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Biomedical subjects

A H Merrill

Publications and source records attributed to A H Merrill.

At least 19 recordsLinked to original sources
PubMed1992

Influences of sphingosine on two-stage skin tumorigenesis in Sencar mice.

Sphingosine (SPH) was studied as an inhibitor of skin tumor promotion in skin cancer initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 12-O-tetradecanoylphorbol-13-acetate (TPA). Two tumorigenesis studies were conducted using female Sencar mice treated with 10 nmol DMBA in 0.2 ml acetone at 8 weeks of age and promoted beginning 1 week later with 3.2 nmol TPA applied twice per week. In the high-dose study, 10 mumol SPH was applied 30 min before each TPA treatment. The low-dose study used 0.5, 0.05, or 0.01 mumol treatments with SPH 30 min before TPA. In the high-dose study SPH treatment alone following initiation by DMBA, and SPH treatment preceding TPA in DMBA-initiated mice accelerated the development of papillomas in comparison with the DMBA/TPA-treated group. The low-dose experiment showed no consistent alteration of tumorigenesis by SPH in the DMBA/TPA-treated groups, and low doses of SPH following DMBA did not promote skin tumorigenesis. SPH treatment did not alter body weight in either experiment.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1992

In vitro toxicology of fumonisins and the mechanistic implications.

The effects of fumonisins B1(FB1), B2(FB2), and the backbone of fumonisin B1 remaining after hydrolysis of the tricarballylic groups with base (HFB1) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK1). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC50 of FB1 = 0.1 microM), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC50 for FB1 = 35 microM), and caused decreased cell proliferation as well. Higher doses (greater than or equal to 70 microM) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.

Animals↗
PubMed1992

Fumonisin inhibition of de novo sphingolipid biosynthesis and cytotoxicity are correlated in LLC-PK1 cells.

Fumonisins are a group of structurally related compounds produced by Fusarium moniliforme. Recently, it has been shown that fumonisins B1 and B2 are the first naturally occurring inhibitors of sphingosine and sphinganine N-acyltransferase (ceramide synthase) in rat primary hepatocytes (Wang et al. J. Biol. Chem. 266, 14, 486-14, 490, 1991). These enzymes are key components in the pathways for de novo sphingolipid biosynthesis and sphingolipid turnover. The results of the present study show that fumonisins B1 and B2 inhibit proliferation and are cytotoxic to LLC-PK1 cells. Concentrations of fumonisin B1 and B2 between 10 and 35 microM inhibited cell proliferation, whereas higher concentrations (greater than 35 microM) killed cells. Inhibition of cell proliferation and cell death were preceded by a lag period of at least 24 hr during which cells appeared to be functioning normally. Cells exposed to fumonisin B1 exhibited normal growth kinetics and morphology soon after fumonisin B1 was removed; thus, the effects of fumonisin B1 were reversible. The EC50 for alterations in sphingolipid biosynthesis was 10 to 15 microM. Inhibition of de novo sphingolipid biosynthesis occurred before inhibition of cell proliferation or cytotoxicity, and the dose response for the decrease in the [3H]sphingosine to [3H]sphinganine ratio at 7 hr closely paralleled the dose response for effects on proliferation and cytotoxicity at 3-5 days. In addition, the level of free sphinganine, and to a lesser extent sphingosine, increased in fumonisin-treated cells in a dose-dependent manner. During the 24-hr lag period preceding inhibition of cell proliferation, the free sphinganine content increased by 12,800% in cells exposed to 35 microM fumonisin B1. Whereas a mechanistic relationship between the inhibition of de novo sphingolipid biosynthesis and inhibition of proliferation and cell death has not been demonstrated, the results of this study support the hypothesis that inhibition of de novo sphingolipid biosynthesis is an early event in the toxicity of fumonisins to LLC-PK1 cells.

Acyltransferases↗
PubMed1992

Increases in serum sphingosine and sphinganine and decreases in complex sphingolipids in ponies given feed containing fumonisins, mycotoxins produced by Fusarium moniliforme.

Consumption of food contaminated with Fusarium moniliforme causes leucoencephalomalacia and hepatotoxicity in horses, pulmonary edema in pigs and liver cancer in rats, and has been correlated with esophageal cancer in humans. The causative agents are thought to be a family of compounds called fumonisins, which have recently been shown to be potent inhibitors of sphingosine (sphinganine) N-acyltransferase. Because inhibition at this step blocks the formation of complex sphingolipids while leading to accumulation of sphinganine, we hypothesized that exposure of animals to fumonisin-contaminated feed might be detected by analyses of serum sphingolipids. Within days of giving ponies feed contaminated with 15 to 44 micrograms/g fumonisin B1, there was an increase in the amount of free sphinganine (and sometimes sphingosine) and a reduction in complex sphingolipids. Free sphinganine and sphingosine decreased when ponies consumed less of the contaminated feed, and increased again when they consumed more fumonisin. When toxicosis was evident as indicated by other serum markers, complex sphingolipids as well as free sphingosine and sphinganine were elevated, probably due to loss of sphingolipids from dying cells. These findings establish that consumption of fumonisin-contaminated feed disrupts sphingolipid metabolism. Because the changes in sphinganine and sphingosine were seen before liver enzymes were noticeably elevated, they may be an early marker of exposure to fumonisins.

Animal Feed↗
PubMed1992

Ceramide: a new lipid "second messenger"?

1-Alpha, 25-dihydroxyvitamin D3, tumor necrosis factor alpha, and gamma-interferon induce sphingomyelin turnover to ceramide in HL-60 cells. The time frame of the turnover is consistent with ceramide formation being a mediator of growth and differentiation; furthermore, differentiation can be induced by addition of exogenous ceramides alone. Hence, the action of the biologically active form of vitamin D and other nutritionally relevant factors may be mediated by lipid second messenger pathways involving sphingolipids.

Animals↗
PubMed1992

Analyses of plasma for metabolic and hormonal changes in rats flown aboard COSMOS 2044.

Plasma samples from rats flown aboard COS-MOS 2044 were analyzed for the levels of key metabolites, electrolytes, enzymes, and hormones. The major differences between the flight group and the synchronous control were elevations in glucose, cholesterol, phosphate, creatinine, blood urea nitrogen, lactate dehydrogenase, and aspartate amino-transferase and decreased levels of thyroxine. Most of these differences were not mimicked by tail suspension of ground-based rats; however, both flight and suspended rats exhibited inhibited testosterone secretion. Corticosterone, immunoreactive growth hormone, and prolactin showed inconsistent differences from the various control groups, suggesting that the levels of these hormones were not due to actual or simulated microgravity.

Adrenal Glands↗
PubMed1992

Differences in glycogen, lipids, and enzymes in livers from rats flown on COSMOS 2044.

Livers from rats flown aboard COSMOS 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis, delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from COSMOS 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for COSMOS 2044.

Amino Acids↗
PubMed1991

Inhibition of sphingolipid biosynthesis by fumonisins. Implications for diseases associated with Fusarium moniliforme.

Culture materials and grains contaminated with certain isolates of Fusarium moniliforme cause equine leucoencephalomalacia, porcine pulmonary edema syndrome, and liver cancer in rats. The causative agents are thought to be a family of compounds called fumonisins, which bear considerable structural similarity to the long-chain (sphingoid) base backbones of sphingolipids. Incubation of rat hepatocytes with fumonisins inhibited incorporation of [14C]serine into the sphingosine moiety of cellular sphingolipids with an IC50 of 0.1 microM for fumonisin B1. In contrast, fumonisin B1 increased the amount of the biosynthetic intermediate sphinganine, which suggests that fumonisins inhibit the conversion of [14C]sphinganine to N-acyl-[14C]sphinganines, a step that is thought to precede introduction of the 4,5-trans double bond of sphingosine (Merrill, A.H., Jr. and Wang, E. (1986) J. Biol. Chem. 261, 3764-3769). In agreement with this mechanism, fumonisin B1 inhibited the activity of sphingosine N-acyltransferase (ceramide synthase) in rat liver microsomes with 50% inhibition at approximately 0.1 microM and reduced the conversion of [3H]sphingosine to [3H]ceramide by intact hepatocytes. As far as we are aware, this is the first discovery of a naturally occurring inhibitor of this step of sphingolipid metabolism. These findings suggest that disruption of the de novo pathway of sphingolipid biosynthesis may be a critical event in the diseases that have been associated with consumption of fumonisins.

Acyltransferases↗
PubMed1991

Functional differences in human neutrophils isolated pre- and post-prandially.

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid-rich meal. There was up to 2-fold greater superoxide generation in response to agonists in leukocytes obtained post-prandially; the maximum increase was observed about 4 h after eating and followed the peak (2-4 h) in serum triglycerides. Neutrophils isolated post-prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

Bacteroides↗
PubMed1991

Long-chain (sphingoid) bases inhibit multistage carcinogenesis in mouse C3H/10T1/2 cells treated with radiation and phorbol 12-myristate 13-acetate.

Sphingosine and other long-chain (sphingoid) bases inhibit protein kinase C, the putative cellular receptor for the tumor promoter phorbol 12-myristate 13-acetate (PMA), and exert potent effects on diverse cell functions. We tested the ability of long-chain bases to modulate multistage carcinogenesis in mouse C3H/10T1/2 cells exposed to gamma-rays and PMA. Sphingosine and sphinganine completely blocked the enhancement of radiation-induced transformation by PMA (promotion) and partially suppressed transformation by radiation alone. N-Acetylsphingosine, a ceramide analog, did not inhibit transformation. Sphingosine was rapidly taken up by the cells and metabolized; hence, the long-chain bases were added daily to achieve prolonged inhibition. Long-chain bases inhibited protein kinase C activity in C3H/10T1/2 cells and suppressed the down-regulation of this enzyme by PMA. Our results establish that long-chain bases are highly effective inhibitors of carcinogenesis in this model. Our results also indicate that the suppressive effects may be mediated, in part, by inhibition of protein kinase C. The data suggest that sphingosine and other long-chain bases derived from complex sphingolipids may act as cancer-preventative agents.

Animals↗
PubMed1991

Cell regulation by sphingosine and more complex sphingolipids.

Sphingolipids have the potential to regulate cell behavior at essentially all levels of signal transduction. They serve as cell surface receptors for cytoskeletal proteins, immunoglobulins, and some bacteria; as modifiers of the properties of cell receptors for growth factors (and perhaps other agents); and as activators and inhibitors of protein kinases, ion transporters, and other proteins. Furthermore, the biological activity of these compounds resides not only in the more complex species (e.g., sphingomyelin, cerebrosides, gangliosides, and sulfatides), but also in their turnover products, such as the sphingosine backbone which inhibits protein kinase C and activates the EGF-receptor kinase, inter alia. Since sphingolipids change with cell growth, differentiation, and neoplastic transformation, they could be vital participants in the regulation of these processes.

Animals↗
PubMed1990

Human acid beta-glucosidase: use of sphingosyl and N-alkyl-glucosylamine inhibitors to investigate the properties of the active site.

Human acid beta-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the beta-glucosidic bonds of glucosylceramide and synthetic beta-glucosides. The specificity of binding to the active site of this enzyme was evaluated using series of inhibitors including synthetic sphingosines, N-alkyl(Cn)-deoxynojirimycins (1,5-dideoxy-5-iminoglucose) and N-Cn-glucosylamines. The sphingosines were rapidly reversible inhibitors with maximal potency (IC50 approximately 78-150 micro M) at chain lengths of 14-18 carbons. The presence of unsaturation between C4 and C5 was required for inhibition of enzyme activity. Neither the nature of this bond (double or triple bond) nor the presence of erythro or threo configurations at C2 influenced inhibitory potency. The N-C10- to N-C14-deoxynojirimycins were rapidly reversible inhibitors with Ki approximately 8.5 nM. In comparison, the 1-amino glucose derivatives, i.e., N-Cn-glucosylamines (n = 12-18), were more potent (IC50 approximatley 0.3-3 nM) and their maximal inhibitory potencies were dependent on time as well as enzyme and substrate concentrations: i.e., the N-C12- to N-C18-glucosylamines were competitive, slow-tight binding inhibitors. Analyses of progress curves at various N-Cn-glucosylamine (n = 14-18) concentrations indicated the formation of rapidly dissociating initial EI collison complex which then undergoes a conformational change to a slowly reversible EI complex. These results were consistent with the long chain N-Cn-glucosylamines being reaction intermediate analogues and with this enzyme's hydrolytic mechanism requiring a conformational change during the transition state.

1-Deoxynojirimycin↗
PubMed1990

An update of the enzymology and regulation of sphingomyelin metabolism.

Sphingomyelin is found in plasma membranes and related organelles (such as endocytic vesicles and lysosomes) of all tissues, as well as in lipoproteins. Abnormalities in sphingomyelin metabolism have been associated with atherosclerosis, cancer and genetically transmitted diseases; however, except for Niemann-Pick disease, little is known about the mechanism for these disorders. Sphingomyelin biosynthesis de novo involves ceramide formation from serine and two mol of fatty acyl-CoA followed by addition of the phosphocholine headgroup. The headgroup appears to come from phosphatidylcholine, but other sources have not been ruled out. Factors that influence the rate of sphingomyelin synthesis include the availability of serine and palmitic acid, plus the relative activities of key enzymes of this pathway. Sphingomyelin turnover involves removal of the headgroup and amide-linked fatty acid by sphingomyelinases and ceramidases, respectively, which have been found in both lysosomes (with acidic pH optima) and plasma membranes (with neutral to alkaline pH optima). The enzymes of sphingomyelin turnover release ceramide and free sphingosine from endogenous substrates, which may have implications for the participation of a sphingomyelin/sphingosine cycle as another 'lipid second messenger' system.

Animals↗
PubMed1990

Characteristics of the growth inhibition and cytotoxicity of long-chain (sphingoid) bases for Chinese hamster ovary cells: evidence for an involvement of protein kinase C.

Long-chain bases are potent inhibitors of protein kinase C and cellular processes mediated by this enzyme. However, when added to cells they usually cause some degree of growth inhibition and cytotoxicity and it is unclear whether this reflects inhibition of protein kinase C or nonspecific detergent effects of these amphipathic compounds. This study examined the effects of sphinganine on Chinese hamster ovary (CHO) cells to gain more insight into these possibilities. Sphinganine concentrations between 0.75 and 4 microM resulted in a combination of growth inhibition and cytotoxicity that correlated with protein kinase C inhibition by five criteria: (1) the effective concentrations were comparable to those for protein kinase C inhibition in vitro and in other intact cells; (2) the structural specificity for the long-chain base moiety paralleled the potency of protein kinase C inhibition; (3) sphinganine blocked changes in protein phosphorylation patterns that occurred in response to phorbol 12-myristate 13-acetate (and vice versa); whereas (4) a mutant cell line that exhibited increased resistance to sphinganine cytotoxicity lacked both phorbol ester- and sphinganine-induced phosphorylation changes and differed somewhat in the behavior of protein kinase C assayed in vitro; and (5) sphinganine did not appear to be acting as a detergent (except at higher concentrations) nor as a lysosomotrophic agent. While the complexity of this cellular behavior mandates caution in interpreting these results, they suggest that the cytotoxicity and growth inhibition may be a consequence of protein kinase C inhibition.

Animals↗
PubMed1990

Modulation of retinoic acid-induced differentiation of human leukemia (HL-60) cells by serum factors and sphinganine.

The human cell line HL-60 was used to investigate the role of protein kinase C in the regulation of retinoic acid-induced maturation of promyelocytic leukemia cells by growth and differentiation factors found in serum. Cells grown in serum-containing medium differentiated less than cells in serum-free medium due to several factors, including albumin binding of retinoic acid. Addition of an inhibitor (sphinganine) of protein kinase C, an enzyme that participates in cellular responses to many serum factors, facilitated the retinoic acid-induced differentiation. Cells treated with both retinoic acid and sphinganine produced more superoxide when stimulated by formylmethionylleucylphenylalanine; hence, this combination generated a more functional population of cells. The ability of sphinganine to promote retinoic acid-induced differentiation suggests that retinoic acid therapy might be improved by the concurrent use of a modulator of protein kinase C activity.

Blood Physiological Phenomena↗
PubMed1990

Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver.

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.

Adenosine Triphosphate↗