The effect of amphotericin b-deoxycholate on proliferation and protein synthesis in human skin fibroblast cultures

Summary Cultures of adult human skin fibroblasts were grown in the presence of the recommended antifungal dose (3 μg per ml) of amphotericin B-deoxycholate. A reduction in cell culture growth, measured as DNA content and protein content per culture, was observed. However, radioisotope incorporation into noncollagen protein and, to a lesser extent, collagen protein was enhanced. These effects were due to amphotericin B, not to deoxycholate. These observations were made under several growth conditions and indicate that cell proliferation or isotope-labeling studies in fibroblasts in the presence of amphotericin B-deoxycholate are susceptible to errors in interpretation. Supported by PHS Grants AM-02456, AM-15312 and AM-17047, by the Kroc Foundation, and by the American Diabetes Association, Washington Affiliate. Recipient of Research Career Development Award AM-47142 from NIAMDD, and to whom requests for reprints should be addressed.     

Persistent biologic actions of insulin on cultured fetal human fibroblasts

Summary Monolayer cultures of fetal human fibroblasts, preincubated in serum-free culture medium overnight (about 18 hr), were incubated with insulin (0.1 to 100 mU per ml), then washed and incubated in an insulin-free medium. The effect of insulin on glucose utilization, uridine incorporation into RNA and leucine incorporation into protein was maintained after removal of insulin and washing. For both glucose utilization and uridine incorporation into RNA, this effect was demonstrated at physiologic levels of insulin (0.1 mU per ml). When anti-insulin serum was added to the cultures after the cell preincubated with insulin were washed, this effect was greatly attenuated. This lasting effect of insulin was probably not due to nonspecifically bound insulin becoming available to the cells. Binding of¹²⁵I-monoiodoinsulin was examined in monolayer cultures of fetal human fibroblasts. When unlabeled insulin was present at about 1 mU per ml concentration, 50% displacement of monoiodoinsulin occured. When fibroblasts were incubated with monoiodoinsulin and then removed from the radioactive medium, initial dissociation of the bound hormone occurred rapidly but then reached a plateau. This prolonged insulin effect appears to result from persistent binding of insulin to its receptor. Supported in part by PHS Grants AM-02456, AM-05020 and AM-15312, by the Kroc Foundation and by the Diabetes Center (AM-17047). Supported in part by Research Career Development Award AM-47142 from NIAMDD.     

Tyrosine aminotransferase deficiency in mink (Mustela vision): A model for human tyrosinemia II

Mink pseudodistemper, a recessive disease associated with high blood tyrosinelevels, is an animal analogue of the human inborn error of metabolism, tyrosinemia II. Affected mink and man have eye and skin lesions. Affected mink have no hepatic tyrosine aminotransferase (TAT) activity, as measured immunologically and biochemically. Hepatic mitochondrial aspartate aminotransferase is increased to 188% of control. This new genetic animal model of TAT deficiency should allow new studies of tyrosine metabolism.Supported by NIH Grants AM-17253 (LAG), 5T32-AM-07093 (LAG), and RCDA AM-0008 (LAG), grants from the Wisconsin State Mink Advisory Board, and a BRSG grant for the Graduate School of the University of Wisconsin, Madison, Publication No. 82 of the dermatology research laboratories of Duke University Medical Center.     

Insulin sensitivity of liver glycogen synthase b into a conversion

Summary Liver glycogen synthase b phosphatase, chromatographically separable from phosphorylase a phosphatase, is decreased in 48-hour alloxan diabetic rats. The phosphatase activities are measured in an in vitro system using exogenous isolated phospho-enzyme as substrates with added phosphatases. Synthase and phosphorylase phosphatases were shown to have differential catalytic properties by their reactivity in the presence of Pi, the heat-stable inhibitor of phosphorylase phosphatase and after incubation with added cAMP-dependent protein kinase.Supported by NIH Grants HD-07788, AM-21149 and, in part, by grants from the Greater St. Louis Diabetic Childrens Welfare Association and the American Diabetes Association, N.Y.     

Osteoid resorption by mononuclear cells in vitro

Summary For the first time, mononuclear cell-mediated ingestion of osteoid in cultures of long bones of fetal rats is described and characterized. The mononuclear cells, located at sites of osteoid deposition, ingest collagen fibrils and clumps of mineral crystals which are segregated within cytoplasmic vacuoles or multivesicular bodies. The ingestion of osteoid continues in cultures treated with agents that normally inhibit osteoclastic bone resorption. Morphologically, the osteoid-containing cells are characterized by a moderate number of mitochondria and short-stranded rough endoplasmic reticulum, a modest Golgi apparatus and variable numbers of vesicles, vacuoles, and multivesicular bodies. The morphologic appearance of the mononuclear cell is consistent with that of a macrophage.This study was supported by NIH Grants DE-04443 and AM-16858     

Localization of serotonin within tanycytes of the rat median eminence

Summary Formaldehyde and glyoxylic acid histochemical methods were employed to examine monoamine fluorescence of the rat median eminence. Tanycytes of the median eminence contained a yellow histofluorescence which was verified with microspectrofluorometry as due to the presence of serotonin. Catecholamine-containing varicosities, arranged in linear profiles throughout the depth of the median eminence, were observed. These linear profiles appeared to follow the contours of serotonin-containing tanycytes. Organculture experiments supported the hypothesis that the serotonin associated with tanycytes is localized within the tanycytes and does not arise from an extrahypothalamic source of nerve terminals. These data provide evidence that a tanycytic catecholamine-indoleamine morphological juxtaposition occurs in a manner reminiscent of that of another circumventricular organ, the pineal.Supported by USPHS Grants NS11642 and AM-19761USPHS Career Development Awardee NS-00259     

Relationship of the central ACTH-immunoreactive opiocortin system to the median eminence and the pituitary gland of the rat

Summary By immunocytochemical methods, the present study describes ACTH-immunoreactive fibers in the pituitary stalk and neural lobe. This opiocortin-hypothalamo-neural lobe projection arises in a bed nucleus of perikarya in the basal hypothalamus, follows supraoptico-hypophyseal fibers in the zona interna of the median eminence, and distributes throughout the neural lobe. No ACTH-immunoreactive fibers project to the zona externa; some are present in the subependymal layer and at the lateral margins of the median eminence. Further studies must identify the role of these fibers in posterior lobe function. It remains also to be determined whether this system terminates upon primary pituitary portal capillaries and delivers opiocortin neuropeptides to the adenohypophysis.Supported by NIH Grants HD-07962, NS-15345 and AM-22029The skillful technical assistance of Donna Wilson, Nancy Dembs and Jay Hocton is thankfully acknowledged     

Transferrin support of stimulated lymphocytes

Summary Transferrin was tested for its ability to replace serum in supporting mitogen and allogeneic cell stimulated human lymphocyte proliferation. Although transferrin, at concentrations greater than 5 μg/ml, was incapable of completely replacing the serum used to support phytohemagglutinin, Concanavalin A, and pokeweed mitogen, stimulated human lymphocytes, in the absence of serum it significantly augmented the proliferative responses observed for mitogen, yet not allogeneically-stimulated cells. Augmentation is not due to a nonspecific protein effect and appears to be independent of the metal content of transferrin. The mechanism of growth support appears to involve an effect of transferrin following the G₁ phase in the initial cell cycle. This work was supported by NIH Grants AM-17554 and AI-05155.     

Characterization of hemoglobin Burke [β107 (G9) Gly→Arg]

Hb Burke [107 (G9) GlyArg] was discovered in a young woman with hemolytic anemia. A substitution in this position has not been previously reported either in the human -chain or in any of the animal -chains so far sequenced. The abnormal hemoglobin shows heat instability and a lowered oxygen affinity. The substitution of a large charged arginine residue for the small glycine residue in the G helix next to a heme contact (Leu-106) may be responsible for these effects. Hb Burke is compared with five other hemoglobins having Gly-Arg substitutions in other parts of the molecule.This work was supported in part by U.S. Public Health Service Grants AM-17850 and AM-18006.     

Altered aggregational properties in a genetic variant of human glucose 6-phosphate dehydrogenase

The Tel Hashomer variant of human G6PD migrates as two prominent components during electrophoresis in several gel systems in which red cell G6PD from other males migrates predominantly as a single band. Since human males normally have but one X-chromosome, the extra band of this variant seemed an exception to earlier biochemical and genetic observations suggesting that human red cell G6PD is determined by a locus on the X chromosome. Results of the present studies indicate that the Tel Hashomer variant is unusually susceptible to the formation of a complex which has a higher molecular weight than normal G6PD and which represents the slow electrophoretic component. The conditions of formation and disruption of this complex in crude and purified Tel Hashomer preparations suggest that it results from the formation of disulfide bridges between molecules of Tel Hashomer G6PD.Supported by U.S. Public Health Service Research Grants AM-11065 and FR-5406 and Research Career Development Award 5 K3 AM 7992.     

Submicroscopic morphology of granular vesicles in sympathetic nerves of rat pineal body

Summary In the rat pineal body sympathetic nerve endings are characterized by numerous agranular and granular vesicles. The latter mainly include vesicles of about 450 Å diameter, each of which is bounded by a trilaminar membrane of about 75 Å total thickness. The cores of these granular vesicles appear structurally similar to the bounding membranes and are formed by a less dense, globular or vacuolar component around which a more dense component is distributed. The relative distributions of these two structural elements result in a variety of submicroscopic configurations which suggest a morphological continuum that may be related to the cyclical uptake, storage and release of biogenic amines. A tripartite classification of granular vesicles, such as proposed previously, is not supported by these data.This investigation was supported by United States Public Health Service Research Grants NB 05175-01 and AM-0699802. — The capable technical assistance of Mrs. Caroline Wilner is gratefully acknowledged.     

Maple syrup urine disease: Branched-chain amino acid concentrations and metabolism in cultured human lymphoblasts

The intracellular concentration of free leucine, isoleucine, and valine and their metabolism were studied in lymphoblast cultures established from peripheral blood of an individual with maple syrup urine disease (MSUD) and a control subject. Branched-chain -keto acid decarboxylase activity in the MSUD cells was 10% or less of the control value as measured by the ability of the cells to release ¹⁴CO₂ from the corresponding [1-¹⁴C]labeled branched-chain amino acid. The intracellular concentrations of free leucine and isoleucine were increased three-fold in MSUD lymphoblasts as compared to control cells. Free valine was present in only trace amounts of less than 0.1 mMin both cell lines. Exposure of normal and mutant cells to a 10 mMload of leucine, isoleucine, and valine resulted in a comparable concentration within cells after 24 hr. Concentrations returned to base values in normal cells 12 hr after removal of load, but leucine remained elevated in MSUD cells after 3 days. Leucine and its keto acid, -ketoisocaproic acid, added to the culture medium gave significant growth inhibition of MSUD lymphoblasts but not of normal cells, in the millimolar range. Isoleucine, valine, and their keto acids had no effect.This investigation was supported in part by Grants AM-13622, AM-05646, and GM-17702 from the United States Public Health Service, Veterans Administration Grant M.R.I.S. No. 3181 to Dr. Nathan Gochman, and grants from the National Foundation and the Kroc Foundation. S. D. S. is a Postdoctoral Research Fellow supported by United States Public Health Service Training Grant AM-05646.     

Uroporphyrinogen III cosynthetase in asymptomatic carriers of congenital erythropoietic porphyria

Mean activity of the enzyme uroporphyrinogen III cosynthetase in hemolysates from five asymptomatic carriers of bovine erythropoietic porphyria was intermediate to the means of the activities in normal controls and affected animals. Similarly, mean cosynthetase activity in hemolysates from eight presumed carriers of human congenital erythropoietic porphyria was lower than the mean for 38 nonporphyric individuals and higher than the activity in hemolysates from patients. These results are consistent with the known autosomal recessive mode of inheritance of the disorder in cattle and the presumed autosomal recessive inheritance in man. The activity of cosynthetase is higher in young red cells than in older ones, which explains the relatively high enzyme activities in blood from human patients with nonporphyric hemolytic disorders accompanied by reticulocytosis. It also explains why cosynthetase activity is higher in newborn porphyric calves than in older porphyric animals, because the former have a period of accelerated erythropoiesis and a high percentage of very young red cells in the circulation.Supported in part by U.S. Public Health Service Grants NB-05367 and AM-10858.     

Localization of tritiated prostaglandin E1 in rat adrenal cortices

Summary To determine whether or not prostaglandins enter adrenocortical parenchymal cells,³H-PGE₁ was injected intravenously into rats. In histological preparations, grains denoting activity were noted in intracellular lipid droplets and nuclei and in sinusoids. At the fine structural level, activity was observed in lipid droplets, mitochondria, the agranular endoplasmic reticulum, nuclei and the plasma membrane. Biochemical lipid analyses of the adrenals revealed activity in the cholesterol and cholesterol ester fractions. Large amounts of unaltered³H-PGE₁ and its degradation products were also present. Compared to the liver, the adrenal was more effective in degrading prostaglandin, when expressed on a weight basis. The possible roles of the organelles in PGE₁ degradation and in prostaglandin-related hormone synthesis are discussed. Supported by N.I.H. Grants AM-09561 and RR-05403.     

扩繁条件对两种AMF菌剂接种势的影响

温室盆栽试验,分别以4种宿主植物和6种基质对两种AMF菌剂AM-1(Glomus intraradices)和AM-2(Glomus spp.)进行了扩繁研究。通过计算菌剂接种势,评价不同扩繁条件下获得菌剂的质量。结果表明,白三叶草和烟草可以作为两种菌剂良好的扩繁宿主,得到菌剂的接种势最高,玉米和高粱次之。当以白三叶草为宿主时,单一蛭石可以作为两种菌剂良好的扩繁基质,得到菌剂的接种势最高,珍珠岩次之,其它基质较差,基质中草炭的含量会影响菌剂扩繁效果。     

Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to eightfold increases in intracellular concentrations of 5-phosphoribosyl 1-pyrophosphate (PRPP). The activities of the enzymes of purine metabolism responsible for production and utilization of PRPP were measured under optimal conditions in each cell line. The activities of adenine phosphoribosyltransferase (APRT), PRPP synthetase, and PRPP amidotransferase were independent of cell density and were not significantly different in the two cell lines. The K _m values of the common substrate, PRPP, were determined in normal lymphoblast extracts for APRT (K _m of 0.033 mM), HGPRT (K _m of 0.074 mM), and PRPP amidotransferase (K _m of 0.3 m M). The relatively low affinity of PRPP amidotransferase for PRPP suggests that deficiency of the HGPRT enzyme with its attendant increase in PRPP concentration should be accompanied by increased in vivo activity of PRPP amidotransferase, the first and presumed rate-limiting enzyme of de novo purine biosynthesis.This work was supported in part by National Institutes of Health Grants AM-05646, AM-13622, and GM-17702.     

Glucose 6-phosphate dehydrogenase in rainbow trout

Electrophoretic analysis of glucose 6-phosphate dehydrogenase from liver and blood of rainbow trout revealed a complex series of bands, which could differ between fish. The partial interconvertible nature of these bands was demonstrated with enzyme that had been incompletely inactivated at pH 8.4. In a single population of 40 fish, a homozygote and a heterozygote for an electrophoretic variant allele were found. We suggest that G6PD in rainbow trout liver and blood is determined by two alleles at a single locus, with posttranslational modification responsible for the complex electrophoretic patterns seen. The basis for this variation appears to be NADH binding to the protein molecule. Another variant and other properties of the enzyme are described.Supported in part by the State of California Department of Mental Hygiene and by USPHS Grants GM-15253, HD-04612, HD-00315, HD-05615, and HL-15125. One of us (S.D.C.) was a Special Postdoctoral Fellow of NIAMD (No. 1F03AM-40, 329-01) during part of this work.     

Histochemical studies of rat ovarian follicular cells in vitro

Summary Membrana granulosa cells were aspirated from large follicles of proestrous rat ovaries and were cultivated as monolayers. For histochemical identification of dehydrogenases, the monolayers were incubated in various steroid substrates, nicotinamide adenine dinucleotide, and Nitro Blue Tetrazolium. The presence of Δ^p5-3β-, 3α-, 17β- and 20α-hydroxysteroid dehydrogenases was demonstrated by the 4th day in vitro and was evident for as long as 20 days. Since none of these hydroxysteroid dehydrogenases is demonstrable in the membrana granulosa of intact follicles, it is concluded that the steroidogenic capacity of the cells, repressed in the preovulatory follicle in vivo, can be expressed upon mechanical removal from the follicle just as steroid synthesis occurs in these cells after normal ovulation. This research was supported in part by a Faculty Research Grant from the Horace H. Rackham School of Graduate Studies, and by United States Public Health Service Grants RR-05383-09 and AM-06918-06.     

13-Methylarachidonic Acid Is a Positive Allosteric Modulator of Endocannabinoid Oxygenation by Cyclooxygenase

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing k_cat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.     

Therapeutic modulation of cannabinoid lipid signaling: Metabolic profiling of a novel antinociceptive cannabinoid-2 receptor agonist

AimsAM-1241, a novel, racemic cannabinoid-2 receptor (CB2) ligand, is the primary experimental agonist used to characterize the role of CB2-mediated lipid signaling in health and disease, including substance abuse disorders. In vivo pharmacological effects have been used as indirect proxies for AM-1241 biotransformation processes that could modulate CB2 activity. We report the initial pre-clinical characterization of AM-1241 biotransformation and in vivo distribution.Main methodsAM-1241 metabolism was characterized in a variety of predictive in vitro systems (Caco-2 cells; mouse, rat and human microsomes) and in the mouse in vivo. Liquid chromatography and mass spectrometry techniques were used to quantify AM-1241 tissue distribution and metabolic conversion.Key findingsAM-1241 bound extensively to plasma protein/albumin. A pharmacological AM-1241 dose (25 mg/kg, i.v.) was administered to mice for direct determination of its plasma half-life (37 min), following which AM-1241 was quantified in brain, spleen, liver, and kidney. After p.o. administration, AM-1241 was detected in plasma, spleen, and kidney; its oral bioavailability was ~ 21%. From Caco-2 permeability studies and microsomal-based hepatic clearance estimates, in vivo AM-1241 absorption was moderate. Hepatic microsomal metabolism of AM-1241 in vitro generated hydroxylation and demethylation metabolites. Species-dependent differences were discovered in AM-1241's predicted hepatic clearance. Our data demonstrate that AM-1241 has the following characteristics: a) short plasma half-life; b) limited oral bioavailability; c) extensive plasma/albumin binding; d) metabolic substrate for hepatic hydroxylation and demethylation; e) moderate hepatic clearance.SignificanceThese results should help inform the design, optimization, and pre-clinical profiling of CB2 ligands as pharmacological tools and medicines.