Showing papers in "Iubmb Life in 1993"

Changes in skeletal muscle, heart and liver mitochondrial electron transport activities in rats and dogs of various ages.

Abstract: We determined skeletal muscle, heart and liver mitochondrial electron transport activities in rats and dogs of various ages. In the skeletal muscle mitochondria, decrease in the activity of complex I was observed in rats aged 28 weeks, and further reduction of the activity was observed in rats aged 55 weeks. A significant decrease in complex IV activity was observed in rats aged 55 weeks. No significant reduction in complex II and III activities were observed in rats aged up to 100 weeks. Significant decreases in complex I and IV activities were observed in heart muscles of rats aged 100 weeks, while no significant changes in the activity of complex I in liver mitochondria were observed in rats aged up to 100 weeks. Similar results were obtained in dogs, i.e., the activity of complex I was the most susceptible to aging among the activities of complexes; and skeletal muscle mitochondria were the most susceptible to aging among the tissues. From our results, involvement of mitochondria in the development of age-related decline in cellular function is especially emphasized in post mitotic cells, and age-associated mitochondrial functional changes are stressed in mitochondrial complexes which contain mitochondrial DNA-encoded subunits.

On the functions of the yeast COX10 and COX11 gene products.

Abstract: COX10 and COX11 are nuclear genes of Saccharomyces cerevisiae whose products are localized in mitochondria and are required for the synthesis of cytochrome oxidase. Genes homologous to COX10 are present in at least four different bacterial cytochrome oxidase operons. The bacterial gene, termed cyoE, has recently been proposed to code for a farnesyl transferase that converts protoheme to heme O (Saiki et al. (1992), Biochem. Biophys. Res. Commun. 189, 1491-1497). In this communication we report that the COX10 protein, like the product of cyoE is needed for heme A synthesis. Analyses of the heme constituents in a cox11 mutant indicate the absence of heme A and presence of a novel heme with chromatographic properties indistinguishable from those of heme O. This evidence suggests that the COX11 protein may be another heme A biosynthetic enzyme involved in forming the formyl group at position 8 of the porphyrin ring.

Inhibition of NF-kappa B DNA binding activity by alpha-tocopheryl succinate.

Abstract: Vitamin E derivatives have been shown to inhibit tumor necrosis factor-induced NF-kappa B activation in human Jurkat T cells. The present report demonstrates that alpha-tocopheryl succinate directly blocks DNA binding activity of activated NF-kappa B, as well as the NF-kappa B activation induced by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. In contrast, alpha-tocopherol, alpha-tocopheryl acetate and 2,2,5,7,8-pentamethyl-6-hydroxychromane have no effects. These observations suggest that the mechanism of alpha-tocopheryl succinate action against NF-kappa B is different from those of other vitamin E derivatives.

Growth of rho 0 human Namalwa cells lacking oxidative phosphorylation can be sustained by redox compounds potassium ferricyanide or coenzyme Q10 putatively acting through the plasma membrane oxidase.

Abstract: Pyruvate is conventionally used as a key growth supplement for mammalian rho 0 cells that lack mitochondrial DNA and are thereby devoid of oxidative phosphorylation. We have tested the proposition that cultured rho 0 human cells can be grown using redox compounds other than pyruvate. The results show that potassium ferricyanide and coenzyme Q10 can each be used to replace pyruvate to support the growth of rho 0 Namalwa cells (a lymphoblastoid cell line). Ferricyanide and coenzyme Q10 have both been reported as substrates for a plasma membrane NADH oxidase system which is capable of re-oxidising cytosolic NADH to NAD+. These compounds are also known to stimulate the activity of this enzyme system. We interpret our data to indicate that redox support for growth of rho 0 human cells can be achieved by external electron acceptors such as ferricyanide (a plasma membrane impermeant compound), or coenzyme Q10 (an integral component of the plasma membrane oxidase), through the enhanced conversion of cytosolic NADH to NAD+. This re-oxidation of NADH enables glycolysis to function efficiently as the sole source of cellular ATP, in the absence of mitochondrial oxidative phosphorylation in rho 0 cells. This has important implications for the development of new strategies for the amelioration of the bioenergy decline that occurs in mitochondrial disease and during the human ageing process.

Failure in detecting mRNA transcripts from the mutated allele in myotonic dystrophy muscle.

Abstract: The expression of myotonin-protein kinase (MT-PK) gene was studied in myotonic dystrophy (DM) muscle and normal controls using a polymerase chain reaction protocol to analyse the 3' intragenic p(CTG) polymorphism. Unaffected individuals show bi-allelic expression, while the sole wild-type allele was transcribed in DM muscle. Our findings support a gene dosage effect in the pathogenesis of the disease.

Purification and characterization of the branched chain alpha-ketoacid dehydrogenase complex from Saccharomyces cerevisiae.

Abstract: Branched chain alpha-ketoacid dehydrogenase complex was purified from Saccharomyces cerevisiae by polyethylene glycol fractionation and chromatography on Sephacryl S-200, DEAE-cellulose and Sepharose CL-2B. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated the enzyme contained subunits of M(r) = 57,000, 52,000, 47,000 and 38,000. The specific activity of the purified enzyme was 0.82 mumol NADH/min/mg protein at 30 degrees C with 16 mM alpha-ketoisovalerate as substrate. The apparent Km values for alpha-ketoisovalerate, alpha-ketoisocaproate and alpha-keto-beta-methylvalerate were 21, 22, and 20 mM, respectively. The preparation was also able to oxidize the intermediates of threonine and methionine metabolism, alpha-keto-gamma-methiolbutyrate and alpha-ketobutyrate, with Km values of 13 and 8 mM, respectively.

Human breast milk contains procathepsin D : detection by specific antibodies

Abstract: The presence of the zymogen of cathepsin D in human milk was detected using antibodies specific for the proenzyme and by the proteolytic activity at low pH. The antibodies were raised against a synthetic propeptide of human cathepsin D and were tested using immunoprecipitations and western blots of samples from different breast cancer cell lines as well as cytosol fractions of human breast cancer tissues. In all experiments these antibodies recognized specifically procathepsin D. Procathepsin D from human milk was partially activated at low pH. The activity was monitored using hemoglobin 14C proteolytic assay, and it was abolished by pepstatin A--a specific inhibitor of aspartic proteinases. Western blots did not reveal presence of cathepsin B or cathepsin H. These data indicate specific secretion of cathepsin D in human breast milk.

Fumarate catabolism in Helicobacter pylori.

Abstract: The metabolism of fumarate by Helicobacter pylori was investigated employing one- and two-dimensional 1H and 13C nuclear magnetic resonance spectroscopy. Metabolically competent cells generated malate and succinate from fumarate as the sole substrate indicating the presence of fumarase and fumarate reductase activities in the bacterium. In incubations of fumarate with cell lysates accumulation of lactate, acetate, formate and alanine was observed after the initial production of malate and succinate. The results indicate the existence of active fumarate catabolism in H. pylori and suggest the possibility of an ATP generating mechanism which may play an important role in the bioenergetics of the bacterium.

New mutation of the myelin P0 gene in a pedigree of Charcot-Marie-Tooth neuropathy 1.

Abstract: P0, the major structural protein of peripheral myelin, is a homophilic adhesion molecule with a single immunoglobulin (Ig) domain, which contains a single N-linked glycosylation site and two cysteines. We have previously reported four different mutations of the myelin P0 gene in four families of Charcot-Marie-Tooth neuropathy type 1 (CMT1). In this study we found a new mutation of the myelin P0 gene in a small family of CMT1. The affected persons had an A - to - G substitution of nucleotide 245 of the myelin P0 gene in one allele, leading to a cysteine substitution for tyrosine82 in the extracellular Ig-domain. An additional cysteine in the extracellular domain may form a disulfide bond and cause an inappropriate change in the tertiary structure of the functional Ig-domain of P0.

Characterization of D-amino acid oxidase from Trigonopsis variabilis.

Abstract: D-amino acid oxidase from Trigonopsis variabilis was purified to homogeneity as a well resolved flavoprotein. Specific activity of pure enzyme was 86.6 U/mg at 30 degrees C and pH 8.5. Optimum pH for enzyme activity was 7.5 and optimum temperature was 55 degrees C. The enzyme is a non-glycosylated homodimer; the protein monomer had a M(r) of 38 +/- 2 kDa and contained one molecule of non covalently bound FAD per mole of monomer. A single molecular form with an isoelectric point of 5.1 was detected in isoelectrofocusing. The A272/A455 ratio as calculated from the absorbance spectrum was 8.4. The enzyme bound competitive inhibitors benzoate and anthranilate giving typical flavin spectral perturbations.

Influence of palm oil or its tocotrienol-rich fraction on the lipid peroxidation potential of rat liver mitochondria and microsomes.

Abstract: The effect of palm oil, a widely used vegetable oil, rich in tocotrienols, on peroxidation potential of rat liver was examined. Long-term feeding of rats with palm oil as one of the dietary components significantly reduced the peroxidation potential of hepatic mitochondria and microsomes. As compared to hepatic mitochondria isolated from rats fed control or corn oil-rich diet, those from palm oil-fed group showed significantly less susceptibility to peroxidation induced by ascorbate and NADPH. However, in microsomes, only NADPH-induced lipid peroxidation was significantly reduced in rats fed palm oil rich-diet. Though the accumulation of thiobarbituric acid reactive substances during ascorbate-induced lipid peroxidation in mitochondria from rats fed corn oil-rich diet supplemented with tocotrienol-rich fraction (TRF) of palm oil was similar to that of control rats, the initial rate of peroxidation was much slower than those from control or corn oil fed diets. Our in vitro studies as well as analyses of co-factors related to peroxidation potential indicated that the observed decrease in palm oil-fed rats may be due to increased amount of antioxidants in terms of tocotrienol as well as decrease in the availability of substrates for peroxidation.

Stimulation of nerve growth factor synthesis/secretion in mouse astroglial cells by coenzymes.

Abstract: We examined the effect of coenzymes such as PQQ, pyrroloquinoline quinone; TOPA, 3-(2,4,5-trihydroxyphenyl)-DL-alanine, and lipoic acid on nerve growth factor (NGF) synthesis in mouse astroglial cells, BALB c/3T3 cells, and WS-1 cells in culture. These coenzymes had a stimulating effect on NGF synthesis without causing cytotoxicity. Especially PQQ showed the strongest activity of promoting NGF synthesis in astroglial cells, whereas lipoic acid had the strongest effect on BALB c/3T3 cells. The activity may not due to the catechol ring or 1,4-benzoquinone ring, but due to the oxidative or reductive activity. These results suggest that these coenzymes may play a role in NGF synthesis and neuronal survival through the stimulating effect of the NGF synthesis in brain and such compounds are good candidates as NGF inducers.

The effect of boron on plasma membrane electron transport and associated proton secretion by cultured carrot cells

Abstract: Plasma membrane electron transport reactions and associated proton secretion were studied in boron-deficient carrot cells. It was found that the hormone-sensitive plasma membrane NADH oxidase was inhibited by boron deficiency and that under such conditions activity could be restored by exogenous boric acid with or without 2,4-dichlorophenoxy acetic acid. Gramicidin, a channel-forming protonophore, further stimulated NADH oxidase by carrot cells. Proton secretion, associated with plasma membrane H(+)-ATPase, was also affected by boron deficiency, but not as severely as ferricyanide-generated proton secretion, reflecting plasma membrane electron transport. The addition of 1 mM boric acid and 1 microM 2,4-dichlorophenoxy acetic acid to carrot cells fully restored the H+ secretion in presence of ferricyanide. The effect of boron deficiency in cultured carrot cells can, therefore, be directly associated with cell growth through its effect on the plasma membrane NADH oxidase and H+ secretion. Ferricyanide provides a probe which activates transmembrane electron transport that is only coupled to proton release when boron is present.

Purification and characterization of human macrophage migration inhibitory factor: evidence for specific binding to glutathione and formation of subunit structure.

Abstract: We cloned cDNA of human macrophage migration inhibitory factor (MIF) from a T-cell line encoding 116 amino acid residues and expressed it in E. coli. MIF specifically bound glutathione (dissociation constant = 600 microM), and the protein was effectively purified by an S-hexylglutathione Sepharose affinity column. MIF was homogeneous on SDS-PAGE and the molecular weight was calculated as 12.5 kDa. The molecular weight was in excellent agreement with that of the primary structure assumed from the cDNA. On the other hand, the molecular weight in the native form calculated by Sephadex G-100 gel chromatography was 24.3 kDa, which suggested that MIF formed a dimeric structure. To further confirm the native molecular weight, we carried out analytical ultracentrifugation. This precise hydrodynamic analysis revealed that the native molecular weight was 24.8 kDa, which confirmed that MIF existed as a homodimeric form.

Cloning and sequence analysis of the major cysteine protease expressed in the trematode parasite Fasciola sp.

Abstract: A cDNA encoding the Fasciola cysteine protease precursor has been cloned and sequenced. The deduced precursor contains 325 amino acid residues (M(r) 36,768) consisting of a signal sequence (pre-region, 15 residues), pro-region (90 residues), and mature protease (220 residues, M(r) 24,371). Cys27, His164, and Asn184 form the catalytic triad in the active site of the mature enzyme, and their adjacent regions are highly conserved. The parasite enzyme showed 50, 44, and 27% amino acid sequence homologies, compared with mammalian lysosomal cathepsins L, H, and B, respectively. N-linked glycosylation sites which are thought to be targeting signals for cathepsins into lysosomes is absent in any region of the Fasciola protease precursor.

Influence of antioxidant vitamins on fatty acid inhibition of lymphocyte proliferation

Abstract: Fatty acids, especially polyunsaturated fatty acids (PUFAs), inhibit a number of lymphocyte functions, including proliferation, cytokine production and cytotoxicity, but their mechanism of action is not known. This study investigated whether fatty acids inhibit lymphocyte proliferation by leading to the production of lipid peroxides, which are known to inhibit the growth of cells. The so-called "thiobarbituric acid-reactive substances" (TBARS) and lipid hydroperoxide contents of lymphocytes (0.75 +/- 0.04 and 1.30 +/- 0.39 nmol/mg protein in fresh cells, respectively) were increased by 48 h culture to 0.96 +/- 0.14 and 3.23 +/- 0.47 nmol/mg protein, respectively. The TBARS content was increased by culture in the presence of 100 microM PUFAs to between 1.46 +/- 0.11 (linoleic acid) and 2.39 +/- 0.31 (docosahexaenoic acid) nmol/mg protein. The lipid hydroperoxide content was increased by culture in the presence of 100 microM PUFAs to between 11.65 +/- 1.12 (linoleic acid) and 22.24 +/- 1.26 (docosahexaenoic acid) nmol/mg protein. These increases were partially prevented by inclusion of 10 microM vitamin E in the culture medium. Vitamin E (1 or 10 microM) enhanced concanavalin A-stimulated rat lymphocyte proliferation by approximately 45%. Vitamin E (10 microM) increased human lymphocyte proliferation by 35%. However, vitamin E did not prevent the inhibitory effects of fatty acids upon lymphocyte proliferation. It is concluded that inhibition of lymphocyte proliferation by fatty acids is not caused by their conversion to peroxidised products.

Determination of halfcystine in proteins as cysteine from reducing hydrolyzates.

Abstract: The separation of cysteine from proline using a variant of the classical cation exchange system for amino acid analysis is described. From reducing hydrolyzates (6 M HCl/0.1% phenol/5% thioglycollic acid, 18 h, 110 degrees C) the halfcystine content of proteins can be accurately determined as cysteine. The system is useful for routine determination of the composition of proteins from a single type of hydrolyzate.

Effect of ageing on rat liver regeneration after partial hepatectomy.

Abstract: The effect of ageing on liver regeneration after two-thirds partial hepatectomy was evaluated using rats of 6 and 60 weeks of age. The induction of thymidylate synthase and thymidine kinase, which are rate-determining enzymes of DNA synthesis in liver regeneration, delayed by 24 h and the maximal activities were significantly lower in old rats. Effects of aging on liver weight and synthesis of DNA, RNA and protein were discussed quantitatively in terms of the relative restoration yield deduced from the comparison with the young animals.

Preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies.

Abstract: Myelin oligodendrocyte glycoprotein, (MOG), a quantitatively minor central nervous system (CNS) myelin component, is a candidate target antigen for autoimmune-mediated demyelination. It is a highly hydrophobic protein present in very small amounts in CNS tissue and thereby difficult to purify. Our aim was to devise a purification procedure that would yield sufficient quantities of highly purified MOG to subsequently test its potential encephalitogenic activity, as well as investigate the humoral and cell-mediated responses to this antigen in naturally occurring and experimentally induced autoimmune demyelinating diseases. MOG was purified from human CNS white matter using immunoaffinity chromatography, a procedure that gave a final yield of MOG corresponding to 0.02% total white matter protein. The final product, which migrated as two bands of molecular weight 28 kDa and 58 kDa, was highly pure as shown also by specific reactivity with monoclonal anti-MOG antibodies on immunoblots in the absence of any detectable reactivity with antibodies specific for myelin basic protein, proteolipid protein and myelin-associated glycoprotein. Partial amino acid sequence was obtained from both MOG bands separated by SDS-PAGE and electroblotted onto PVDF. The sequence of the first 17 N-terminal amino acids had approximately 55% homology with the reported rat MOG sequence deduced from the cloned cDNA sequence; small internal sequences obtained showed also very high homology. Our purified MOG preparations have been used to investigate T cell response to MOG by peripheral blood lymphocytes of multiple sclerosis patients and to induce a relapsing remitting demyelinating disease in Lewis rats.

Tumor necrosis factor-alpha is a direct hepatocyte mitogen in the rat.

Abstract: We previously reported that tumor necrosis factor-alpha (TNF-alpha) increases in vivo hepatocyte mitoses and liver regeneration in rats. In the present studies, we used in vitro hepatocyte cultures to determine whether TNF-alpha itself was mitogenic or whether other cytokines (IL-1 beta and IL-6) that share similar actions with TNF-alpha might be involved. Hepatocytes were cultured with TNF-alpha (4, 40 or 400 U/ml), IL-1 beta (0.1, 1 and 10 ng/ml) or IL-6 (0.2, 2, or 20 ng/ml) for 24 h and 3 d. Incorporation of [3H]thymidine was increased significantly by TNF-alpha (40 and 400 U/ml) at 3 d. Both IL-1 beta and IL-6 at all concentrations significantly decreased [3H]thymidine incorporation at 3 d. These results indicate that TNF-alpha has a direct mitogenic action on hepatocytes. In contrast, IL-1 beta and IL-6 appear to suppress DNA synthesis in hepatocytes.

Effect of beta-sitosterol on uterine biochemistry: a comparative study with estradiol and progesterone.

Abstract: Administration of estradiol/progesterone to ovariectomized animals significantly increased the uterine weight, RNA, DNA and protein concentrations. Similarly, administration of beta-sitosterol alone or in combination with estradiol caused a marked increase in the above parameters and the maximum influence was evident only after median and high dose treatments. However, administration of median/high dose of beta-sitosterol along with progesterone accentuated only the RNA and protein concentrations but exerted an inhibitory effect on sitosterol-induced increment in uterine weight and DNA concentrations.

Transnitrosation as a predominant mechanism in the hypotensive effect of S-nitrosoglutathione.

Abstract: S-Nitrosoglutathione, a possible intermediate in the pharmacological mechanisms of many vasodilators, undergoes hemolysis at pH 7.4 and 37 degrees C to give oxidized glutathione and nitric oxide with a second-order rate constant of approximately 3 x 10(-4) M-1sec-1. At the dosages normally employed, this reaction is, therefore, too slow to be an obligatory step in the pharmacological mechanisms of those, usually, fast-acting drugs. Transfers of the nitroso moiety to another thiol or to certain hemoproteins are, however, both much faster and could be involved in those pharmacological mechanisms. Intravenously administered S-nitrosoglutathione reduced the blood pressure of anesthesized dogs and monkeys to the same extent and with essentially the same rapid onset and dissipation as sodium nitroprusside, which is the fastest-acting of those vasodilators.

Effects of triiodothyronine on DNA synthesis and differentiation markers of normal human osteoblast-like cells in vitro.

Abstract: To study the direct effects of thyroid hormones on human osteoblasts we examined the effects of triiodothyronine (T3) on proliferation and differentiation of human osteoblast-like (hOB) cells in vitro. T3 increased 3H-thymidine incorporation in DNA of hOB cells (p < 0.05, n = 10). Half maximal effects obtained at a T3 concentration of 1-10 nM which lies within the physiological concentration of the hormone. In addition, T3 increased alkaline phosphatase production (p < 0.05, n = 13) and inhibited procollagen type I carboxyterminal propeptide (PICP) production (p < 0.05, n = 13). T3 interaction with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) was also studied. 1,25-(OH)2D3 (10(-9)M) alone doubled AP production and induced osteocalcin expression by hOB cells. Concurrent addition of T3 and 1,25-(OH)2D3 did not further increase production of AP, PICP or osteocalcin by hOB cells. In conclusion, T3 exerts significant effects on osteoblast proliferation and differentiation, suggesting that human osteoblasts are targets for thyroid hormones.

Catalytic activity of elastase in reverse micelles.

Abstract: The activity of porcine pancreatic elastase has been studied in reverse micelles formed by AOT (sodium bis(2-ethylhexyl) sulfosuccinate) in isooctane. For the two substrates succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide and succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, the catalytic constant, kcat, in reverse micelles increases with increasing wo until, at high wo, the value of kcat measured in bulk buffer solution is approached (wo = [H2O/]AOT]). In analogy to alpha-chymotrypsin--and in apparent contrast to many other enzymes--elastase does not show a maximum in the kcat-wo profile. Within the wo range of 8 to 35, for both substrates, the Michaelis constant Km (as expressed relative to the total volume of the solution, Km,overall) increases with increasing wo.

Effect of iron on the growth and siderophore production of mycobacteria.

Abstract: To gain a better understanding of the role of iron in the pathogenesis of tuberculosis, the growth and production of siderophores were studied in the presence of different concentrations of free iron in vitro with M. smegmatis and virulent, avirulent and low virulent strains of M. tuberculosis. Increase in the concentrations of iron caused an appreciable increase in the growth (as assessed by cell dry-weight and log viable counts) of all 4 strains. This was, however accompanied by a significant decrease in the production of both exochelins and mycobactins, suggesting that these siderophores are necessary only under iron-deficient conditions. The growth and production of siderophores were significantly higher with the virulent strain of M.tuberculosis than with the avirulent (or) the low virulent strains.

"Stimulation of dihydropyridine-sensitive Ca2+ influx in cultured myoblasts by 1,25(OH)2-vitamin D3".

Abstract: The rapid, non-genomic effects of 1,25(OH)2-vitamin D3 [1,25(OH)2D3] on calcium influx in cultured neonate rat and embryonic chick myoblasts were investigated. The hormone stimulated 45Ca uptake in a time (1-10 min) and dose (10(-11)-10(-7) M)-dependent manner. The effects of 1,25(OH)2D3 on both rat and chick myoblasts were mimicked by the Ca(2+)-channel agonist BAY K8644 and effectively suppressed by the antagonist nifedipine. 1,25(OH)2D3 induction of Ca2+ uptake was dependent on the presence of extracellular calcium, since it was abolished by prior addition of EGTA and was restored upon the addition of 1 mM Ca2+ Cell membrane depolarization with high K+, similarly to the hormone, elicited a rapid increase in Ca2+ uptake (+75% above control). These results suggest the modulation of Ca2+ influx through competent calcium channels in rat and chick embryo myoblasts by 1,25(OH)2D3.

Effect of furfural on plasmid DNA.

Abstract: Furfural is a dietary mutagen and is present in various food products and beverages. We have previously shown that it induces single strand breaks in double stranded DNA that occur mainly in AT sequences. In this paper, we have examined the mutagenic effect of furfural induced lesions in the plasmid pBR322. Plasmid DNA was treated with furfural before transformation in E. coli. There was a progressive decrease in the transformation capacity of the plasmid both as a function of furfural concentration and time of reaction. Several mutants were isolated from plasmid preparations whose transforming capacity had been decreased by treatment with furfural. Depending on the concentration of furfural used, these were found to have undergone DNA alterations including deletions. Experiments on the conservation of EcoRI cleavage site in mutant plasmids indicated that repair of furfural damaged plasmid DNA takes place on propagation in host cells.

Down-regulation in the production of matrix metalloproteinase 1 by human aortic intimal smooth muscle cells.

Abstract: Effects of dexamethasone, retinoic acid, prostaglandin E2 (PGE2), and Iloprost as a agonist of prostacyclin (A-PGI2) on DNA synthesis and production of a precursor of matrix metalloproteinase 1 (tissue procollagenase/proMMP-1) by human aortic smooth muscle cells were investigated. When after treatment with platelet-derived growth factor (PDGF), these agents were added to the cultures, DNA synthesis and production of proMMP-1 were inhibited in a dose-dependent manner. These results suggest that these agents are negative regulators of PDGF. Since these agents are present in the blood or produced in the blood wall, in addition, since PDGF plays the most important role in the process of atherosclerosis, we propose that these agents function in vivo as a systems of protection against atherosclerosis.

Pyruvate is a lipid precursor for rat lymphocytes in culture: evidence for a lipid exporting capacity.

Abstract: Since acetyl-CoA produced through pyruvate dehydrogenase reaction is poorly oxidized by the Krebs cycle in rat lymphocytes, the fate of acetyl units was investigated in these cells. The results presented here show that 24-h cultured lymphocytes actively synthesize lipids from [3-14C]pyruvate. Furthermore, a considerable amount of these lipids have shown to be exported into the culture medium. Experiments with [1-14C] acetate as a lipid precursor showed a close similarity with the rates of incorporation of [3-14C] pyruvate into the same lipid fractions. Treatment of lymphocytes with the mitogen concanavalin A (Con A) markedly enhanced [1-14C] acetate incorporation into a variety of lipids, but the lectin did not affect [3-14C] pyruvate incorporation. The results suggest that lymphocytes convert pyruvate into lipids via the acetyl-CoA pathway and that Con A interferes in lymphocyte lipogenesis but does not seem to affect the pyruvate dehydrogenase reaction. The ability to incorporate pyruvate into certain lipids may have an important role for the rapidly dividing capacity of lymphocytes since the human cancer strain HeLa 155 (a quickly proliferating cell line) also exhibits this feature by converting much more [3-14C] pyruvate into lipids than do lymphocytes. In addition, comparative experiments with lymphocytes, peritoneal macrophages and HeLa cells indicate that pyruvate may provide precursors for cells with active lipid producing and exporting capacities.

The major fat-globule membrane proteins, bovine components 15/16 and guinea-pig GP 55, are homologous to MGF-E8, a murine glycoprotein containing epidermal growth factor-like and factor V/VIII-like sequences.

Abstract: The glycoproteins, Components 15/16 and GP 55, were partially purified from bovine and guinea-pig fat-globule membrane (FGM), respectively. Peptides prepared from immunoblots of Component 16 and GP 55, were shown by sequencing, to be similar to mouse MFG-E8 and a human milk protein of M(r) 46,000. Both of these latter proteins are associated with the FGM and contain epidermal growth factor-like domains and/or factor V/VIII-like sequences. These data complement earlier biochemical studies on the biosynthesis, tissue distribution and topology of Components 15/16 and GP 55, and indicate that MFG-E8-like proteins are expressed on the apical surfaces of mammary epithelial cells and are secreted both in a soluble form and as major components of the FGM in diverse species. The possible function of these proteins in lactation and in mammary and neonatal development is discussed.