies showed that the H(2)-producing pyruvate lyase reaction is the major route of H(2) and CO(2) production. An active pyruvate-(14)CO(2) exchange reaction was demonstrable with cell extracts. The (14)C-glucose growth studies indicated that formate, as well as CO(2), arises from the 3 and 4 carbon positions of glucose. A formate-producing pyruvate lyase system was not demonstrable either by pyruvate-(14)C-formate exchange or by net formate formation from pyruvate. Growth studies with unlabeled glucose and labeled (14)CO(2) or (14)C-formate suggest that formate arises from the 3 and 4 carbon positions of glucose by an irreversible reduction of CO(2). The results of the studies on the time course of formate production showed that formate production is a late function of growth, and the rate of production, as well as the total amount produced, increases as the glucose concentration available to the organism increases.

Bacteria↗
PubMed1991

Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and pH: definition of the formate regulon.

The products of a minimum of 15 genes are required for the synthesis of an active formate-hydrogenlyase (FHL) system in Escherichia coli. All are co-ordinately regulated in response to variations in the oxygen and nitrate concentration and the pH of the culture medium. Formate is obligately required for transcriptional activation of these genes. Analysis of the transcription of one of these genes, hycB linked to the lacZ reporter gene, revealed that oxygen and nitrate repression of transcription could be relieved completely, or partially in the case of nitrate, either by the addition of formate to the medium or by increasing the copy number of the gene encoding the transcriptional activator (fhlA) of this regulon. These studies uncovered a further level of regulation in which the transcription of hycB was reduced in cells grown on glucose. This effect was most clearly seen in aerobically grown cells when formate was added externally. Addition of cAMP overcame this glucose repression, which could be shown to be mediated by the cAMP receptor protein. These results would be consistent with the transport of formate being regulated by catabolite repression. Moreover, the repression of transcription through high pH also could be partially overcome by addition of increasing concentrations of formate to the medium, again being consistent with regulation at the level of formate import and export. Taken together, all these observations indicate that it is the intracellular level of formate that determines the transcription of the genes of the formate regulon by FhlA. This represents a novel positive feedback mechanism in which the activator of a regulon induces its own synthesis in response to increases in the concentration of the catabolic substrate, and this in turn is governed by the relative affinities of FhlA and the three formate dehydrogenase isoenzymes for formate.

Anaerobiosis↗
PubMed1990

Phorbol diesters stimulate the accumulation of phosphatidate, phosphatidylethanol, and diacylglycerol in three cell types. Evidence for the indirect formation of phosphatidylcholine-derived diacylglycerol by a phospholipase D pathway and direct formation of diacylglycerol by a phospholipase C pathway.

The effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), on phospholipid degradation was investigated in three cell lines of dissimilar origin, Madin-Darby canine kidney cells (MDCK), rat aorta smooth muscle cells (RASM), and bovine pulmonary artery endothelial cells (BPAE). In cells prelabeled with [3H]myristic acid, which is predominantly incorporated into phosphatidylcholine (PC), TPA treatment (80 nM) in the absence or presence of ethanol (2%) in the culture medium resulted in either the rapid generation of [3H]phosphatidate (PA) or the sustained accumulation of [3H]phosphatidylethanol (PEt), respectively. Increases in [3H]PA and [3H]PEt were paralleled by quantitative decreases in cellular [3H]PC radioactivity. TPA-induced [3H]PEt formation occurred in a similar fashion, irrespective of the presence of Ca2+ in the culture medium. The experiments demonstrate that TPA elicits PC degradation by phospholipase D (PLD) in cells of diverse origin. Data from further experiments revealed a complex relationship between TPA-induced [3H]PA and [3H]diacylglycerol (DG) generation in the three cell lines that was suggestive of dual pathways for the generation of [3H]DG. Experiments to discern the pathways for TPA-induced, PC-derived DG were conducted by comparing the variation of [3H]PA and [3H]DG formation in the absence and in the presence of increasing ethanol concentrations in the culture medium. With increasing amounts of ethanol, the formation of [3H]PA decreased at the expense of [3H]PEt formation, and depending upon the pathway operable, the amount of [3H]DG formed was either decreased, indicative of indirect formation of DG via PA phosphohydrolase, or not modified, indicative of DG formation by a direct phospholipase C (PLC) pathway. Increasing the concentration of ethanol in the medium blocked TPA-induced [3H]DG generation in MDCK cells in a concentration-dependent manner, while the formation of [3H]PEt increased at the expense of [3H]PA formation. In BPAE cells the presence of ethanol likewise reduced TPA-elicited formation of DG. Conversely, in two smooth muscle cell lines, RASM and A-10, ethanol was without influence on TPA-induced formation of [3H]DG, although [3H]PEt was generated at the expense of [3H]PA. In RASM cells prelabeled with [3H]choline, TPA induced the release to the medium of [3H]choline and [3H]phosphocholine, indicative of both PLD and PLC activation. These results show that TPA elicits DG formation from PC in MDCK cells predominantly by an indirect pathway, whereas in arterial smooth muscle cells DG is formed in part by the direct action of PLC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

The formate dehydrogenase involved in electron transport from formate to fumarate in Vibrio succinogenes.

1. The formate dehydrogenase of Vibrio succinogenes, which is involved in electron transport with fumarate as terminal acceptor, was solubilized with Triton X-100 and purified some 200-fold by means of chromatography on hydroxyapatite, sucrose-density-gradient centrifugation and chromatography on DEAE-Sephadex. Gel filtration failed to increase the specific acitivity of the enzyme while gel electrophoresis in the presence of dodecylsulfate revealed that 73% of the protein of the preparation consisted of a polypeptide of Mr 110 000. The Mr of the functional enzyme was found to be 263 000 on the basis of the Stokes radius (5.8 nm) and the sedimentation coefficient (11.3 S). 2. The preparation contained 9 micronmol molybdenum/g protein and about 170 mumol iron-sulfur/g protein. The contents of b and c cytochromes varied and were lower than that of molybdenum. The low-potential cytochrome b [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 497-506] present in the preparation was reduced by formate. 3. The preparation catalyzed the reduction of a variety of dyes by formate, but not of NAD, FMN, ferredoxin or oxygen. The reduction of CO2 or bicarbonate by reduced methyl viologen was not catalyzed. The reaction with benzyl viologen obeyed the rate law consistent with a ping-pong mechanism. The Km for formate was 1.5 mM at infinite concentration of benzyl viologen while that for benzyl viologen was 0.53 mM at infinite formate concentration. Enzymic activity was inhibited by azide, KCN and HgCl2, but not by 4-chloromercuriphenylsulfonate or 2-(n-nonyl)-4-hydroxyquinoline-N-oxide, both of which inhibit overall electron transport. The inhibition by azide was competitive with formate; the Ki was 45 micron. 4. The midpoint potential of the low-potential cytochrome b of the membrane fraction was shifted -40 mV by the presence of 2-(n-nonyl)-4-hydroxyquinoline-N-oxide. 5. It is concluded that the formate dehydrogenase of V. succinogenes is isolated as a dimer consisting of two identical subunits of Mr 110,000, each of which carries one atom of molybdenum and iron-sulfur groups. The low-potential cytochrome b is the direct acceptor for the electrons of formate dehydrogenase in the electron transport of formate-fumarate reduction of V. succinogenes. Inhibition of electron transport of the membrane fraction between formate dehydrogenase and menaquinone by 2-(n-nonyl)-4-hydroxyquinoline-N-oxide [Kröger, A. and Innerhofer, A. (1976) Eur. J. Biochem. 69, 487-495] is caused by the inhibitor binding to the low-potential cytochrome b.

Aldehyde Oxidoreductases↗
PubMed1976

Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli.

When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.

Aldehyde Oxidoreductases↗
PubMed1991

Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase.

Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme. A ping-pong Bi Bi kinetic mechanism was observed. The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM. The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1. Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism. Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data. The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate. This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction. The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5. Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM. Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.

Azides↗
PubMed1987

The role of formate and S-adenosylmethionine in the reversal of nitrous oxide inhibition of formate oxidation in the rat.

Studies have been performed in rats in order to test whether methionine reverses the inhibition of formate oxidation produced by nitrous oxide by virtue of the conversion of methionine to formate. At a dose of methionine (100 mg/kg, 671 mumol/kg) that completely reverses the nitrous oxide inhibition of formate oxidation no significant conversion of the methyl group, carboxyl, or backbone of methionine to formate was apparent. No increases in hepatic formate levels were seen after the administration of 671 mumol/kg methionine or ethionine, and formate treatment did not alter the rate of 14CO2 formed after methionine was administered labeled in the methyl, carboxyl, or backbone position. The reversal of nitrous oxide inhibition of formate oxidation was found to correlate temporally with either S-adenosylmethionine levels after methionine administration or S-adenosylethionine levels following ethionine treatment. After methionine or ethionine administration, elevated hepatic steady state levels of tetrahydrofolate were observed and were coincident with elevated S-adenosylmethionine or S-adenosylethionine. Since formate oxidation rates are dependent on the hepatic tetrahydrofolate level, the mechanism of methionine reversal of nitrous oxide inhibition appears to be related to effects of hepatic S-adenosylmethionine which are important in maintaining and regulating tetrahydrofolate, rather than formate generation from methionine.

Adenosine↗
PubMed1990

Physiological de novo thyroid hormone formation in primary culture of porcine thyroid follicles: adenosine 3',5'-monophosphate alone is sufficient for thyroid hormone formation.

We describe a method of culturing intact porcine thyroid follicles for physiological de novo thyroid hormone formation; the roles of cAMP and protein kinase-C in thyroid hormone formation were also studied. Thyroid follicles were obtained by digesting minced porcine thyroid tissue with 0.04% collagenase and cultured in Coon's Modified Ham's F-12 medium supplemented with 0.5% calf serum, 0.5 mU/ml TSH, other standard hormones, and 3 antibiotics (6H medium). On the fourth day of culture, 6000-8000 follicles/well were plated in 12-well culture dishes. On the sixth day, thyroid hormone formation was carried out by incubating thyroid follicles with 0.5 microM KI in the presence of 6H medium for 2 days in a 5% CO2-95% air incubator at 37 C. To examine the effects of cAMP and protein kinase-C on de novo thyroid hormone formation, follicles were incubated with KI in the presence of 1-2.5 mM (Bu)2cAMP, 10 microM forskolin, 2 microM prostaglandin E2 (PGE2), or 0.5-1 microM 12-O-tetradecanoylphorbol-13-acetate in TSH-free medium for 2 days. The amount of newly formed thyroid hormone was measured by RIA of T3 content in the Pronase digest of thyroid follicular cells. Thyroid follicles cultured in 6H medium had normal polarity of the membrane, determined by electron microscope, and thyroid cAMP was responsive to the alteration of TSH. In this culture system cAMP alone was sufficient to form thyroid hormone. 12-O-Tetradecanoylphorbol-13-acetate, a protein kinase-C stimulator, disrupted thyroid follicles and inhibited cAMP-mediated thyroid hormone formation. The integrity of follicular structure was also required for thyroid hormone formation in this culture system. This study introduces perhaps the most physiological culture system for de novo thyroid hormone formation. Our data provide direct evidence that thyroid hormone formation is linked to cAMP and that the protein kinase-C system acts as an inhibitor of thyroid hormone formation.

Animals↗
PubMed1969

Lipid peroxide formation in microsomes. Relationship of hydroxylation to lipid peroxide formation.

1. Aminopyrine strongly inhibits NADPH-induced lipid peroxide formation in rat liver microsomes, but ascorbate-induced peroxidation is inhibited to a smaller extent. 2. Aminopyrine oxidation is stimulated by Mg(2+) but inhibited by Ca(2+). Concentrated solutions (10mm) of iron-chelating agents inhibit aminopyrine oxidation, but the more dilute solutions (0.5mm) of chelators that block lipid peroxide formation do not inhibit aminopyrine oxidation. Microsomes prepared from sucrose-EDTA homogenates rapidly oxidize aminopyrine, but do not form lipid peroxide when incubated with ascorbate or NADPH. 3. Aminopyrine oxidation is strongly inhibited by p-chloromercuribenzoate, less by iodoacetamide and weakly by N-ethylmaleimide. The site of action of these compounds is considered to be a ferredoxin-type protein. GSH and cysteine also inhibit. 4. Other drugs oxidized by microsomes such as caffeine, phenobarbitone and hexobarbitone had either no or little effect on lipid peroxide formation, but codeine inhibited. 5. Most aliphatic hydrocarbons, alcohols, ketones and aldehydes did not affect lipid peroxide formation, but chloroform and carbon tetrachloride inhibited. 6. Many aromatic compounds inhibited lipid peroxide formation. Only aromatic acids were without any effect and phenols and amines were very strong inhibitors. 7. Induction of lipid peroxide formation in microsomes by incubation with ascorbate or NADPH or by treatment with ionizing radiation leads to a sharp decline in the ability of microsomes to oxidize aminopyrine or hydroxylate aniline. 8. It is considered that the two processes of hydroxylation and lipid peroxide formation are closely linked in microsomes. They probably depend on the same electron-transport chain, and peroxide formation, which involves membrane disintegration, may be part of the normal membrane remodelling process.

Aminopyrine↗
PubMed1976

Rosette formation between human lymphocytes and sheep erythrocytes. Inhibition of rosette formation by specific glycopeptides.

Rosette formation with unsensitized sheep erythrocytes is a characteristic of human thymus dependent lymphocytes. Release of glycopeptides from the sheep erythrocyte by trypsin reduces rosette formation. These tryptic glycopeptides inhibit rosette formation by untrypsinized sheep erythrocytes; this suggests that rosetting is mediated by erythrocyte surface glycopeptides. To investigate the molecular nature of this interaction, we examined the abilities of various model compounds to act as haptenic inhibitors of rosette formation. Inhibition is given by glycopeptides bearing oligosaccharide units rich in sialic acid, galactose, N-acetylglucosamine, and mannose linked to asparagine residues through glycosylamine bonds. Among compounds tested, fetuin glycopeptide is most effective, but human transferrin glycopeptide and human erythrocyte glycopeptide I also inhibit rosette formation. Other compounds including human erythrocyte glycopeptide II, human IgG glycopeptide, lacto-N-neotetraose, 3'- and 6'-sialyllactose show no significant inhibition. Neither sialic acid, galactose, manose, nor N-acetyl-glucosamine alone inhibits rosette formation. Stepwise degradation of fetuin glycopeptide established the galactose residues as important determinants of inhibitory activity. Fetuin glycopeptide blocks rosette formation when added to a suspension of human lymphocytes and sheep erythrocytes or when preincubated with human lymphocytes, but not when preincubated with sheep erythrocytes. Studies of the binding of [3H] fetuin glycopeptide to normal lymphocytes demonstrate 7.5 x 10(6) saturable binding sites per cell. No saturable binding of this compound to sheep erythrocyte membranes is observed. Compared to normals, lymphocytes from patients with chronic lymphatic leukemia demonstrate decreased fetuin glycopeptide binding with a mean of 0.9 x 10(6) sites per cell. This decreased binding correlates with the impaired ability of these cells to form rosettes. The data suggest that fetuin glycopeptide inhibits rosette formation by binding to the thymus-dependent cell where competition occurs with sheep erythrocytes for specific lymphocyte surface receptors.

Animals↗
PubMed1977

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation.

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 mumoles/min X mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10(-7) M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibitedan antagonistic effect in this organism. Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accellerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cyclindrosporum and to form an active formate dehydrogenase. The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.

Aldehyde Oxidoreductases↗
PubMed1986

Transient kinetics of formation of phosphorylated intermediate (EP) by fragmented sarcoplasmic reticulum from bullfrog skeletal muscle: ligand addition sequence-dependent rate of EP formation.

In order to obtain a better understanding of Ca2+-activated ATP hydrolysis by sarcoplasmic reticulum, the transient kinetics of phosphorylated intermediate (EP) formation was examined with different sequences of addition of Ca2+ and ATP to GEDTA-added (Ca2+-free) fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. With a short delay line (10-20 ms), the addition sequence of ATP followed by Ca2+ gives rise to a faster EP formation without any lag time. In the reverse sequence of ligand addition, a lag time of 2.5-3 ms was consistently observed irrespective of ATP concentration, and the rate of EP formation was lower. As the preincubation time with Ca2+ became longer, the rate constant for EP formation and the maximum level of EP attainable increased even in the presence of fixed concentrations of Ca2+ and ATP. The rate constant per the unit concentration of EP, however, remained constant, indicating second-order kinetics between Ca2+-activated FSR and ATP. With a preincubation time of 83.6 ms, no lag time was observed. In the addition sequence of ATP and 83.6 ms later Ca2+, a burst in EP formation was observed, which was followed by a usual exponential time-course of EP formation. Similar determinations in the presence of various concentrations of ATP led us to the following estimates of the rates of the reactions leading to EP formation at 15 degrees C. (formula: see text)

Adenosine Triphosphate↗
PubMed1981

Catechol estrogen formation by brain tissue: a comparison of the release of tritium from [2-3H]estradiol with [6,7-3H]2-hydroxyestradiol formation from [6,7-3H]estradiol by rabbit hypothalami in vitro.

In the course of establishing an assay for estrogen-2-hydroxylase activity, a detailed comparison was made between the formation of tritiated water (3H2O) and [6,7-3H]2-hydroxyestradiol (2-OHE2) by rabbit hypothalami in vitro from 2-3H- and 6,7-3H-labeled estradiol, respectively. The amounts of both 3H2O and [6,7-3H]2-OHE2 formed were stimulated several-fold by the nonionic detergent Tween-80. Maximum activity for both functions was associated with the soluble fractions (S2, 17,500 X g supernatant, for tritium release; S3, 100,000 X g supernatant, for 2-OHE2 formation). In contrast, maximal 3H2O formation by rat liver was associated with the microsomal (P3, 100,000 X g pellet) fraction and was virtually abolished by Tween-80. The amount of 3H2O formed exceeded, up to severalfold, the amount of 2-OHE2 produced under all conditions examined and in all subcellular fractions. The bulk of the excess 3H2O formation, unrelated to the production of 2-OHE2, could be eliminated by adding ascorbic acid (10 mM) to the incubation medium. However, a second, smaller component of spurious 3H2O release could not be suppressed. This component was responsible for a persistent lack of stoichiometry between the formation of 3H2O and 2-OHE2, with the former exceeding the latter by up to 2-fold. This discrepancy was unaffected by ascorbic acid (up to 20 mM), unlabeled 2-OHE2 (up to 10 microM), and reducing the temperature of incubation from 37 to 30 C, measures that prolonged the t1/2 of 2-OHE2 during incubation with hypothalamic tissue from under 3 min to over 100 min. These findings 1) raise doubts about the validity of using 3H2O formation from [2-3H]estradiol as a quantitative index of estrogen-2-hydroxylase activity, and 2) establish conditions under which further metabolism of 2-OHE2 is inhibited, thereby making it practical to quantify enzyme activity on the basis of the amount of catechol estrogen formed. Evidence is also presented that the release of 3H2O from [2-3H]estradiol by hypothalamic tissue, unrelated to 2-OHE2 formation, may be enzymatically mediated.

Animals↗
PubMed1990

[Effects of attitude formation, persuasive message, and source expertise on attitude change: an examination based on the Elaboration Likelihood Model and the Attitude Formation Theory].

The purpose of this study was to investigate how attitude change is generated by the recipient's degree of attitude formation, evaluative-emotional elements contained in the persuasive messages, and source expertise as a peripheral cue in the persuasion context. Hypotheses based on the Attitude Formation Theory of Mizuhara (1982) and the Elaboration Likelihood Model of Petty and Cacioppo (1981, 1986) were examined. Eighty undergraduate students served as subjects in the experiment, the first stage of which involving manipulating the degree of attitude formation with respect to nuclear power development. Then, the experimenter presented persuasive messages with varying combinations of evaluative-emotional elements from a source with either high or low expertise on the subject. Results revealed a significant interaction effect on attitude change among attitude formation, persuasive message and the expertise of the message source. That is, high attitude formation subjects resisted evaluative-emotional persuasion from the high expertise source while low attitude formation subjects changed their attitude when exposed to the same persuasive message from a low expertise source. Results exceeded initial predictions based on the Attitude Formation Theory and the Elaboration Likelihood Model.

Adult↗