Presence Of Exogenous Substrate Research Articles

Monoamine oxidase-A promotes protective autophagy in human SH-SY5Y neuroblastoma cells through Bcl-2 phosphorylation

Monoamine oxidases (MAOs) are located on the outer mitochondrial membrane and are drug targets for the treatment of neurological disorders. MAOs control the levels of neurotransmitters in the brain via oxidative deamination and contribute to reactive oxygen species (ROS) generation through their catalytic by-product H2O2. Increased ROS levels may modulate mitochondrial function and mitochondrial dysfunction is implicated in a vast array of disorders. However, the downstream effects of MAO-A mediated ROS production in a neuronal model has not been previously investigated.In this study, using MAO-A overexpressing neuroblastoma cells, we demonstrate that higher levels of MAO-A protein/activity results in increased basal ROS levels with associated increase in protein oxidation. Increased MAO-A levels result in increased Lysine-63 linked ubiquitination of mitochondrial proteins and promotes autophagy through Bcl-2 phosphorylation. Furthermore, ROS generated locally on the mitochondrial outer membrane by MAO-A promotes phosphorylation of dynamin-1-like protein, leading to mitochondrial fragmentation and clearance without complete loss of mitochondrial membrane potential. Cellular ATP levels are maintained following MAO-A overexpression and complex IV activity/protein levels increased, revealing a close relationship between MAO-A levels and mitochondrial function. Finally, the downstream effects of increased MAO-A levels are dependent on the availability of amine substrates and in the presence of exogenous substrate, cell viability is dramatically reduced.This study shows for the first time that MAO-A generated ROS is involved in quality control signalling, and increase in MAO-A protein levels leads to a protective cellular response in order to mediate removal of damaged macromolecules/organelles, but substrate availability may ultimately determine cell fate. The latter is particularly important in conditions such as Parkinson's disease, where a dopamine precursor is used to treat disease symptoms and highlights that the fate of MAO-A containing dopaminergic neurons may depend on both MAO-A levels and catecholamine substrate availability.

A novel light-dependent activation of DAGK and PKC in bovine photoreceptor nuclei

In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-32P]ATP or [3H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKβ content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.

P8 Hydrogen sulfide production during colonic inflammation occurs primarily via a pyridoxal-5′-phosphate (P5P)-independent pathway

Hydrogen sulfide (H 2 S) is an important endogenous anti-inflammatory mediator, which has been shown to promote resolution of colitis and healing of ulcers. H 2 S synthesis was believed to occur primarily via two P5P-dependent enzymes (cystathionine- β -synthase (CBS) and cystathionine- γ -lyase (CSE)). In the present study, we examined the possible contribution of a 3 rd pathway for H 2 S synthesis, in the healthy and inflamed colon, which involves the enzymes cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Methods The capacity of tissue to produce H 2 S was measured from homogenized tissue in the presence of exogenous substrate and/or inhibitors using a zinc-trapping assay. Production of H 2 S via the CAT-3MST pathway was explored using the substrate α -ketoglutarate and the CAT inhibitors l -aspartate and O -carboxymethyl-hydroxylamine hemihydrochloride (CHH). Colitis was induced in rats by intracolonic administration of dinitrobenzene sulfonic acid (DNBS). Rats were euthanized at different time points up to 28 days post-DNBS for measurement of H 2 S synthesis. To assess the contributions of different colonic layers to total colonic H 2 S synthesis, healthy and inflamed colonic sections collected on day 3 after DNBS-instillation were dissected to separate mucosal and smooth muscle layers. Diseased colonic sections were further dissected at the edges of the ulcer yielding distinct ulcerated mucosal and smooth muscle layers. Results Colonic H 2 S synthesis via the CAT-3MST pathway was 5-fold greater than that produced via the previously studied pathways (CBS and CSE) (225.2 ± 58.9 nmol/g/h vs. 41.8 ± 21 nmol/g/h respectively; p 2 S synthesis via CAT-3MST markedly increased (>4-fold) compared to controls (963.6 ± 115.0 nmol/g/h vs. 225.2 ± 58.9 nmol/g/h, respectively; p 2 S synthesis were observed when inflammation was most robust (3 days post DNBS) and were suppressed by both l -aspartate and CHH (1132.3 ± 87.7 nmol/g/h control vs. 424.2 ± 153.4 nmol/g/h; p p 2 S synthesis decreased to control levels. Furthermore, the ability of the ulcerated mucosa to produce H 2 S via the CAT-3MST pathway was markedly increased when compared to smooth muscle layers (both healthy and ulcerated) and the mucosa from healthy colons. The capacity of the ulcerated mucosa to produce H 2 S via the CBS/CSE pathways also increased significantly when compared to smooth muscle sections (both healthy and ulcerated) and healthy mucosal layers. Conclusions These data suggest that in both the healthy and inflamed colon CAT and 3MST represent the primary pathway for H 2 S synthesis. The increased production of H 2 S via the CAT-3MST enzymes appears to be occurring at the site of mucosal ulceration. Interestingly, the CBS/CSE pathways also make important contributions to H 2 S synthesis at the ulcerated mucosa. Further understanding of the pathways for H 2 S production in the colon can present new opportunities for modulating colonic H 2 S production, which may have important implications in the development of novel therapies for inflammatory bowel disease.

Melanization of flavonoids by fungal and bacterial laccases

Laccase activity in plants results in the formation of a number of brown pigments, often referred to as tannins. Laccase-dependent pigment production is also catalogued in numerous fungal and bacterial species. The laccase of the haploid yeast Cryptococcus neoformans forms melanin-like pigmentation outside the cell wall in the presence of exogenous substrates. While this process is a contributing factor to its virulence in humans, the evolutionary intent for the laccase function remains a mystery. We show here that C. neoformans and Bacillus subtilis have the ability to create melanin-like pigments from a variety of flavonoid molecules across a range of conformations, preferring those with 3',4'-dihydroxylations. Since flavonoids are ubiquitous plant molecules and often-considered antimicrobial agents, we postulate that they are the intended natural targets of laccase activity and result in the formation of a defensive melanin-like coat. These results suggests a new mechanism by which flavonoid-melanin formation may occur, using not only A- and C-ring linkages, but also monomer links through the B-ring of the flavonoid structure. We also show that resveratrol and other non- and mono-hydroxylated polyphenol substrates have the ability to restrict pigment formation and may be potent inhibitors of laccase activity.

Characteristics of the succinate transport into Saccharomices cerevisiae cellsafter prolonged cold preincubation

A nonconventional approach to the measurement of succinate transport through plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport through plasmalemma. Impermeable specific inhibitor of plasmalemma dicarboxylate transporter was employed as a tool to optimize conditions for the transport activity assay. For this purpose yeast culture was grown in synthetic medium under selected conditions. After aerobic preincubation of S. cerevisiae cells at 0 degree C (instead of incubation at 15 degrees C), the rate of endogenous respiration decreased substantially and was stabilized during measurements at a level that was five times lower than oxidation rates in the presence of exogenous substrate. This approach allowed for the reproducible determination of K(M) for the dicarboxylate transporter (7.3 +/- 2.1 mM) within a half-hour period. The advantages and drawbacks of this fast, but indirect, assay of slow substrates transport into the cell are compared with conventional methods.

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in l-dopa, methyl-dopa, (−)-epinephrine or (−)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by l-dopa. Electron microscopy indicated that l-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase.

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) beta 2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) β2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Complement C5b-9-Mediated Arachidonic Acid Metabolism in Glomerular Epithelial Cells: Role of Cyclooxygenase-1 and -2

In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which is partially mediated by eicosanoids. This study addresses the role of cyclooxygenase (COX)-1 and -2 in C5b-9-mediated eicosanoid production in GEC. Unstimulated rat GEC in culture primarily express COX-1. When stimulated with sublytic C5b-9, COX-2 was significantly up-regulated, whereas COX-1 was not affected. Compared with control, complement-treated GEC produced 32% more prostaglandin (PG) E(2) in the presence of exogenous substrate, and the increase was abolished with the COX-2-selective inhibitor, NS-398. Release of arachidonic acid from GEC phospholipids via C5b-9-induced activation of cytosolic phospholipase A(2) was associated with a marked stimulation of PGE(2) production, which was inhibited by 60% with NS-398. The results in cultured GEC were extended to GEC injury in vivo by examining COX-1 and -2 expression in PHN. Glomeruli from rats with PHN expressed significantly more COX-1 and COX-2, as compared with normal rats. PGE(2) production in glomeruli of rats with PHN was about twofold greater than in control glomeruli, and the increase was partially inhibited with NS-398. Thus, in GEC in culture and in vivo, C5b-9-induced eicosanoid production is regulated by both isoforms of COX. The inducible COX-2 may be an important novel mediator of C5b-9-induced glomerular injury.

What is the signal for the posttranslational arginylation of proteins?

The N-terminal, posttranslational arginylation of proteins is ubiquitous in eukaryotic cells. Previous experiments, using purified components of the reaction incubated in the presence of exogenous substrates, have shown that only those proteins containing acidic residues at their N-terminals are arginylation substrates. However, data from experiments that used crude extracts of brain and nerve as the source of the arginylating molecules, suggest that the in vivo targets for arginylation are more complex than those demonstrated using purified components. One of the proposed functions for arginylation is as a signal for protein degradation and proteins that have undergone oxidative damage have been shown to be rapidly degraded. In the present experiments we have tested the hypothesis that the presence of an oxidatively damaged residue in a protein is a signal for its arginylation. These experiments have been performed by adding synthetic oxidized peptides to crude extracts of rat brain, incubating them with [3H]Arg and ATP and assaying for arginylated peptides using RP-HPLC. Results showed that while the oxidized A-chain of insulin was arginylated in this system, confirming previous experiments, other peptides containing oxidized residues were not. When a peptide containing Glu in the N-terminus was incubated under the same conditions it too was not a substrate for arginylation. These findings show that neither the presence of an N-terminal acidic residue nor an oxidized residue alone are sufficient to signal arginylation. Thus, another feature of the oxidized A-chain of insulin is required for arginylation. That feature remains to be identified.

Apoptogenic Ganglioside GD3 Directly Induces the Mitochondrial Permeability Transition

Early events in apoptotic cascades initiated by ceramides or by activation of the surface receptor CD95 (Fas/APO-1) include the formation of ganglioside GD3. GD3 appears to be both necessary and sufficient to propagate this lipid-mediated apoptotic pathway. Later events common to many apoptotic pathways include induction of the mitochondrial permeability transition (PT) and cytochrome c release, which in turn triggers downstream caspases and cell death. The links between GD3 formation and downstream stages of apoptosis are unknown. We report that ganglioside GD3 directly induces the PT in isolated rat liver mitochondria at 30-100 microM in the presence of exogenous substrate (succinate) and at approximately 3 microM in the absence of exogenous substrate. In contrast, other gangliosides tested (e.g. GM1) have only weak stimulatory effects in the presence of succinate and protect against PT induction in the absence of respiratory substrates. GD3-mediated induction of PT was antagonized by known PT inhibitors, namely cyclosporin A, ADP, trifluoperazine, and Mg(2+). GD3 induced PT even in the presence of submicromolar Ca(2+); GD3 is therefore the first biological PT inducer identified that does not require elevated Ca(2+). Exposure to GD3 also led to mitochondrial cytochrome c release. In contrast, C(2)-ceramide, which can initiate the lipid-mediated apoptotic cascade in susceptible cells, failed to either induce PT or release cytochrome c. These observations suggest that GD3 propagates apoptosis by inducing the PT and cytochrome c release. This model provides a mechanistic link between the earlier and later stages of CD95-induced/ceramide-mediated apoptosis.

Changes in the forward and reverse metabolism of aromatizable androgens during the development of large ovarian cysts in the pregnant rat.

Twice-daily treatments with subovulatory doses of hCG result in the development of large ovarian cysts that possess the capacity to produce preovulatory amounts of estradiol (E2) in the presence of exogenous substrate (Biol Reprod 1991; 45:34-42). To determine the effects of prolonged stimulation by subovulatory doses of LH-like activity on the ability of ovarian follicles to produce aromatizable androgens and their metabolites and on the capacity of similar follicles to metabolize exogenous androstenedione (A4) and testosterone, pregnant rats were treated with either 0 (control), 1, or 3 IU hCG twice daily for 9 days, beginning on Day 13 of pregnancy. The largest follicles or cysts in the ovaries of these animals on Days 15, 17, 19, and 22 were incubated for 4 h in the presence of 1) medium alone, 2) 1 mM cAMP, 3) 800 ng/ml A4 with or without cAMP, or 4) 800 ng/ml testosterone with or without cAMP. In the presence or absence of cAMP, follicular incubates from controls displayed limited amounts of A4, testosterone, E2, estrone (E1), and 5alpha-reduced androgen accumulation compared to incubates from rats treated with hCG. By Day 22, follicular incubates from rats treated with 3 IU hCG contained more A4 than incubates from animals treated with 1 IU hCG. However, on each day tested, incubates from rats treated with 1 IU hCG displayed at least as much, and usually more, testosterone, E2, E1, and 5alpha-reduced androgens as did incubates from rats treated with 3 IU hCG. Follicles from rats treated with 3 IU hCG lost their ability to respond to cAMP with increased steroidogenesis. The capacity of follicles from controls and from rats treated with 3 IU hCG to metabolize exogenous A4 to testosterone, E2, and 5alpha-reduced androgens was maintained between 32 and 37 ng of steroid/4 h from Day 15 to Day 22, whereas the capacity of follicles from rats treated with 1 IU hCG to metabolize A4 ranged from 68 to 92 ng of steroid/4 h. Similar patterns for E2 and 5alpha-reduced steroid production were observed when exogenous testosterone was used as substrate. Follicles from all in vivo treatment groups displayed similar capacities to reverse-metabolize exogenous testosterone to A4 and E1 on Day 15 of pregnancy. This capacity increased dramatically, from 29 to 75 ng of steroid/4 h, between Days 15 and 17 for follicles from rats treated with 3 IU hCG. A similar increase was not observed for follicular incubates from rats treated with 1 IU hCG until Day 22 of pregnancy, and was never observed for follicles from controls. In summary, prolonged exposure to stimulation by subovulatory doses of LH-like activity differentially affects the forward and reverse metabolism of aromatizable androgens by ovarian cysts that are developing in the pregnant rat. The limited ability of large, hCG-induced cysts to forward-metabolize A4 and the apparent increased capacity of these follicles to reverse-metabolize testosterone to A4 indirectly support the notion that follicular 17-ketosteroid reductase and 17beta-hydroxysteroid dehydrogenase activities may be differentially regulated during the induction of ovarian cysts in response to prolonged stimulation by gonadotropins in the anovulatory environment of the pregnant rat.

Isolation and Sequencing of Two cDNA Clones Encoding Rat Spleen Cathepsin E and Analysis of the Activation of Purified Procathepsin E

Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase consisting of two identical subunits with a molecular mass of approximately 42 kDa and has a unique subcellular distribution in various rat tissues. In this study, we determined the complete amino acid sequence of rat spleen CE and examined the activation mechanism of the proenzyme purified from this tissue. Two cDNA clones encoding rat CE, termed pTNO5 and pTN1, were isolated. Comparison of the amino acid sequences predicted from the respective cDNA sequences revealed that they were essentially identical, except that pTN1 lacked a sequence corresponding to residues Tyr 293-Pro 325 of the longer cDNA clone (pTN05). Based on the structural analysis of purified enzyme forms, the CE precursor was found to comprise a signal peptide, a prosequence, and a mature protein region of 19, 39, and 337 (pTN05) or 304 (pTN1) residues, respectively. Despite a high degree of similarity in the overall structure of CE between rat and other mammalian species, the first 11 residues in the NH 2-terminal sequence of rat mature enzyme were significantly different from those of other species. The purified pro-CE was analyzed for its conversion to the mature form. The results indicated that the maximal conversion occurred at pH 3.0-4.0 in a temperature- and time-dependent manner by autocatalytic cleavage at the site between Phe 39-Ser 40. This conversion was highly dependent on the protein concentrations of pro-CE and delayed by the presence of exogenous substrates, suggesting the predominance of intermolecular reaction for its conversion to the mature form.

Interleukin-1-induced activation of a protein kinase co-precipitating with the type I interleukin-1 receptor in T cells.

We have investigated the possibility of a protein kinase participating in the signal transduction mechanisms of the interleukin-1 (IL-1) type I receptor (IL-1RI). Our data show that a protein kinase was co-precipitated with the IL-1RI from the two murine T helper cell lines D10N and EL-4. The kinase activity was detected in an in vitro kinase assay performed with the immuno beads in the presence of exogenous substrates. IL-1 treatment of the cells resulted in a rapid activation of this protein kinase in a concentration-dependent manner. Both forms of IL-1, IL-1 alpha and IL-1 beta, induced this kinase activity, whereas the IL-1 receptor antagonist (IL-1ra) was inactive. In excess IL-1ra competitively antagonized IL-1 stimulation. In the in vitro kinase assay the exogenous substrates myelin basic protein and histone H1 were phosphorylated, whereas casein or heat-shock protein HSP27 were not accepted, reflecting a certain selectivity of this protein kinase. The IL-1RI co-precipitable protein kinase showed a serine/threonine specificity and was inhibited by staurosporine, but not by inhibitors specific for protein tyrosine kinase or protein kinase C. These results show that a serine/threonine protein kinase directly interacts with the IL-1RI at the plasma membrane level of T helper cells forming a novel type of IL-1 inducible signaling complex. This protein kinase may resemble the link coupling the plasma membrane IL-1 receptor to cytosolic downstream elements in the IL-1 signaling pathway.

Purification and characterization of casein kinase II from lactating rabbit mammary gland

1. 1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono Q steps. 2. 2. Its K m for ATP was 2.22 μM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II K i = 5 and 1 mM respectively). 3. 3. MG-CK II autophosphorylated on its α-, α '- and β-subunits. The β-subunit auto-phosporylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration. 4. 4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

Relative Roles of Theca and Granulosa Cells in Ovarian Follicular Steroidogenesis in the Amphibian, Rana nigromaculata

Using frog ovarian follicles in vitro we assessed the role of the theca/epithlium (THEP) layer and granulosa cells in the production of steroids. The THEP layer and granulosa cell-enclosed oocytes (GCEOs) were separated from ovarian follicles of the frog, Rana migromaculata, by microdisxection. Intact follicles (IFs), THEP layer, or GCEOs were cultured for 6 hr in amphibian Ringer's in the presence or absence of various steroid precursors (100 ng/ml) or of frog pituitary homogenate (FPH, 0.05 gland/ml). The amounts of progesterone (P 4), 17α-hydroxyprogesterone (17α-OHP 4), androstenedione (AD), testosterone (T), and estradiol (E 2) secreted into the medium by the different types of follicular components during culture were measured by radioimmunoassay. Addition of pregnelone resulted in a marked increase in P 4 concentrations produced by GCEOs (1858 pg/follicle) and IFs (1549 pg/follicle), but a much smaller increase in P 4 occurred in the THEP layer (427 pg/follicle). Likewise, addition of P 4 stimulated greater production of 17α-OHP 4 by GCEOs (659 pg/follicle) and by IFs (877 pg/follicle) than by the THEP layer (330 pg/follicle). Exogenous 17α-OHP 4 markedly increased AD levels secreted by GCEOs (1139 pg/follicle) and by IFs (841 pg/follicle), but not by the THEP layer (<15 pg/follicle) and by IFs (841 pg/follicle), but not by the THEP layer (<15 pg/follicle). In contrast, AD addition resulted in the secretion of markedly elevated levels of T by the THEP layer (2326 pg/follicle) and IFs (3652 pg/follicle) as compared to the GCEOs (408 pg/follicle). Addition of T markedly increased E 2 concentrations produced by GCEOs (378 pg/follicle) and by IFs (673 pg/follicle), but not by the THEP layer (<12 pg/follicle). Without addition of FPH or steroid precursors, none of the follicular components examined produced significant amounts of the steroids measured (<20 pg/follicle). Denuded oocytes incubated in the presence of exogenous substrates or FPH secreted nearly nondetectable amounts of steroids. Theses data suggests that the main sites for synthesis of P 4, 17α-OHP 4, AD, and E 2 are granulosa cells and those of T are the theca/epithelium layer in amphibian ovarian follicles.

Synthesis of phosphatidylinositol 4,5-bisphosphate in the endoplasmic reticulum of Chinese hamster ovary cells.

We have investigated the intracellular localization and synthesis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in Chinese hamster ovary (CHO) cells by analyzing membrane fractions that were obtained by sucrose density gradient centrifugation. After labeling the cells for 24 h with [3H]inositol, the bulk of [3H] PtdInsP2 was found in the plasma membrane fraction, yet this lipid was also distinctly present in the microsomal fraction (20% of total cellular [3H]PtdInsP2). To determine the origin of this microsomal PtdInsP2, gradient fractions from unlabeled CHO cells were incubated with [3H]inositol in the presence of an ATP-generating system. Under these conditions of labeling, [3H]PtdIns was exclusively present in the microsomal fractions and found to be partially converted to [3H] phosphatidylinositol 4-phosphate ([3H]PtdInsP) and phosphatidylinositol 4-phosphate ([3H]PtdInsP) and [3H]PtdInsP2. The ability of microsomes to synthesize PtdInsP and PtdInsP2 was confirmed by assaying the gradient fractions for PtdIns and PtdInsP kinase activity using endogenous substrate and [gamma-32P]ATP. In the presence of exogenous substrate and Triton X-100, PtdInsP kinase activity was particularly high in the plasma membrane fractions. When phosphoinositide synthesis was studied in permeabilized CHO cells under conditions of sustained membrane vesicle flow (Helms, J. B., Karrenbauer, A., Wirtz, K. W. A., Rothman, J. E., and Wieland, F. T. (1990) J. Biol. Chem. 265, 20027-20032), no lag-time could be detected between the synthesis of [3H]PtdIns and the formation of [3H]PtdInsP2. Moreover, when lipid transport pathways were blocked in these permeabilized cells either by omission of membrane-free cytosol, addition of GTP gamma S and brefeldin A, or temperature block, PtdInsP2 formation still occurred at normal levels. These results strongly suggest that PtdInsP2 can be formed at the site of PtdIns synthesis, i.e. the endoplasmic reticulum (ER). The relationship between PtdInsP2, generated in the ER, and PtdInsP2 present in the plasma membrane, remains to be established.

The effect of inorganic phosphate on chick ( Gallus domesticus) heart mitochondrial volume and cation content

1. 1. In the absence of exogenous Ca(II), P i induces a swelling change that is kinetically first order with k = 1.08 ± 0.1 min −1. The first-order rate constant is independent of [P i] over the range 0.5–45 mM. 2. 2. In the presence of exogenous substrate, the volume change induced by P i is monophasic and can be reversed by ADP. 3. 3. The swelling process and the approach to steady state is accompanied by controlled losses of both K + and Mg(II) from within the mitochondria. 4. 4. The loss of K + is biphasic as a function of time with K 1 = 14.1 ± 1.6 and k 2 = 4.4 ± 0.34 nmol min −1 mg mitochondria −1. 5. 5. The loss of Mg(II) is monophasic and the rate at which this cation is released decreases as a function of time. Ca(II) fluxes are not involved in the volume occurring secondary to P i uptake. 6. 6. In the absence of exogenous substrate, P i induces a triphasic change in mitochondrial volume. 7. 7. The sequence of volume changes corresponds to an initial first-order swelling secondary to the addition of P i, a contraction apparently triggered by the loss of approximately 85% of total intra-mitochondrial Mg(II), and a second larger swelling phase that cannot be reversed with ADP. 8. 8. The P i-induced swelling of chick heart mitochondria is not inhibited by EGTA and does not depend on the provision of exogenous Ca(II). 9. 9. The Ca(II) and Mg(II) ions released from within the mitochondria are responsible for activating divalent cation-dependent ATPases which cosediment with isolated chick heart mitochondria.

Mass spectrometric evidence for the conversion of exogenous adrenate to dihomo-prostaglandins by seminal vesicle cyclo-oxygenase : A comparative study of two animal species

We present conclusive mass spectrometric evidence for the synthesis of 1 a,1 b-dihomoprostaglandin E 2 and 1 a,1 b-dihomoprostaglandin F 2α by sheep seminal vesicle microsomes and swine whole vesicular homogenates. Swine microsomes prepared according to standard procedure were catalytically inactive, possibly due to autoinactivation of the cyclo-oxygenase caused by metabolism of endogenous substrate released during the isolation procedure. Cyclo-oxygenase activity could be demonstrated, however, when the porcine vesicles were homogenized in the presence of exogenous substrate. Chemical and mass spectrometric evidence presented here allow unequivocal structural assignments.

Substrate-level energy dependence of acid secretion in the isolated human gastric mucosa

The substrate-level energy dependence of acid secretion was investigated in the human gastric mucosa in vitro using biopsy specimens obtained during fiberoptic gastroscopy in symptomatic patients. The oxygen consumption and the accumulation of aminopyrine were used as indexes of secretory activity. Basal and histamine-stimulated oxygen uptake by fundic biopsy specimens were not affected by medications (pethidine and diazepam) administered during gastroscopy. Under substrate-depleted conditions, the oxygen consumption and the aminopyrine accumulation of fundic mucosa were not significantly increased by histamine. Stimulation of functional activities by gastric secretagogues was observed only in the presence of exogenous substrates. The ability of various substrates to support acid formation in the presence of gastric secretagogues was evaluated. Carbohydrates were found to be effective substrates in supporting the functional responses of the tissue, with glucose being the most effective. Propionate, butyrate, β-hydroxybutyrate, and octanoate were ineffective as substrates. Glucose by itself, but not butyrate, significantly increased oxygen uptake and aminopyrine accumulation. The results suggest that the human gastric mucosa, in vitro, has an absolute requirement for metabolic substrates to support secretory responses and that carbohydrates seem to be the preferential substrates.