Evidence for a new biologic pathway of androstenedione synthesis from 11-deoxycortisol

17-Hydroxyprogesterone is a well-known precursor of androstenedione in adrenal biosynthesis. This study using sheep adrenal incubations demonstrates that 11-deoxycortisol, the precursor of cortisol synthesis, also can be a precursor of androstenedione. Indeed, our data show that androstenedione synthesis is negatively correlated to the synthesis of cortisol and cortisone. This fact allowed us to infer that this new pathway is closely related to the activity of the 11 beta-hydroxylase that is responsible for the synthesis of cortisol. Indeed, when the activity of this enzyme is impaired, 11-deoxycortisol follows the pathway that leads to androstenedione synthesis in the adrenals. This pathway could explain, at least in part, the marked increase of androstenedione observed in congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency.     

Inhibition of simian virus 40 DNA synthesis in Yaba virus-preinfected cells.

The effect of Yaba virus preinfection on DNA synthesis in SV40-infected Jinet cells was studied. Time-course synthesis studies were conducted using the incorporation of labeled thymidine. Yaba virus preinfection resulted in the inhibition of SV40 DNA synthesis when the elapsed time between Yaba virus and SV40 infections was three days. This inhibition was demonstrated by hybridization studies and sedimentation analysis. In addition, the usual stimulation of cellular DNA synthesis induced by SV40 infection was inhibited. This inhibition occurred at a time in Yaba virus infection when no cytoplasmic Yaba virus-specific DNA synthesis occurred.     

Convergent synthesis of aminomethylene peptidomimetics

This protocol describes a convergent synthesis of reduced amide bond peptidomimetics using thioacid-terminated peptides and aziridine-containing peptide conjugates. This approach could be used to produce peptides and proteins with modified backbones. The peptide conjugates are made using readily available aziridine aldehydes. The convergent synthesis of peptidomimetics is demonstrated through the preparation of long and short peptide fragments with an aminomethylene group incorporated within them. This transformation is amenable to the synthesis of peptides with reduced amide bonds at cysteine and alanine. The procedure describes the preparation of each component used and highlights the ease of synthesis of aminomethylene peptidomimetics, and takes about 3 d to complete.     

Replication timing and cell differentiation

Cell differentiation may depend in part upon a type of unbalanced growth in which several cell cycles occur with a reduced level of total protein synthesis. During this period the synthesis of the chromatin protein HMG-I/Y is reduced since its synthesis is correlated with that of total protein. The synthesis of histone H1 shows less reduction since its synthesis is entrained with that of DNA. This greater reduction of HMG-I/Y than of histone H1 is thought to delay or prevent replicon initiations within AT-enriched isochores. This shifts their time of replication from early to late S phase. This may restrict certain pathways of cell differentiation in multipotent progenitor cells and allow one particular type of differentiation.     

Dual effect of protein kinase C on the induction of DNA synthesis by colcemid in G0-arrested human diploid fibroblasts

G0-arrested human diploid fibroblasts, TIG-1, was stimulated to induce DNA synthesis by serum, epidermal growth factor (EGF), colchicine, colcemid, or 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of DNA synthesis was mediated by protein kinase C (PKC) when stimulated with TPA but not when stimulated with other agents. When TPA-stimulated cells were immediately treated with colcemid, induction of DNA synthesis was reduced. This reduction diminished when colcemid was added more than 6 h after TPA treatment. Conversely, when colcemid-stimulated cells were treated with TPA, induction of DNA synthesis was also reduced. This reduction was enhanced when the interval between the addition of two stimulants was extended. PKC-deprivation abolished both stimulatory and inhibitory effects of TPA on DNA synthesis. Staurosporine blocked an induction of DNA synthesis by TPA but appeared to be ineffective on the inhibitory action of TPA on DNA synthesis by colcemid. These results suggest that the inhibitory effect of TPA on the induction of DNA synthesis by colcemid is mediated by down regulation-sensitive and staurosporine-insensitive PKC.     

Studies on the control of ribosomal RNA synthesis in HeLa cells.

In many eucaryotic systems protein synthesis is coupled to ribosomal RNA synthesis such that shut-down of the former causes inhibition of the latter. We have investigated this stringency phenomenon in HeLa cells. The protein synthesis inhibitors cycloheximide and puromycin cause inactivation of both processes but valine starvation totally inhibits only the processing of 45-S RNA. DNA-dependent RNA polymerases from A, B and C (or I, II and III respectively) were extracted, separated partially by DEAE-cellulose chromatography and their activity levels determined. These do not decrease significantly during inhibition of protein synthesis. To find out whether or not form A is bound to its template under these conditions, proteins were removed from chromatin with the detergent sarkosyl. This does not affect bound RNA polymerase. Inhibition of protein synthesis caused up to 50% reduction in endogenous alpha-amanitin-insensitive chromatin-RNA-synthesising activity. This reduced level of activity was not affected by sarkosyl treatment. Levels in normal cells were stimulated. This result indicates that the form A RNA polymerase is not bound to its template when protein synthesis is inhibited.     

丙交酯合成研究进展

介绍了丙交酯合成研究的最新进展,比较了不同催化体系和不同合成工艺对丙交酯收率的影响,并对丙交酯合成的发展前景进行了展望。     

Practical and convenient synthesis of coumarins from phenols and propiolic acid esters

This protocol describes the synthesis of 6,7-methylenedioxy-4-phenylcoumarin from sesamol and ethyl phenylpropiolate using a Pd(OAc)2 catalyst to illustrate coumarin synthesis. This procedure is simple and easy and can be applied to the synthesis of other coumarins that have electron-rich phenol groups. The reaction is conducted by stirring a solution of Pd(OAc)2, sesamol and ethyl phenylpropiolate in trifluoroacetic acid at room temperature (15-20 degrees C) under atmospheric conditions. This protocol can be completed in 3 d.     

Polyamine-mediated control of mammalian S-adenosyl-L-methionine decarboxylase expression: effects on the content and translational efficiency of the mRNA

The expression of mammalian AdoMet decarboxylase, a key enzyme in polyamine synthesis, was shown to be regulated by polyamines at two different levels. Polyamine depletion of Ehrlich ascites tumor cells induced a marked compensatory increase in the synthesis of the enzyme, as measured by 35S-methionine pulse-labeling and immuno-precipitation. This increase in synthesis rate was counteracted by provision of spermidine, which reduced the synthesis of AdoMet decarboxylase to an undetectable level. Northern analysis revealed a nearly 2-fold increase in the amount of AdoMet decarboxylase mRNA when the putrescine and spermidine content was depleted. This increase in AdoMet decarboxylase mRNA content cannot account for the more than 5-fold increase in synthesis rate, indicating a feedback regulation also at the level of mRNA translation.     

The biology and chemistry of antifungal agents: A review

In recent years their has been an increased use of antifungal agents and has resulted in the development of resistance to drugs. Currently, use of standard antifungal therapies can be limited because of toxicity, low efficacy rates. Different types of mechanisms contribute to the development of resistance to antifungals. This has given raise to search for a new heterocycle with distinct action or multitargeted combination therapy. This review addresses the areas such as the underlying mechanisms, eight different targets such as ergosterol synthesis, chitin synthesis, ergosterol disruptors, glucan synthesis, squalene epoxidase, nucleic acid synthesis, protein synthesis, microtubules synthesis. The clinically employed drugs along with the current research work going on worldwide on different heterocycles are discussed. In recent advances various heterocycles including imidazole, benzimidazole etc., twenty three scaffolds and their lead identification are discussed.     

Fibronectin and proteoglycan synthesis in long term cultures of cartilage explants in Ham's F12 supplemented with insulin and calcium: effects of the addition of TGF-beta

Canine cartilage explants were maintained in a basal medium supplemented with a commercially available supplement (ITSCR+) which includes insulin for up to 12 days in culture. During this time it was found that proteoglycan synthesis, as measured by 35SO4 incorporation into high molecular weight proteoglycans, was maintained at levels comparable to those at Day O. This is in substantial agreement with the results of McQuillan et al. (1) for bovine cartilage explants. Since the basal medium which we used, Ham's F12, is low in calcium, we found that supplementation with additional calcium also was needed for maintenance of proteoglycan synthesis. This defined medium was not adequate to prevent a decrease in fibronectin, total protein, and collagen synthesis relative to Day O levels. The addition of transforming growth factor-beta (TGF-beta) at 2 and 10 ng/ml to the defined medium not only prevented the decline in fibronectin synthesis but progressively increased the rate of fibronectin synthesis until the Day O levels were exceeded by an average of fourfold. This TGF-beta-induced increase in fibronectin synthesis was contrasted with the increase in fibronectin synthesis previously reported for degenerated cartilage of osteoarthritic joints (2,3), and possible implications for understanding the disease were discussed.     

Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds

The interaction of fatty-acid synthesis with starch synthesis has been studied in intact amyloplasts isolated from floral buds of cauliflower (Brassica oleracea L.). These amyloplasts perform acetate-dependent fatty acid synthesis at maximum rates only at high external ATP concentrations. Neither pyruvate nor malate inhibit acetate-dependent fatty-acid synthesis. In contrast, acetate is inhibitory to the low pyruvate-dependent fatty acid synthesis. These observations indicate that neither pyruvate nor malate are used as natural precursors of fatty-acid synthesis. In contrast to fatty-acid synthesis, the rate of glucose-6-phosphate-dependent starch synthesis is already saturated in the presence of much lower ATP concentrations. Rising rates of starch synthesis influence negatively the process of acetate-dependent fatty acid synthesis. This inhibition appears to occur under both limiting and saturating concentrations of external ATP, indicating that the rate of ATP uptake is limiting when both biochemical pathways are active. The rate of starch synthesis is modulated specifically by the concentration of 3-phosphoglycerate in the incubation medium. This observation leads to the conclusion that the activity of ADP-glucose pyrophosphorylase is of primary importance for the control of both, starch and fatty-acid synthesis. Using the modified approach of Kacser and Burns (1973; Symp. Soc. Exp. Biol.27, 65–104) we have quantified the contribution of the rate of starch synthesis to the control of the metabolic flux through fatty-acid synthesis.Abbreviations ADPGlc-PPase ADPglucose pyrophosphorylase - Glc6P glucose-6-phosphate - PGA 3-phosphoglyceric acid     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Dependence of Ribonucleic Acid Synthesis on Continuous Protein Synthesis in Yeast

Using an auxotrophic strain of Saccharomyces cerevisiae, we examined the kinetics of ribonucleic acid (RNA) synthesis following inhibition of protein synthesis caused by amino acid starvation or cycloheximide. Removal of a required amino acid immediately stopped net protein synthesis. After a brief lag, RNA synthesis also ceased. Cycloheximide, a ribosome-inhibiting drug, also immediately halted net protein synthesis. Again RNA synthesis stopped after a brief lag. Although cycloheximide and amino acid starvation affect different steps in protein biosynthesis, both inhibited RNA synthesis in identical fashion. This indicates that amino acids do not play a unique role in the control of RNA production in rapidly growing yeast; rather, it suggests that RNA synthesis is responsive to the overall rate of protein synthesis itself.     

New temperature-sensitive mutants of Saccharomyces cerevisiae affecting DNA replication

Summary We have isolated new mutants of the yeast Saccharomyces cerevisiae that are defective in mitotic DNA synthesis. This was accomplished by directly screening 1100 newly isolated temperature-sensitive yeast clones for DNA synthesis defects. Ninety-seven different mutant strains were identified. Approximately half had the fast-stop DNA synthesis phenotype; synthesis ceased quickly after shifting an asynchronous population of cells to the restrictive temperature. The other half had an intermediate-rate phenotype; synthesis continued at a reduced rate for at least 3 h at the restrictive temperature. All of the DNA synthesis mutants continued protein synthesis at the restrictivetemperature. Genetic complementation analysis of temperature-sensitive segregants of these strains defined 60 apparently new complementation groups. Thirty-five of these were associated with the fast-stop phenotype, 25 with the intermediate-rate phenotype. The fast-stop groups are likely to include many genes whose products play direct roles in mitotic S phase DNA synthesis. Some of the intermediate-rate groups may be associated with S phase as well. This mutant collection should be very useful in the identification and isolation of gene products necessary for yeast DNA synthesis, in the isolation of the genes themselves, and in further analysis of the DNA replication process in vivo.     

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1 mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early develpment. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. This increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.     

Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis.     

The role of phosphatidylcholine and choline metabolites to cell proliferation and survival

The reorganization of metabolic pathways in cancer facilitates the flux of carbon and reducing equivalents into anabolic pathways at the expense of oxidative phosphorylation. This provides rapidly dividing cells with the necessary precursors for membrane, protein and nucleic acid synthesis. A fundamental metabolic perturbation in cancer is the enhanced synthesis of fatty acids by channeling glucose and/or glutamine into cytosolic acetyl-CoA and upregulation of key biosynthetic genes. This lipogenic phenotype also extends to the production of complex lipids involved in membrane synthesis and lipid-based signaling. Cancer cells display sensitivity to ablation of fatty acid synthesis possibly as a result of diminished capacity to synthesize complex lipids involved in signaling or growth pathways. Evidence has accrued that phosphatidylcholine, the major phospholipid component of eukaryotic membranes, as well as choline metabolites derived from its synthesis and catabolism, contribute to both proliferative growth and programmed cell death. This review will detail our current understanding of how coordinated changes in substrate availability, gene expression and enzyme activity lead to altered phosphatidylcholine synthesis in cancer, and how these changes contribute directly or indirectly to malignant growth. Conversely, apoptosis targets key steps in phosphatidylcholine synthesis and degradation that are linked to disruption of cell cycle regulation, reinforcing the central role that phosphatidylcholine and its metabolites in determining cell fate.     

Translation of tubulin messenger ribonucleic acid.

Tubulin messenger RNA has been partially purified from embryonic chick brain. This messenger has been shown to be polyadenylated and capable of directing tubulin synthesis in an heterologous cell-free protein synthesizing system. Phosphocellulose fractions of IF-3 derived from embryonic leg muscle or brain were tested for their effect on tubulin and myosin synthesis in vitro. Phosphocellulose fraction four from either tissue source stimulates tubulin synthesis three fold. Myosin synthesis is enhanced significantly only by the muscle subfraction. This result suggests the existence of specific factors in muscle for the translation of the myosin messenger.