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Biomedical subjects

R D Nolan

Publications and source records attributed to R D Nolan.

At least 37 records · Page 2Linked to original sources
PubMed1983

Cyclo-oxygenase products of arachidonic acid metabolism in rat osteoblasts in culture.

The metabolism of arachidonic acid to its cyclo-oxygenase products was studied in monolayer cultures of osteoblast-rich rat calvarial cells and of clonal cell lines from a rat osteogenic sarcoma, enriched in the osteoblast phenotype. Prostanoids were measured by radioimmunoassay after extraction of media and fractionation by high pressure liquid chromatography. In both normal and malignant osteoblasts the major cyclooxygenase product was 6-oxo-prostaglandin F1 alpha, the hydration product of prostacyclin, with lesser amounts of prostaglandin E2 and prostaglandin F2 alpha. No significant thromboxane B2 was detected. Prostaglandins are thought to have a local role in the regulation of bone resorption. These results point to the possible importance of prostacyclin either in bone resorption or in some other local function, e.g., regulation of bone blood flow.

6-Ketoprostaglandin F1 alpha↗
PubMed1983

The interactions of [14C]cefotetan with penicillin binding proteins of a wide variety of Gram-positive and gram-negative species.

Membrane preparations were made from a number of Gram-positive and Gram-negative bacterial strains. The ability of the membrane-bound penicillin-binding proteins (PBP) in these preparations to bind [14C]cefotetan has been studied. Increasing amounts of unlabelled benzylpenicillin were added and subsequently [14C]cefotetan was introduced to determine its ability to bind to additional proteins or to displace the pre-bound benzylpenicillin. In the Gram-positive series some quantitative binding differences between the two labelled compounds could be seen. However, in the majority of Gram-negative organisms tested cefotetan exhibited greatest affinity for PBP 3 and none for PBP 2. No new PBPs were detected using [14C]cefotetan, suggesting that the compound exerts its antibacterial effect by a mechanism similar to the other cephamycins. The high minimal inhibitory concentrations (MICs) of cefotetan found for the Gram-positive genera tested are reflected in only one organism by the failure to bind of some of its PBPs. In the majority of cases, permeability is assumed to play the dominant role in the relatively low sensitivity of these organisms to cefotetan.

Anti-Bacterial Agents↗
PubMed1983

Conversion of triostins to quinomycins by protoplasts of Streptomyces echinatus.

Protoplasts of Streptomyces echinatus have been used to investigate the biosynthesis of echinomycin (quinomycin A). It has been shown that this organism has the capacity to convert a series of triostins to the corresponding quinomycins by a mechanism involving methylation. Evidence is presented which suggests that triostin A is the natural precursor of echinomycin. Conversion of tetra-N-demethyl analogues of triostin A to corresponding analogues of echinomycin was not detected.

Anti-Bacterial Agents↗
PubMed1982

The pericardium as a source of prostacyclin in the dog, ox and rat.

Cyclo-oxygenase products of arachidonic acid metabolism formed by the pericardium and epicardial surface of dog heart were identified and quantitated by radioimmunoassay after separation by high-pressure liquid chromatography. Pieces of parietal pericardium, of dog, ox and rat, when incubated in vitro produced mainly 6-keto-PGF1 alpha, with lesser amounts of PGE2, PGF2 alpha and thromboxane B2. Biosynthesis of all prostanoids increased during incubation of the parietal pericardium of each species with arachidonic acid, but 6-keto-PGF1 alpha was still the major metabolite. When slices of dog heart were incubated with arachidonic acid (1 microgram/ml) the rates of 6-keto-PGF1 alpha formation by the parietal pericardium was much greater than that of the myocardium and endocardium. Epicardial slices appeared to be intermediate in 6-keto-PGF1 alpha formation. The hearts of anesthetized dogs were also irrigated in situ with Krebs' solution, and during the first 5 min of epicardial irrigation the pericardial fluid leaving the heart again contained high levels of 6-keto-PGF1 alpha, with lesser amounts of the other prostanoids. Addition of arachidonic acid (3 micrograms/ml) to the irrigating fluid caused an increase in all measured prostanoid levels, although 6-keto-PGF1 alpha remained the predominant metabolite. In contrast, intravenous infusion of isoproterenol selectively increased the release of 6-keto-PGF1 alpha from the irrigated heart. It is concluded that the pericardium and epicardium continuously release prostacyclin into the pericardial fluid, and that the increased release of this substance observed when cardiac workload increases derives mainly from these membranous sources. This raises the interesting possibility that pericardial prostacyclin might influence coronary vascular tone and chemoreflexes which arise from the epicardium during myocardial ischemia.

6-Ketoprostaglandin F1 alpha↗
PubMed1981

Prostacyclin (PGI2) release accompanying angiotensin conversion in rat mesenteric vasculature.

The relationship between angiotensin conversion and release of prostaglandins (PGs) were studied in isolated, perfused, mesenteric vasculature of rats. PGs in the mesenteric effluent were detected by bovine coronary artery and rat stomach strip, and by radioimmunoassay of 6-oxo-PGF1 alpha and PGE2. Angiotensin II (AII) was assayed simultaneously using rabbit aorta. Both angiotensin I (AI) and AII released a prostacyclin (PGI2)-like substance, but little PGE2 from the mesentery, AII being 5 times more potent. Indomethacin (2.8 micrometers) abolished angiotensin-induced release of PGI2. Of the AI (0.2--1.5 nmol) injected into the perfusion medium, 3--8% was converted to AII in passage through the mesentery. Captopril (4 micrometers), infused through the mesentery, inhibited AI-induced PGI2 release, but did not affect release induced by AII. Release of the PGI2-like substance by higher doses of AI (2--20 nmol) in the presence of captopril was always accompanied by concomitant contraction of rabbit aorta, indicating residual conversion of AI. Infusion of (Sar1, Ala8)AII (3--30 nM) through the mesentery abolished PGI2 release by both peptides. By choosing doses of AI and AII which produce equivalent amounts of AII in the mesenteric effluent, it appeared that AII generated locally in the mesenteric vasculature was a more effective stimulus for PGI2 generation than AII in the perfusion fluid. There is no evidence for intrinsic activity of AI, and release of the PGI2-like substance appears to be mediated through an AII receptor.

Angiotensin I↗
PubMed1981

Stimulation of prostacyclin release from the epicardium of anaesthetized dogs.

1 The generation of prostanoids in the hearts of anaesthetized dogs was studied by irrigating in situ the epicardial surface with Krebs solution. Prostanoids were measured by direct bioassay on smooth muscles and by radioimmunoassay of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha) and prostaglandin E2 (PGE2) in the epicardial irrigation fluid. 2 The epicardial irrigation fluid contained a prostacyclin-like substance, as indicated by the bioassay tissues, and immunoreactive 6-oxo-PGF1 alpha; PGE2-like materials were also detected. By both methods the output of the prostacyclin-like substance, which decreased with time of epicardial irrigation, was increased by manipulating the heart and by adding arachidonic acid (3 microgram/ml), and decreased by adding indomethacin (1 microgram/ml) to the irrigation fluid. 3 Bioassayed prostacyclin and immunoreactive 6-oxo-PGF1 alpha in the epicardial irrigation fluid increased by about 3-5 ng/ml during and after infusion of isoprenaline (0.1 microgram kg-1 min-1). The substance was not released by isoprenaline when indomethacin was added to the irrigation fluid, or when propranolol (0.5 mg/kg) was given intravenously. 4 Aortic constriction, bilateral carotid artery occlusion and intravenous angiotensin infusion all increased output of the prostacyclin-like substance into the epicardial irrigation fluid. The output was abolished by treating the heart with indomethacin (10 mg/kg intravenously or 1 microgram/ml epicardially). 5 The prostacyclin-like substance was also released by all of the above stimuli after the parietal pericardium had been removed and replaced by a plastic sheet. 6 It is concluded that prostacyclin is continually released from tissues close to the epicardial surface and from the pericardium, and that prostacyclin generation increases when cardiac workload increases. Prostacyclin of epicardial or pericardial origin might therefore contribute to metabolic regulation of coronary blood flow.

Angiotensins↗
PubMed1979

Comparison of the penicillin-binding proteins of different strains of Neisseria gonorrhoeae.

This study was undertaken to determine if the categorization of Neisseria gonorrhoeae strains into disseminated gonococcal infection (DGI) and non-DGI types was also paralleled by some common characteristic in their penicillin-binding proteins (PBPs). Fluorography of sodium dodecyl sulfate-containing polyacrylamide gels, on which the [14C]penicillin-labeled PBPs had been separated, was used to visualize the PBPs. No common characteristic PBP for genital or DGI strains was observed. Apart from PBPs 1 and 4, which were observed in all cases, there was an apparently random distribution of other PBPs in both types of N. gonorrhoeae. It was concluded from these data that the penicillin susceptibility associated with most DGI strains of N. gonorrhoeae cannot be correlated with any specific changes in their PBP patterns.

Bacterial Proteins↗
PubMed1978

The isolation and characterization of elongation factor eEF-Ts from Krebs-II mouse-ascites-tumor cells and its role in the elongation process.

A factor having activity similar to that described in other systems for the eukaryotic elongation factor eEF-Ts was isolated from the heavy, aggregate form of eEF-TH (formally named EF-1H). This protein has a molecular weight of 52000 under native conditions and of 25500 under denaturing conditions. It has been shown to stimulate eEF-Tu-dependent aminoacyl-tRNA binding to ribosomes and therefore eEF-Tu/eEF-G-dependent polyphenylalanine synthesis by ribosomes and was found to stimulate GDP-GTP exchange in eEF-Tu . GDP complexes. In the course of this work, it was also demonstrated that the removal of deacylated tRNA from the ribosome is a GTP-dependent process. This report, therefore, adds further support to the concept that a third elongation factor, eEF-Ts, may be common to all systems in the eukaryotic domain.

Animals↗
PubMed1977

Further evidence that elongation factor 1 remains bound to ribosomes during peptide chain elongation.

This paper describes three types of experiments which indicate that the binding sites for elongation factor 1 (EF-1) and elongation factor 2 (EF-2) on ascites cell ribosomes are not identical and perhaps not even overlapping. The experimental evidence presented includes direct competitive binding of labeled elongation factors to ribosomes as well as the influence of pokeweed antiviral protein and Escherichia coli anti L7/L12 proteins on the binding and function of the two factors. It is further shown that EF-1beta from Artemia salina does not function in displacing EF-1 from mouse ascites tumor cell ribosomes. These results also support our recently proposed model that EF-1 remains bound to the ribosome during the peptide chain elongation cycle.

Animals↗
PubMed1976

Functional identity of the monomeric and multiple forms of elongation-factor 1 from Krebs-II mouse ascites-tumor cells.

Highly purified 3H-labelled elongation factor 1 (EF-1) from Krebs II mouse ascites tumour cells was separated into biologically active monomeric and aggregate forms of the enzyme by either gradient centrifugation or gel filtration. When corrected for their content of inactive enzyme both forms of the factor were found to be equally active whether tested in the binding or synthesis reaction. The only form of the enzyme found bound to ribosomes was the monomer; it was therefore concluded that the aggregate form of the enzyme must first dissociate before it reacts with the ribosome. The stoichiometry of the aminoacyl-tRNA binding reaction to ribosomes in the presence of guanosine nucleotides was also studied. It was found that one molecule of aminoacyl-tRNA and of Guo-5'-P2-CH2-P is bound per molecule of EF-1 bound to the ribosome. Following interaction with a release from, the ribosomes, EF-1 was found to be predominantly monomeric.

Animals↗
PubMed1976

The binding of tritiated elongation-factors 1 and 2 to ribosomes from Krebs II mouse ascites-tumore cells. The influence of various antibiotics and toxins.

The effect of a number of different antibiotics and toxins on the capacity of Krebs II mouse ascites ribosomes to bind 3H-labelled elongation factors (EF-1 and EF-2) has been examined. It was found that abrin and ricin inhibit the binding of EF-2, while diphtheria toxin, sparsomycin, streptovitacin A, and cycloheximide had essentially no effect on its binding. Of the other compounds examined, sparsomycin was unique in its capacity, under some circumstances, to significantly affect the binding of aminoacyl-tRNA and EF-1 to ribosomes. Fusidic acid appears to nonspecifically enhance the binding of both EF-1 and EF-2.

Abrin↗
PubMed1976

A new concept of the function of elongation factor 1 in peptid chain elongation.

An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments in which mixtures of [3H]EF-1, ribosomes from Drebs II ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound.

Animals↗
PubMed1975

The binding of tritiated elongation factors 1 and 2 to ribosomes from Krebs II mouse ascites tumor cells.

Tritiated elongation factors 1 and 2 (EF-1 and EF-2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors. The following results were obtained. 1. EF-1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl-tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF-1. If GTP is replaced by Guo-5'-P2-CH2-P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques. 2. Likewise EF-2 binding to ribosomes can only be detected in the presence of GUO-5'-P2-CH2-P. 3. The affinity of ribosomes for EF-2 appears to be higher than for EF-1: PREINCUBATION OF RIBOSOMES WITH EF-2 inhibits the subsequent attachment of EF-1 almost completely. EF-1 prebound to ribosomes in the presence of GUO-5'-P2-CH2-P, POLY(URIDYLIC ACID) AND Phe-tRNA-Phe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2. 4. Although EF-2 binding to ribosomes precludes any stable association between ribosomes and EF-1 it does not prevent the insertion of aminoacyl-tRNA into the ribosomal A-site. The attachment of aminoacyl-tRNA under these conditions enhances the binding of EF-2 to the ribosome. 5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2. 6. A-site ribosomes display a strong preference for the attachment of EF-2 and bind EF-1 only very poorly. The reverse is true for P-site ribosomes which are good substrates for the binding of EF-1 and bind EF-2 less efficiently than A-site ribosomes. These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2.

Animals↗