Prostaglandin synthesis in rat embryo tissue: The effect of non-steroidal anti-inflammatory drug in vivo and ex vivo

Aspirin and salicylate are well-known but poorly understood teratogens in laboratory animals. Because aspirin inhibits PG synthesis, we systematically examined PG synthesis in rat embryo homogenates, the inhibition of PG synthesis $\text{in vivo}$ and $\text{ex vivo}$ by various non-steroidal anti-inflammatory drugs, and tested the hypothesis that the inhibition of PG synthesis is responsible for aspirin-induced limb defects in rats. We report that embryonic rat homogenates synthesis 6-keto-PGF_1α, PGE, and PGF in large amounts from endogenous substrate, that aspirin and other non-steroidal anti-inflammatory drugs inhibit PG synthesis $\text{in vitro}$ but not necessarily $\text{in vivo}$, and that contrary to our original hypothesis, the inhibition of PG synthesis is likely not responsible for aspirin-induced limb defects in rats.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Protein synthesis in mitochondrial and microsomal fractions from rat brain and liver after acute or chronic ethanol administration

Incorporation of C¹⁴ Leucine was determined $\text{in vitro}$ or $\text{in vivo}$ in isolated mitochondria and microsomes of rat brain and liver after acute or chronic ethanol administration $\text{in vivo}$.The protein synthesis in mitochondrial and microsomal preparation was inhibited respectively by chloramphenicol and cycloeximide, specific inhibitors for the two systems tested. The experimental data demonstrate that the $\text{in vitro}$ protein synthesis in both systems, mitochondrial and microsomal, is strongly affected only after chronic treatment which produces significant activation at the mitochondrial and microsomal level in the liver and an inhibition on the same systems of the brain.The data for $\text{in vivo}$ protein synthesis instead show strong inhibition after acute administration, except for brain mitochondria, which are practically unaffected, while after chronic treatment no significant alterations are observed.     

Unscheduled DNA synthesis in isolated hepatic nuclei after treatment of rats with methylnitrosourea in vivo

Administration of the carcinogenic methylating agent, methylnitrosourea, to rats caused a significant increase in endogenous DNA synthesis assayed subsequently in isolated hepatic nuclei $\text{in}\text{vitro}$. DNA synthesis was related directly to the dose of carcinogen and inversely to the interval between treatment and isolation of nuclei. This synthesis appears to represent the continuation $\text{in}\text{vitro}$ of unscheduled, reparative DNA synthesis initiated in damaged cells $\text{in}\text{vivo}$.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Regulation of the basal and cyclic AMP-stimulated rates of glycogen synthesis in Escherichia coli by an intermediate of purine biosynthesis

In $\text{Escherichia}\text{coli}$an abrupt increase in the rate of glycogen synthesis occurs at the onset of total nitrogen starvation. We present here both $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$ data indicating that this increase occurs because of the loss of a nitrogen-containing intermediate of purine biosynthesis (apparently 5-aminoimidazole-4-carboxamide ribonucleotide) that inhibits glycogen synthesis. We also show that this inhibitory intermediate antagonizes the stimulation of glycogen synthesis by 3′,5′-cyclic AMP. The uncovering of the regulation of glycogen synthesis by this inhibitor apparently provides the first link in understanding the 23-year-old observation of a reciprocal relationship between growth rate and glycogen accumulation in $\text{E.}\text{coli}$.     

Enzyme secretion in E. coli K12 : studies on alkaline phosphatase synthesis using an unsaturated fatty-acid auxotroph.

The role of unsaturated fatty-acid starvation, and of the substitution of $\text{trans}$ for $\text{cis}$ fatty acids in the membrane phospholipid, on the secretion of alkaline phosphatase, has been investigated. A system in which alkaline phosphatase synthesis was initiated by a temperature shift has been used to obviate possible complications resulting from phosphate depletion. In contrast to earlier reports, we find (a) there is very little effect of unsaturated fatty-acid starvation on the synthesis of alkaline phosphatase; (b) the synthesis of both β-galactosidase and alkaline phosphatase synthesis was severely reduced below 27–30°C in cells grown on $\text{trans}\text{Δ}{}^9$ 16:1 fatty-acid, compared to cells grown on the $\text{cis}\text{Δ}{}^9$ 16:1 analogue. Thus no $\text{preferential}$ effect on alkaline phosphatase synthesis was observed.     

Increased de novo phospholipid biosynthesis in the stimulated rat exocrine pancreas

The $\text{hisU1820}$ mutant (TA799) of $\text{Salmonella}\text{,}\text{typhimurium}$ shows a substantial increase in the levels of ppGpp (MSI) and of pppGpp (MSII) during several types of metabolic shifts. Noticeable amounts of ppGp (MSIII) are also present post-carbon/energy source downshifts and temperature up-shifts. The increased levels of these guanosine polyphosphates were observed despite the absence of the expected reduction in RNA synthesis upon a nutritional downshift. We, therefore, suggest that the $\text{hisU}$ mutation causes an increase in the accumulation of MSI and MSII; and that ppGpp alone is not sufficient to promote restriction of RNA synthesis during a nutritional transition.     

Regulation of argF mRNA synthesis,performed in vitro

The specific synthesis of $\text{arg}$F mRNA directed by the $\text{arg}$F gene carried on the specialized transducing bacteriophage $\text{λh80C}{}_1\text{857darg}$F, performed $\text{in vitro}$, is described with the use of an S180 extract from a strain carrying $\text{arg}$R^?. Synthesis of $\text{arg}$F mRNA is biphasic at approximately 7 minutes. The regulation of $\text{arg}$F mRNA synthesis by the specific arginine holorepressor present in an S180 extract prepared from a strain carrying the $\text{arg}$R⁺ allele is described.     

Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

Lytic enzymes in sporulating Bacillus subtilis

Two specific lytic enzymes were found in sporulating $\text{B. subtilis}$ cells: a N-acetyl muramyl L-alanine amidase and a $\text{γ-D-glutamyl-(L)}\text{meso}$ diaminopimelyl endopeptidase. Both enzyme activities were measured using radioactive synthetic substrates. They are low in vegetative cells and increase during sporulation. The highest rates of increase are concomitant with cortex formation. In a mutant with delayed sporulation enzyme synthesis is also delayed. We suggest that both enzymes play a role in the synthesis of the specific cortex peptidoglycan.     

Identification of a phi X174 coded protein involved in the inhibition of beta-galactosidase synthesis in Escherichia coli

The synthesis of β-galactosidase (EC 3.2.1.23: β-D-galactoside galactohydrolase) in $\text{Escherichia}\text{coli}$ is repressed as a result of infection with single-stranded DNA phage ØX174. An $\text{amber}$ mutant in ØX174 cistron A, which codes for two proteins, does not inhibit the enzyme synthesis while $\text{amber}$ mutants in all other genes do cause repression. A mutant near the amino-terminal end of cistron A, which produces the small 35,000 molecular weight cistron A polypeptide, also inhibits the synthesis of β-galactosidase. Inhibition is also observed in an $\text{Escherichia}\text{coli}\text{rep}$ mutant which does not support the replication of replicative-form DNA. Exogenous nucleotide bases and cyclic 3′,5′-adenosine monophosphate (cyclic AMP) do not have any effect on the degree of repression.     

Regulation of the Escherichiacoli tryptophan operon by readthrough of UGA termination codons

The regulation of the synthesis of $\text{trp}$ operon enzymes was studied in streptomycin-resistant $\text{Escherichia}\text{coli}$ mutants temperature-sensitive for UGA suppression by normal tRNA^Trp. Our mutants carry a $\text{trp}\text{R}{}^{\mathrm{+}}$ allele that when transferred to a different genetic background causes repression of trp operon enzyme synthesis at both low (35°C) and high (42°C) temperatures; however, in our mutants with an excess of tryptophan and at increased temperatures $\text{trp}$ enzyme synthesis is derepressed. Based on our results and the sequence data of the $\text{trp}$R gene [Singleton et al. (1980) Nucleic Acids Res., 8, 1551–1560], we offer a model for the involvement of the limited misreading of UGA codons by normal charged tRNA^Trp in the autogenous regulation of the $\text{trp}$R gene expression. The UGA readthrough process may be a regulatory amplifier of the effect of tryptophan starvation.     

Responsibility of 16S RNA for the stimulation of polypeptide synthesis by spermidine.

From the studies on the spermidine stimulation of polyphenylalanine synthesis catalyzed by $\text{E. coli}$ 50S and reconstituted 30S particles containing 16S RNA and 30S ribosomal proteins from $\text{E. coli}$ and $\text{B. thuringiensis}$ in different kinds of combinations, it is concluded that 16S RNA is mainly responsible for the stimulation of polypeptide synthesis by spermidine.     

The influence of mitochondria on ribosomes. Cycloheximide resistance of ribosomes from petite mutants of saccharomyces cerevisiae

The influence of cycloheximide on amino acid incorporation into protein of standard strain ? + and cytoplasmic mutant ? ? of $\text{S.}\text{cerevisiae}$ was determined $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$. $\text{In}\text{vivo}$ cycloheximide at the concentration which inhibits protein synthesis in ? + strain by over 60% has litle or no effect in mutant ? ? strain. $\text{In}\text{vitro}$ cycloheximide in the range of 0.05 to 0.3 ug/ml of incubation medium inhibits polyphenylalanine synthesis in ? + strain by 50% and in ? ? strain by less than 10%. Similar resistance to this antibiotic are shown in standard strain grown in anaerobic conditions. It has been found that the resistance to cycloheximide is associated with changes in cytoplasmic ribosomes and may depend on the integrity of mitochondrial system.     

Effect of methylglyoxal bis (guanylhydrazone) (MGBG) in vivo on the decarboxylation of s-adenosylmethionine and synthesis of spermidine in the rat and guinea pig.

The rates of decarboxylation of $\text{S}$-adenosylmethionine and synthesis of spermidine have been measured in extracts of liver, kidney and brain of the rat and guinea pig after intraperitoneal injection of MGBG, both before and after dialysis. The rate of decarboxylation of $\text{S}$-adenosylmethionine paralleled that of spermidine synthesis in all of the tissues investigated, even when spermidine synthesis was measured using preformed decarboxylated $\text{S}$-adenosylmethionine as substrate instead of $\text{S}$-adenosylmethionine itself. MGBG inhibited CO₂ production and spermidine synthesis to a similar extent in extracts of liver and kidney of both the rat and the guinea pig. After dialysis, a similar increase in both CO₂ production and spermidine synthesis was noted in these extracts. No effects on CO₂ production or spermidine synthesis were noted on extracts of brain of the rat or guinea pig, either before or after dialysis. When MGBG was injected intracisternally, CO₂ production and spermidine synthesis by extracts of brain were inhibited to the same extent, and after dialysis a similar increase in CO₂ production and spermidine synthesis was observed. These results indicate that the effects of MGBG are essentially the same in brain as they are in liver and kidney, and the MGBG injected intraperitoneally does not pass into the brain.