Precursor-product relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium.

Exponential biosynthesis and excretion of lipoteichoic acid (LTA) during the exponential phase of growth, and continued synthesis and excretion during valine starvation of Streptococcus faecium (S. faecalis ATCC 9790), were shown. During exponential growth, extracellular LTA (LTAx) accounted for approximately 13% of the total LTA in cultures, whereas during valine starvation, this percentage increased to approximately 60% within 4 h. LTAx was present in a low-molecular-weight, apparently deacylated form, whereas intracellular (LTAi) was present primarily in an apparently high-molecular-weight, acylated and micellar form. Experiments utilizing chases of either fully equilibrated or short pulses of [14C]- or [3H]glycerol were used to demonstrate that LTAx was derived directly from LTAi.     

Effect of cerulenin on cellular autolytic activity and lipid metabolism during inhibition of protein synthesis in Streptococcus faecalis.

Cellular autolytic activity as well as lipid and lipoteichoic acid metabolism have been studied in cultures of Streptococcus faecalis receiving various combinations of the following treatments: chloramphenicol addition, starvation for an essential amino acid (valine), and cerulenin treatment. Lipoteichoic acid initially accumulated in chloramphenicol-treated and amino acid-starved cells and decreased relative to the cellular mass in cerulenin-treated cells. The relative phosphatidylglycerol content of amino acid-starved cultures or of cultures treated with either antibiotic rapidly decreased upon initiation of each treatment. In all cases, cerulenin initially stimulated diphosphatidylglycerol synthesis. Pretreatment of cultures with cerulenin prevented the inhibition of cellular synthesis autolysis normally observed during chloramphenicol treatment, but did not affect amino acid starvation-induced inhibition of autolytic activity. Variations in the levels of the nonionic lipid fraction, predominantly diglycerides, correlated best with the patterns of autolytic activity observed during chloramphenicol treatment, whereas variations in the levels of diphosphatidylglycerol and lipoteichoic acid correlated best with the patterns of autolytic activity observed during amino acid starvation. Components of the nonionic lipid fraction were demonstrated to inhibit autolytic activity 50% in whole cell and in cell wall assays at 60 and 120 nmol/mg (dry weight), respectively.     

Membrane lipoteichoic acid of Streptococcus pyogenes and its stabilized L-form and the effect of two antibiotics upon its cellular content.

Membrane lipoteichoic acid continues to be synthesized by an osmotically fragile, stabilized L-form of Streptococcus pyogenes. Chromatographic and electrophoretic comparisons indicate that the lipid componenent of lipoteichoic acid in this L-form and its parental streptococcus is glycerophosphoryldiglucosyl diglyceride and not phosphatidylkojibiosyl diglyceride. Based upon dry weight determinations, the yield of lipoteichoic acid from the L-form is 0.19%, as compared with 0.97% from the streptococcus. When grown with bacitracin the L-form contains the same amount of teichoic acid as when grown without this antibiotic; however, its lipoteichoic acid content is reduced by 85%. Similarly, the L-form grown with novobiocin for 10 h contains only 17% of the teichoic acid found in control cells.     

Inhibition of cholesterol absorption by SCH 58053 in the mouse is not mediated via changes in the expression of mRNA for ABCA1, ABCG5, or ABCG8 in the enterocyte

Intestinal cholesterol absorption is a major determinant of plasma low density lipoprotein-cholesterol (LDL-C) concentrations. Ezetimibe (SCH 58235) and its analogs SCH 48461 and SCH 58053 are novel potent inhibitors of cholesterol absorption whose mechanism of action is unknown. These studies investigated the effect of SCH 58053 on cholesterol metabolism in female 129/Sv mice. In mice fed a low cholesterol rodent diet containing SCH 58053, cholesterol absorption was reduced by 46% and fecal neutral sterol excretion was increased 67%, but biliary lipid composition and bile acid synthesis, pool size, and pool composition were unchanged. When the dietary cholesterol content was increased either 10- or 50-fold, those animals given SCH 58053 manifested lower hepatic and biliary cholesterol concentrations than did their untreated controls. Cholesterol feeding increased the relative mRNA level for adenosine triphosphate-binding cassette transporter A1 (ABCA1), ABC transporter G5 (ABCG5), and ABC transporter G8 (ABCG8) in the jejunum, and of ABCG5 and ABCG8 in the liver, but the magnitude of this increase was generally less if the mice were given SCH 58053. We conclude that the inhibition of cholesterol absorption effected by this new class of agents is not mediated via changes in either the size or composition of the intestinal bile acid pool, or the level of mRNA expression of proteins that facilitate cholesterol efflux from the enterocyte, but rather may involve disruption of the uptake of luminal sterol across the microvillus membrane.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

HMG-CoA reductase inhibitors perturb fatty acid metabolism and induce peroxisomes in keratinocytes.

Topical lovastatin stimulates epidermal fatty acid synthesis in vivo; therefore, studies were undertaken to examine the effects of HMG-CoA reductase inhibitors on fatty acid metabolism in cultured keratinocytes. When exposed to fluindostatin or lovastatin for greater than or equal to 24 h, keratinocytes in serum-free media accumulated nile red-fluorescent lipid droplets. By 72 h, the triacylglycerol and phospholipid content were increased 2.5- and 1.3-fold, respectively. Reductase inhibitors (1-10 microM) increased fatty acid synthesis approximately 1.5-fold; increased synthesis was noted only after greater than 15 h exposure and was distributed among phospholipids and triacylglycerols. Oxidation of [14C]palmitate to CO2 was decreased greater than 50% in inhibitor-treated cultures, and label accumulated in triacylglycerols. Inhibitor-treated keratinocytes exhibited increased numbers of peroxisomes, using diaminobenzidene ultracytochemistry. Peroxisomal hyperplasia was also demonstrated by increased catalase activity (1.5- to 2.5-fold), increased dihydroxyacetone phosphate acyltransferase activity (1.4-fold) and increased peroxisomal (KCN-insensitive) fatty acid oxidation (1.4-fold) in inhibitor-treated cultures. Thus HMG-CoA reductase inhibitors increase fatty acid synthesis, induce triacylglycol and phospholipid accumulation, and induce peroxisomes in cultured keratinocytes. Coincubations with either low density lipoproteins or 25-hydroxycholesterol prevented both the peroxisomal hyperplasia and increased fatty acid synthesis, suggesting that these effects of reductase inhibitors may be linked to their effects on the cholesterol biosynthetic pathway.     

Nisin resistance of Streptococcus bovis

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 microM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 microM nisin was added, but resistant cells retained potassium even after addition of 10 microM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.     

Methyl jasmonate modulated biotransformation of phenylpropanoids to vanillin related metabolites using Capsicum frutescens root cultures.

Normal root cultures of Capsicum frutescens biotransform externally fed precursors, like caffeic acid and veratraldehyde, to vanillin and other related metabolites. The bioconversion of caffeic acid to further metabolites--viz. vanillin, vanillylamine, vanillic acid--was shown to be elicited by treating the cultures with 10 microM methyl jasmonate (MJ). Root cultures treated with MJ accumulated (1.93 times) more of vanillin (20.2 microM on day-3) than untreated ones. A concomitant increase in enzymatic activity of caffeic acid O-methyl transferase (CAOMT, EC 2.1.1.68) was obtained in MJ treated cultures, compared to untreated cultures. After 24 h of MJ treatment, a 13.7-fold increase in CAOMT activity was recorded in root cultures of C. frutescens. Cultures treated with veratraldehyde accumulated more vanillin (78 microM) than caffeic acid fed cultures, 6 days after precursor addition. Capsaicin did not accumulate even after addition of precursors. The efficiencies of biotransformation with caffeic acid and veratraldehyde were 2.2% and 9% with respect to vanillin formation, indicating a possible diversion of the phenylpropanoid pathway towards other secondary metabolites.     

Binding of Streptococcal Lipoteichoic Acid to Fatty Acid-Binding Sites on Human Plasma Fibronectin

The ability of Streptococcus pyogenes lipoteichoic acid and palmitic acid to bind to purified human plasma fibronectin was investigated. Initial studies indicated that intact fibronectin formed soluble complexes with lipoteichoic acid, resulting in a change in the mobility of fibronectin in an electrical field. Fibronectin covalently linked to agarose beads bound radiolabeled lipoteichoic acid in the acylated form but not in the deacylated form. An 18-M excess of fibronectin inhibited binding of lipoteichoic acid to the immobilized protein by 92%. Fibronectin-bound [(3)H]lipoteichoic acid could be specifically eluted with unlabeled lipoteichoic acid, as well as by fatty acid-free serum albumin. Serum albumin, which is known to contain fatty acid-binding sites capable of binding to the lipid moieties of lipoteichoic acid, inhibited the binding of lipoteichoic acid to fibronectin in a competitive fashion. The fibronectin-bound lipoteichoic acid could be eluted by 50% ethanol and various detergents but not by 1.0 M NaCl, various amino acids, or sugars. Similarly, radiolabeled palmitic acid adsorbed to fibronectin could be eluted with 50% ethanol but not with 1.0 M NaCl. Fibronectin adsorbed to a column of palmityl-Sepharose was eluted with 50% ethanol in 0.5% sodium dodecyl sulfate but not with 1.0 M NaCl or 1% sodium dodecyl sulfate alone. The binding of lipoteichoic acid to fibronectin followed first-order kinetics and was saturable. A Scatchard plot analysis of the binding data indicated a heterogeneity of lipoteichoic acid-binding sites similar to that previously found for serum albumin. Nevertheless, fibronectin contains at least one population of high-affinity binding sites for lipoteichoic acid. The binding affinity (nKa approximately 250 muM(-1)) is 2 orders of magnitude greater than the binding affinity of serum albumin. These data suggest that human plasma fibronectin contains specific binding sites for fatty acids and that lipoteichoic acid binds to these sites by way of its glycolipid moiety.     

The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus

Pulse-chase experiments with [2-3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol leads to (glycerophospho)25-diglucosyldiacylglycerol + 25 diacylglycerol. Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor. Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative. In contrast to normal growth, energy deprivation caused an immediate switch-over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.     

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.     

Separation of penicillin G from phenylacetic acid in a supported liquid membrane system

The separation of penicillin G (Pen G) from phenylacetic acid (PAA) by use of a supported liquid membrane (SLM) system with Amberlite LA-2 dissolved in 1-decanol, supported on a microporous polypropylene membrane, was studied. The results show that the individual permeability of each component in mixture was lower than that in a single compartment system and, it suggests a strong transport competition between Pen G and PAA. The SLM system in this study proved to be a promising process for the selective separation of Pen G from PAA. The maximum separation factor was found to be 1.8 under a liquid membrane resistance controlled mechanism. (c) 1994 John Wiley & Sons, Inc.     

Nisin Resistance of Streptococcus bovis

The growth of Streptococcus bovis JB1 was initially inhibited by nisin (1 μM), and nisin caused a more than 3-log decrease in viability. However, some of the cells survived, and these nisin-resistant cells grew as rapidly as untreated ones. To see if the nisin resistance was merely a selection, nisin-sensitive cells were obtained from agar plates lacking nisin. Results indicated that virtually any nisin-sensitive cell could become nisin-resistant if the ratio of nisin to cells was not too high and the incubation period was long enough. Isolates obtained from the rumen were initially nisin sensitive, but they also developed nisin resistance. Nisin-resistant cultures remained nisin resistant even if nisin was not present, but competition studies indicated that nisin-sensitive cells could eventually displace the resistant ones if nisin was not present. Nisin-sensitive, glucose-energized cells lost virtually all of their intracellular potassium if 1 μM nisin was added, but resistant cells retained potassium even after addition of 10 μM nisin. Nisin-resistant cells were less hydrophobic and more lysozyme-resistant than nisin-sensitive cells. Because the nisin-resistant cells bound less cytochrome c, it appeared that nisin was being excluded by a net positive (i.e., less negative) charge. Nisin-resistant cells had more lipoteichoic acid than nisin-sensitive cells, and deesterified lipoteichoic acids from nisin-resistant cells migrated more slowly through a polyacrylamide gel than those from nisin-sensitive cells. These results indicated that lipoteichoic acids could be modified to increase the resistance of S. bovis to nisin. S. bovis JB1 cultures were still sensitive to monensin, tetracycline, vancomycin, and bacitracin, but ampicillin resistance was 1,000-fold greater.     

Gentamicin induces Jun-AP1 expression and JNK activation in renal glomeruli and cultured mesangial cells

Reactive oxygen species (ROS) mediate MC contraction, proliferation and apoptosis induced by gentamicin (G) in vitro and in vivo. Sustained increases in cytosolic free calcium, increased iNOS expression and elevated nitric oxide (NO) production are associated with MC apoptosis in vitro. As NO strongly activated c-Jun N-terminal kinase (JNK) and increased AP1 expression, and these two factors are involved in MC proliferation in vitro, we have measured Jun-AP1 expression in rat glomeruli from G-treated rats, and the effect of G on Jun-AP1 expression and JNK activity in cultured MC. Moreover, we studied the expression of inducible (iNOS) and constitutive (cNOS) NO synthases in rat glomeruli. Glomeruli were obtained from rats treated with G (100 mg/kg body weight/day) along 6 days, and MC primary cultures were evaluated after 24, 48 and 72 h incubation with 10(-5) M G. G induced an increase in the expression of iNOS, cNOS and Jun-AP1 in rat glomeruli and in MC cultures. Moreover, G activated JNK; JNK activation was reduced by co-incubation with the calcium channel blocker verapamil and with the ROS scavengers superoxide dismutase and catalase. These results strongly suggest a role for reactive oxygen/nitrogen species produced by increased NOS activity in G-induced MC activation. These reactive oxygen molecules and increased intracellular free calcium may mediate the increase in Jun-AP1 expression and JNK activation induced by G treatment in MC.     

Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: further characterization and effect of nafenopin, a peroxisome proliferator

The addition of 2% dimethyl sulfoxide to adult rat hepatocytes cultured in a chemically defined medium at Day 1 after cell plating resulted in maintenance of the cytochrome P-450 content and the cyanide-insensitive palmitoyl-CoA beta-oxidation activity at 66 and 70% of the initial Day 1 values. The addition of phenobarbital, 3-methylcholanthrene, or nafenopin from Day 3 to Day 6 increased the contents of cytochrome P-450 to 128, 239, and 251%, respectively, compared to untreated controls at Day 3. In addition, nafenopin also caused a pronounced and time-dependent increase in palmitoyl-CoA beta-oxidation activity but was found to have only a weak stimulating effect on replicative DNA synthesis (2-fold) when compared to that of epidermal growth factor (6.5-fold). In the presence of dimethyl sulfoxide the hepatocyte cultures could be kept alive for more than 1 month. Exposure of such cultures to nafenopin from Day 1 do Day 37 resulted in survival which was even better than that of their untreated counterparts. This effect was accompanied by the appearance of abundant endoplasmic reticulum membranes and an increased number of peroxisomes.     

Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.     

The effect of monensin on beta-hexosaminidase transport in normal and I-cell fibroblasts.

The carboxylic ionophore, monensin, blocks the migration of glycoprotein-containing vesicles from the Golgi region to the plasma membrane in fibroblasts resulting in an accumulation of secretory products in the Golgi cisternae. Treatment of cultured I-cell fibroblasts with monensin (0.5 muM) decreased the abnormal excretion of beta-hexosaminidase to 40% of untreated cultures within 15 min. A corresponding intracellular accumulation of the enzyme to greater than 200% of untreated cultured by 24 h was also observed. A small intracellular accumulation and slightly enhanced excretion of beta-hexosaminidase occurred in treated normal fibroblasts cultures. The intra- and extra-cellular distribution of newly synthesized beta-hexosaminidase in both monensin-treated normal and I-cell fibroblasts were electrophoretically indistinguishable from the four bands characteristic of I-cell intracellular beta-hexosaminidase. The excreted enzyme from both cultures was found to be a low- or no-uptake form. This form of beta-hexosaminidase may have been excreted from a secondary route preceding the site of the monensin effect. The similar findings in monensin-treated normal and I-cell cultures suggest that the subcellular site of the biochemical defect in I-cell disease is at a location after the site of the monensin effect i.e. late in the Golgi region or at a post-Golgi-region location.     

Human bone cell enzyme expression and cellular heterogeneity: correlation of alkaline phosphatase enzyme activity with cell cycle

Alkaline phosphatase, long implicated in biomineralization, is a feature of the osteoblast phenotype. Yet in cultured bone cells, only a fraction stain positive histochemically. To determine whether osteoblast enzyme expression reflects cellular heterogeneity with respect to cell cycle distribution or length of time in culture, the activities of alkaline phosphatase, tartrate-resistant and -sensitive acid phosphatases, and non-specific esterases were assayed kinetically and histochemically. In asynchronous subconfluent cultures, less than 15% of the cells stained positive and assayed activity was 0.04 IU/10(6) cells/cm2. After 1 week, the percent of alkaline phosphatase positive-staining cells increased 5-fold, while activity increased 10-fold. Non-specific esterases and tartrate-sensitive acid phosphatase were constitutive throughout time in culture, whereas tartrate-resistant acid phosphatase activity appeared after 2 weeks. Cell cycle analysis of human bone cells revealed a growth fraction of 80%, an S phase of 8.5 h, G2 + 1/2 M of 4 h, and a G1 of 25-30 h. In synchronous cultures induced by a thymidine-aphidicolin protocol, alkaline phosphatase activity dropped precipitously at M phase and returned during G1. A majority of the alkaline phosphatase activity lost from the cell surface at mitosis was recovered in the medium. Tartrate-sensitive acid phosphatase and non-specific esterase levels were relatively stable throughout the cell cycle, while tartrate-resistant acid phosphatase activity was not assayable at the density used in synchronous cultures. From these data, variations in alkaline phosphatase activity appear to reflect the distribution of cells throughout the cell cycle.     

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine(6)-oxytocin and penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs.

Six [Pen(6)]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa(1)]oxytocin, [dPen(1)]oxytocin, [5-t-BuPro(7)]oxytocin, [Mpa(1), 5-t-BuPro(7)]oxytocin and [dPen(1), 5-t-BuPro(7)]oxytocin. When tested in the uterotonic test in vitro [Pen(6)]oxytocin, [Pen(6), 5-t-BuPro(7)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen(1), Pen(6)]Oxytocin and [dPen(1), Pen(6), 5-t-BuPro(7)]oxytocin were found to be pure antagonists with similarly high pA2 values of approximately 8.2. Replacement of proline by 5-tert-butylproline increased binding affinity by a factor of two in [Pen(6)]oxytocin and had no influence on the binding affinity of [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. Assignment of the proton signals for prolyl amide cis- and trans-isomers by NMR experiments in water indicated that the Pen(6)-5-tert-BuPro(7) peptide bond cis-isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5-tert-butylproline(7) analogs of [Pen(6)]oxytocin, [Mpa(1), Pen(6)]oxytocin and [dPen(1), Pen(6)]oxytocin. This augmentation in cis-isomer population was correlated with a 21-fold reduction in the agonistic potency and 2-fold augmentation in antagonistic potency for [Pen(6), 5-t-BuPro(7)]oxytocin relative to [Pen(6)]oxytocin. Augmentation of cis-isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa(1), Pen(6)]oxytocin to [Mpa(1), Pen(6), 5-t-BuPro(7)]oxytocin. In the potent oxytocin antagonist, [dPen(1), Pen(6)]oxytocin, substitution of 5-tert-butylproline for proline augmented the cis-isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen(6)]oxytocin and [Pen(6), 5-t-BuPro(7)]oxytocin analogs 1-6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis-isomer population caused by 5-tert-butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.