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PubMed1994

Induction of dolichyl-saccharide intermediate biosynthesis corresponds to increased long chain cis-isoprenyltransferase activity during the mitogenic response in mouse B cells.

There are large increases in the rates of Glc3-Man9GlcNAc2-P-P-Dol (Oligo-P-P-Dol) biosynthesis and protein N-glycosylation during the proliferative response of murine B lymphocytes (B cells) to bacterial lipopolysaccharide (LPS). To learn more about the regulation of dolichyl-saccharide biosynthesis, the possible relationships between developmental changes in specific steps in dolichyl phosphate (Dol-P) and N-acetyl-glucosaminylpyrophosphoryldolichol (GlcNAc-P-P-Dol) biosynthesis and the induction of Oligo-P-P-Dol biosynthesis were investigated. These studies describe an impressive induction of long chain cis-isoprenyltransferase (cis-IPTase) activity, an enzyme system required for the chain elongation stage in de novo Dol-P synthesis, which corresponded to the striking increase in the rate of Oligo-P-P-Dol biosynthesis in LPS-activated B cells. The cellular level and specific activity of cis-IPTase increase 15-fold in LPS-treated cells with relatively unaltered affinity for isopentenyl pyrophosphate. The rates of Dol-P and Oligo-P-P-Dol synthesis increased substantially when cis-IPTase activity was induced, suggesting a regulatory relationship between the level of cis-IPTase activity and lipid intermediate synthesis. Distinctly different developmental patterns were observed for cis-IPTase and HMG-CoA reductase activity, and when sterol biosynthesis was drastically inhibited by lovastatin, the rate of synthesis of Dol-P was slightly higher in the presence of the drug. Modest elevations in the cellular levels of dolichol kinase, Dol-P phosphatase, and UDP-GlcNAc:Dol-P N-acetylglucosaminylphosphoryltransferase (L-G1PT) activities were also observed, but these changes were relatively small compared with the increases in cis-IPTase activity and the rates of Dol-P, Gl-cNAc-P-P-Dol, and Oligo-P-P-Dol synthesis. The expression of the L-G1PT gene is an early event in the developmental program for Oligo-P-P-Dol synthesis, but GlcNAc-P-P-Dol formation is apparently not rate-limiting. In summary, large increases in cis-IPTase activity and the rate of Dol-P biosynthesis appear to play a key regulatory role in the induction of Oligo-P-P-Dol biosynthesis during the proliferative response of B cells to LPS, and the biosynthetic pathways for Dol-P and cholesterol are regulated independently in dividing B cells.

Alkyl and Aryl Transferases↗

PubMed1998

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells.

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.

Animals↗

PubMed1978

Glucose stimulated proinsulin biosynthesis rates of turn off after cessation of the stimulus.

Proinsulin biosynthesis was stimulated progressively to a plateau level at 45 min by 5.5 mmol/1 glucose. The rate of biosynthesis promptly decreased following cessation of either 15 or 60 min exposure to this concentration of glucose. Actinomycin D had no effect on the rate of decline. In contrast, exposure to 20 mmol/1 glucose caused a prolonged increase in proinsulin biosynthesis which was still apparent at 75 min. After removal of the stimulus there was an initial decrease in the rate of proinsulin biosynthesis followed by persistently elevated rates. When proinsulin biosynthesis was stimulated by 20 mmol/1 glucose in the presence of actinomycin D, there was a rapid switch off of biosynthesis and no persistent effect. Thus, two controls for regulation of proinsulin biosynthesis can be characterised: a prompt, rapidly reversible stimulation in response to low or high glucose concentrations and a persistent stimulation in response to high glucose concentrations. These two effects may represent the translational and transcriptional effects of glucose respectively.

Animals↗

PubMed2026

Initiation of Hybrid Polyketide-Nonribosomal Peptide Biosynthesis via Two Distinct Pathways in C. elegans.

Nemamide A and B are hybrid polyketide-nonribosomal peptides that are produced by the PKS-1-NRPS-1 enzymatic assembly line in the canal-associated neurons (CANs) of the nematode Caenorhabditis elegans. These signaling molecules promote survival during and recovery from starvation-induced larval arrest. Here, using genome editing and targeted metabolomics, we probed the roles of the different domains of PKS-1 in the initiation of nemamide biosynthesis. We showed that the first four domains of PKS-1 are not required for the biosynthesis of the triene-containing nemamide A, but are required for the biosynthesis of the tetraene-containing nemamide B. By targeting genes that are highly expressed in the CANs, we identified two additional enzymes that participate in the biosynthetic pathway: the peroxisomal carnitine O-octanoyl transferase CROT-1, which is required for the biosynthesis of nemamide A, and the enoyl-CoA hydratase ECH-7, which is required for the biosynthesis of nemamide B. We heterologously expressed CROT-1 and showed that it prefers hexanoyl-CoA and octanoyl-CoA as substrates, converting them to the corresponding carnitine esters. According to our model, ECH-7 is needed to supply the starter unit for nemamide B biosynthesis, which is loaded onto the first carrier protein of PKS-1 and extended by the first module, thereby installing the double bond that is unique to nemamide B. Meanwhile, CROT-1 is needed to supply the starter unit for nemamide A biosynthesis, which is loaded onto the second carrier protein of PKS-1. Our data suggest that the biosynthetic pathways of nemamide A and B are under the control of two different initiation mechanisms and, thus, that the production of these two secondary metabolites may be independently regulated.

Animals↗

PubMedDate not supplied

[Regulatory role of amino acids in protein biosynthesis; effect of various factors].

The paper embraces data available in literature and the results of the author's investigations which show synergism and antagonism interrelations between certain amino acids in the processes of transmembrane transport, amino acylation of transfer RNA and incorporation into protein. These interrelations may lead to activation and inhibition of the protein biosynthesis. It is established that an excess of any amino acid created with its administration into the organism induces the inhibition of biosynthesis and activity of the corresponding aminoacyl-tRNA-synthetase (ARSase), while deficiency of an amino acid intensifies the biosynthesis of the corresponding ARSase. Homogeneous crystalline proteins, such as aldolase of rabbit skeletal muscles, collagen I of rat skin, globin of chicken blood and others, are used as an example to show that as a result of feeding of the amino acid excess to animals, especially against a background of protein deficiency, the biosynthesis intensity changes and proteins with other primary structure and properties are synthetized. This testifies to that amino acids being substrates in the protein biosynthesis are regulators in this process. It is established that the biosynthesis of proteins with other primary structure under conditions of complete fasting, protein deprivation, feeding of an excess of certain amino acids to animals against a background of protein deficiency, atherosclerosis and other extremal states of the organism is not a result of erroneous incorporation of amino acids but is the process of regular, specific and stable character for each state and may be predicted. The biosynthesis of the protein with other primary structure under the effect of extremal conditions is caused, apparently, by capability to the changes of the proteinsynthetizing system.

Amino Acid Sequence↗

PubMed1981

Effect of cations on insulin biosynthesis in mouse pancreatic islets.

Rates of incorporation of L-phenyl(2,3-3H)alanine into insulin and total protein in mouse pancreatic islets were measured. Glucose-stimulated total protein synthesis and insulin biosynthesis are depressed when the medium is deprived of Na+, K+ and Mg++ whereas the same processes are totally independent of extracellular Ca++. Excess Ca++ (15 mM) has no effect on insulin biosynthesis but 12 mM Mg++ impairs glucose-stimulated insulin biosynthesis preferentially. 40 mM K+ inhibits glucose-stimulated insulin biosynthesis as well as total protein synthesis. Omission of K+ inhibits glucose-stimulated insulin biosynthesis preferentially. 10(-4) M ouabain inhibits both glucose-stimulated total protein synthesis and insulin biosynthesis. When Ca++ is replaced by 2 mM Ba++ glucose-stimulation of both total protein synthesis and insulin synthesis is abolished. Preferentially inhibited insulin biosynthesis in the presence of very low Na+ (24 mM) is seen not only with glucose but also with leucine, N-acetylglucosamine and mannose as stimulants, suggesting that the sodium-sensitive step is localized distal to the process of glucose recognition by the beta-cell.

Animals↗

PubMed1996

Acetylcholine receptor clustering associates with proteoglycan biosynthesis in C2 variant and heterkaryon muscle cells.

Several lines of evidence have suggested roles for proteoglycans (PGs) in acetylcholine receptor (AChR) clustering on muscle cells. One line of evidence comes from the correlation between a defect in the biosynthesis of glycosaminoglycans (GAGs), the defining carbohydrates of PGs, and the failure of spontaneous AChR clustering in the S27 cell line, a genetic variant of the C2 muscle cell line. Two approaches were used in the present study to investigate whether GAG and AChR clustering defects are causally linked. First, the formation of AChR clusters was examined in two more variant lines, S11 and S26, also isolated from the C2 muscle cell line on the basis of deficiencies in GAG biosynthesis. S11 and S26, like S27, are also defective in AChR clustering. Ion exchange analysis of the GAGs made by the S11, S26, and S27 lines revealed that the defects in GAG biosynthesis differ between the three lines. Second, heterokaryon myotubes formed between pairs of the GAG defective variants were tested for complementation in both AChR clustering and GAG biosynthesis. AChR clusters were conspicuous on individual heterokaryon myotubes, and GAG biosynthesis was restored to near wild type levels in the heterokaryon cultures. Complementation in GAG biosynthesis corroborates the biochemical data that the relevant mutations in the genetic variants are in different genes and establishes that the defects are not dominant. The consistent correlation between GAG defects and the failure of AChR clustering across three independent genetic variants and the complementary association of GAG biosynthesis with AChR clustering in heterokaryon myotubes argues against a chance association of the two phenotypes and for a causal relationship between PGs and AChR clustering. A prominent chondroitin sulfate peak correlated with AChR clustering in the heterokaryon cultures. This is consistent with earlier results suggesting that chondroitin sulfate in general is required for the spontaneous clustering of AChRs in C2 cultures and further suggests that a particular chondroitin sulfate proteoglycan may be essential for the clustering process.

Animals↗

PubMed1991

Modulation of tissue plasminogen activator biosynthesis by phosphatidylinositol liposomes in human fetal lung fibroblasts.

Phosphatidylinositol (PI) liposomes at 40 microM increased tissue plasminogen activator (t-PA) biosynthesis by human fetal lung fibroblasts IMR-90 (FLF), after 5 days of incubation by 7.4 +/- 1.4 times of the control level. Other phospholipid liposomes, such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylglycerol (PG), had no effect on t-PA biosynthesis by FLF. The induction of t-PA biosynthesis by PI liposomes was inhibited by specific inhibitors of phosphoinositide pathway: gentamycin and lithium chloride. Thus, gentamycin inhibited the effect of PI liposomes on t-PA biosynthesis by 76% (P less than 0.001), while it had no effect on control FLF. Likewise, lithium chloride inhibited t-PA biosynthesis of both PI-treated and control FLF by greater than 84%. The induction of t-PA biosynthesis by PI liposomes was dependent on RNA transcription and independent of DNA biosynthesis.

Aphidicolin↗

PubMed2001

Inhibition of cholesterol biosynthesis in cultured fibroblasts by D003, a mixture of very long chain saturated fatty acids.

The present study was undertaken to investigate the effects of D003, a mixture of very long chain saturated fatty acids isolated and purified from sugar cane wax, on cholesterol biosynthesis in cultured fibroblasts. Cholesterol biosynthesis is regulated through feedback regulation of at least two sequentially acting enzymes, 3-hydroxy-3-methyl coenzyme A (HMG-CoA) synthase and reductase. They are up-regulated when sterol levels fall and down-regulated when sterol levels rise. The exposure of cultured fibroblasts to a lipid-depleted medium (LDM) and D003 (0.05-50 microg ml(-1)) for 12 h inhibited, in a dose-dependent manner, cholesterol biosynthesis from 14C-labelled acetate (33-68%). The addition of D003 at concentrations inhibiting cholesterol biosynthesis from labelled acetate significantly decreased incorporation of radioactivity from 3H2O into sterols, but not from 14C-mevalonate. These data indicate that D003 inhibits cholesterol biosynthesis by interfering with early steps of cholesterol biosynthetic pathway. We reasoned that D003 acts directly on HMG-CoA reductase, the main regulatory enzyme of cholesterol biosynthetic pathway. However, when enzyme activity was measured in cell extracts in the presence of various concentrations of D003 (0.5-50 microg ml(-1)), reductase activity was not inhibited. Thus, there was no evidence for a competitive or non-competitive inhibition of enzyme activity by D003. Treatment with D003 significantly suppressed (68%) the enzyme up-regulation when cells were cultured in LDM, which suggests a depression of de novo synthesis of HMG-CoA reductase and/or a stimulation of its degradation. However, since the suppressive action of D003 on cholesterol biosynthesis was observed in metabolic conditions under which synthase up-regulation was also enhanced, we cannot rule out a possible effect of D003 on HMG-CoA synthase. Thus, further studies are needed to clarify the precise mechanism of the inhibitory effect of D003 on cholesterol biosynthesis.

Animals↗

PubMed2007

Identification of three Zwittermicin A biosynthesis-related genes from Bacillus thuringiensis subsp. kurstaki strain YBT-1520.

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.

Bacillus thuringiensis↗

PubMed2002

Candicidin biosynthesis in Streptomyces griseus.

The biosynthesis of the aromatic polyene macrolide antibiotic candicidin, produced by Streptomyces griseus IMRU 3570, begins with a p-aminobenzoic acid (PABA) molecule which is activated to PABA-CoA and used as starter for the head-to-tail condensation of four propionate and 14 acetate units to produce a polyketide molecule to which the deoxysugar mycosamine is attached. Using the gene coding for the PABA synthase ( pabAB) from S. griseusIMRU 3570 as the probe, a 205-kb region of continuous DNA from the S. griseus chromosome was isolated and partially sequenced. Some of the genes possibly involved in the biosynthesis of candicidin were identified including part of the modular polyketide synthase (PKS), genes for thioesterase, deoxysugar biosynthesis, modification, transport, and regulatory proteins. The regulatory mechanisms involved in the production of candicidin, such as phosphate regulation, were studied using internal probes for some of the genes involved in the biosynthesis of the three moieties of candicidin (PKS, aromatic moiety and amino sugar). mRNAs specific for these genes were detected only in the production medium (SPG) but not in the SPG medium supplemented with phosphate or in the inoculum medium, indicating that phosphate represses the expression of genes involved in candicidin biosynthesis. The modular architecture of the candicidin PKS and the availability of the PKSs involved in the biosynthesis of three polyene antibiotics (pimaricin, nystatin, and amphotericin B) shall make possible the creation of new, less toxic and more active polyene antibiotics through combinatorial biosynthesis and targeted mutagenesis.

4-Aminobenzoic Acid↗

PubMed1984

Changes in prostacyclin and thromboxane biosynthesis and their catabolic enzyme activity in kidneys of aging rats.

The effects of aging on the prostacyclin and thromboxane biosynthesis and prostaglandin catabolic enzyme activity in rat kidney were investigated. The prostacyclin biosynthesis, using arachidonic acid as substrate, was the greatest in young kidneys (2 months old) and then progressively decreased in mature (12 months old) and old (24 months old) kidneys, while thromboxane biosynthetic activity showed no significant change as a function of age. When prostaglandin H2 was used as substrate, the prostacyclin and thromboxane biosynthesis showed similar results as when arachidonic acid was used as substrate; the prostacyclin biosynthesis progressively decreased and thromboxane biosynthesis showed no significant change as a function of age. The fatty acid cyclooxygenase in kidney was measured by a specific radioimmunoassay. No significant change in renal fatty acid cyclooxygenase as a function of age was found. Thus, we concluded that the progressive decrease in renal prostacyclin biosynthesis as a function of age is due to a defect in prostacyclin synthetase in aged kidneys. The prostaglandin catabolic enzyme, NAD+-dependent 15-hydroxyprostaglandin dehydrogenase, in kidneys was also investigated. The enzyme activity progressively decreased as a function of age, which suggested a decrease in the metabolism of thromboxane A2 in aged kidneys. The present results, indicating a decrease in renal prostacyclin biosynthesis and renal 15-hydroxyprostaglandin dehydrogenase activity with aging, might contribute to a plausible explanation of the progressive decrease in renal functions in the elderly.

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Effects of methylmercury and trimethyltin on cardiac, platelet, and aorta eicosanoid biosynthesis and platelet serotonin release.

The effects of two organometals on heart, platelet, and aorta prostaglandin biosynthesis were examined in vitro. Methylmercuric chloride (MMC, 39-796 nM) increased the biosynthesis of thromboxane A2 (TxA2) and prostacyclin (PGI2) in the heart and stimulated the biosynthesis of PGI2 in incubates of aorta rings. The aorta biosynthesis of PGI2 was monitored by its inhibition of platelet aggregation and serotonin [5-hydroxytryptamine (5-HT)] secretion, while the metabolites 6-keto-PGF1a and TxB2 were quantified by radioimmunoassay. In platelet experiments, low concentrations of MMC (5 microM) enhanced aggregation to adenosine diphosphate (ADP) and at high concentrations MMC (50-100 microM) directly stimulated aggregation and 5-HT secretion. These effects of MMC were inhibited by nonsteroidal antiinflammatory drug, thromboxane synthetase, and phospholipase A2 inhibitors. Trimethyltin (TMT), another highly toxic organometal, had no effect on prostaglandin biosynthesis in either heart or aorta incubates. TMT did not increase platelet aggregation responses to ADP, nor did it directly trigger aggregation. At moderate concentrations, TMT (20-45 microM) slightly depressed ADP aggregation; however, this was paradoxically associated with increased 5-HT secretion. In platelets pretreated with either NDGA or ASA, TMT in the absence of aggregation enhanced 5-HT secretion in response to ADP. TMT, unlike MMC, did not stimulate platelet TxB2 biosynthesis from exogenous [3H]arachidonic acid, whereas MMC stimulates heart, vascular, and platelet eicosanoid biosynthesis. TMT, unlike MMC, does not directly activate the arachidonic acid cascade.

6-Ketoprostaglandin F1 alpha↗

PubMed1985

Effects of riboflavin deficiency upon prostaglandin biosynthesis in rat kidney.

The effects of riboflavin deficiency on the activity in vitro of prostaglandin synthetase were determined in rat kidney homogenates. For a period of two to five months, weaning rats were fed either a diet deficient in riboflavin or equal amounts of a diet identical in composition except for the addition of riboflavin at four times the RDA for this vitamin. In further experiments, each group of rats was treated for 10 days with either an inhibitor of cyclooxygenase (flurbiprofen) or buffer. Following sacrifice, prostaglandin biosynthesis in vitro was measured both in the absence and presence of reduced glutathione, and subsequently in the presence of reduced glutathione with and without flurbiprofen. Reaction products were extracted from supernatant solutions with diethylether, and the PGE2 and PGF2 alpha formed were measured by radioimmunoassay. Dietary riboflavin deficiency increased biosynthesis rates in vitro of both PGE2 and PGF2 alpha in rat renal medulla and papilla. When both control and riboflavin deficient rats were treated with flurbiprofen for a 10 day period, PGE2 biosynthesis in vitro was markedly inhibited. This inhibition of PGE2 biosynthesis was partially overcome by the addition of reduced glutathione in vitro. The addition of flurbiprofen in vitro to samples containing reduced glutathione prevented the restoration of PGE2 biosynthesis by the latter. The rate of prostaglandin biosynthesis in kidney homogenates from riboflavin deficient rats remained higher than that of controls with each experimental manipulation. These data in their entirety suggest a possible role for riboflavin in the regulation of renal prostaglandin biosynthesis in the rat.

Animals↗

PubMed2006

Effect of melanin on netilmicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

It is known that various drugs form complexes with melanins and that melanins are abundant constituents of the inner ear. In this study, we determined whether the aminoglycoside antibiotic, netilmicin, interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. The obtained results indicate that netilmicin forms stable complexes with melanin characterized by the association constants K(1) approximately 10(6)M(-1) and K(2) approximately 10(3)M(-1). We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for aminoglycoside-induced inhibition of collagen biosynthesis. We found that netilmicin strongly induced inhibition of prolidase activity (IC(50)<5microM) and collagen biosynthesis (IC(50) approximately 10microM). At 10microM concentration of netilmicin, prolidase activity in human skin fibroblasts was inhibited by about 80% and DNA biosynthesis-only by about 25%. Melanin at 100microg/mL produced about 30% inhibition of collagen biosynthesis and about 30% inhibition of prolidase activity in cultured fibroblasts. However, the addition of melanin (100microg/mL) to netilmicin-treated cells (10microM) restored the prolidase activity in fibroblasts to almost 100% of control values and partially reversed the inhibitory action of the drug on collagen and DNA biosynthesis. The data suggest that the ability of netilmicin to form stable complexes with melanin may prevent its toxicity on prolidase activity and collagen biosynthesis.

Anti-Bacterial Agents↗

PubMed1997

On the mechanism of the losartan-mediated inhibition of phosphatidylcholine biosynthesis in H9c2 cells.

Phosphatidylcholine is the major phospholipid in mammalian tissues and the biosynthesis of phosphatidylcholine in H9c2 cells was previously shown to be stimulated by angiotensin II. In this study, we used the potent AT1 receptor antagonist, losartan, to determine if the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis was mediated by AT1 receptors. H9c2 cells were incubated with angiotensin II in the absence or presence of various concentrations of losartan. The cells were then incubated with [methyl-3H]choline for an additional 60 min and the radioactivity incorporated into phosphatidylcholine and its choline-containing metabolites determined. Losartan at concentrations which block AT1 receptors did not effect phosphatidylcholine biosynthesis mediated by angiotensin II. In contrast, higher concentrations of losartan inhibited radioactivity incorporated into phosphatidylcholine and its metabolites and this was due to a losartan-mediated reduction in choline uptake. Kinetic studies revealed that the losartan-mediated inhibition of choline uptake was competitive. High concentrations of losartan caused a translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic (inactive) to the membrane (active) fraction likely as a compensatory mechanism for the losartan-mediated reduction in new phosphatidylcholine biosynthesis. Incubation of cells with PD123319, a potent AT2-receptor antagonist, did not block the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis. The results suggest that angiotensin II stimulates phosphatidylcholine biosynthesis independent of AT1- and AT2-receptor activation and losartan inhibits phosphatidylcholine biosynthesis by reducing choline uptake in H9c2 cells.

Angiotensin II↗

PubMed2001

Melanin potentiates daunorubicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

One of the recognized side effects of antineoplastic anthracyclines is poor wound healing, resulting from an impairment of collagen biosynthesis. The most affected tissue is skin. The mechanism underlying the tissue specificity of the side effects of anthracyclines has not been established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanins are abundant constituents of the skin, we determined whether daunorubicin interacts with melanin and how this process affects collagen biosynthesis in cultured human skin fibroblasts. Results indicated that daunorubicin forms complexes with melanin. Scatchard analysis showed that the binding of daunorubicin to melanin was heterogeneous, suggesting the presence of two classes of independent binding sites with K1 = 1.83 x 10(5) M(-1) and K2 = 5.52 x 10(3) M(-1). The number of strong binding sites was calculated as n1 = 0.158 micromol/mg of melanin and the number of weak binding sites as n2 = 0.255 micromol/mg of melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracycline-induced inhibition of collagen synthesis. We found that daunorubicin induced inhibition of prolidase activity (IC50 = 10 microM), collagen biosynthesis (IC50 = 70 microM) and DNA biosynthesis (IC50= 10 microM) in human skin fibroblasts. Melanin (100 microg/ml) by itself produced about 25% inhibition of DNA synthesis and prolidase activity but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to daunorubicin-treated cells (at IC50 concentration) augmented the inhibitory action of daunorubicin on collagen and DNA biosynthesis without having any effect on prolidase activity. The same effect was achieved when the cells were treated with daunorubicin at one-fourth of the IC50 given at 0, 6, 12 and 18 h during a 24-h incubation. The data suggest that the melanin-induced augmentation of the inhibitory effects of daunorubicin on collagen and DNA biosynthesis may result from: (i) accumulation of the drug in the extracellular matrix, (ii) gradual dissociation of the complex, and (iii) constant action of the released drug on cell metabolism. The phenomenon may explain the potential mechanism for the organ specificity of daunorubicin-induced poor wound healing in patients administered this drug.

Antibiotics, Antineoplastic↗

PubMed2002

Melanin potentiates gentamicin-induced inhibition of collagen biosynthesis in human skin fibroblasts.

One of the recognized side effects of gentamicin is ototoxicity. The mechanism underlying the organ specificity of this side effect of gentamicin has not been fully established. In view of the fact that a number of pharmacologic agents are known to form complexes with melanin and melanin is an abundant constituent of the inner ear tissues, we determined whether gentamicin interacts with melanin and how this process affects the biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that gentamicin forms complexes with melanin. The amount of gentamicin bound to melanin increases with increasing of initial drug concentration. The Scatchard plot analysis of drug binding to melanin showed that at least two classes of independent binding sites are implicated in gentamicin-melanin complex formation: one class with an association constant K(1) approximately 4 x 10(3) M(-1), and the second class with an association constant K(2) approximately 3 x 10(2) M(-1). The number of total binding sites (n(1)+n(2)) was calculated as about 1.36 micromol gentamicin per 1 mg melanin. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for gentamicin-induced inhibition of collagen biosynthesis. We found that gentamicin-induced inhibition of prolidase activity (IC(50) approximately 100 microM) and collagen biosynthesis (IC(50) approximately 100 microM). At this concentration of gentamicin, DNA biosynthesis in human skin fibroblasts was inhibited only by about 30%. Melanin at 100 microg/ml produced about 25% inhibition of DNA synthesis and about 30% inhibition of prolidase activity, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to gentamicin-treated cells (100 microM) augmented the inhibitory action of gentamicin on collagen and DNA biosynthesis and partially reversed its inhibitory effect on prolidase activity. A melanin-induced augmentation of the inhibitory effects of gentamicin on collagen and DNA biosynthesis may explain the mechanism for the organ specificity of gentamicin-induced hearing loss in patients administered this drug.

Anti-Bacterial Agents↗