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Biomedical subjects

J E Midgley

Publications and source records attributed to J E Midgley.

At least 37 records · Page 2Linked to original sources
PubMed1982

A comparison of free T4 and the ratio of total T4 to T4-binding globulin in serum through pregnancy.

Serum free T4 concentrations have been variously reported as either constant or falling in pregnancy. In this study, 122 serum samples from apparently normal pregnancies were used to derive euthyroid ranges throughout pregnancy for T4 and free T4 (measured by the Amerlex kits) and for T4-binding globulin (TBG). The mean free T4 concentration fell with increasing gestational age, the range of values in the third trimester being narrower than the non-pregnant normal range. Evidence is reviewed which shows that the Amerlex free T4 assay is unaffected by the elevation of TBG in non-pregnant subjects. The decline in mean free T4 as pregnancy proceeds is in good agreement with the change in free T4 predicted by the falling T4/TBG ratio.

Alpha-Globulins↗
PubMed1982

An improved method for the estimation of relative binding constants of t4 and its analogues with serum proteins.

A new method is reported for the determination of the binding constants of homologous ligands relative to that of the parent ligand. The technique minimizes the effects of random and bias errors inherent in absolute methods and obviates the need to quantitate the radiolabelled tracer concentration. In addition, it is likely to be of general applicability for monovalent proteins. The method has been applied in the determination of the binding constant (relative to that of T4) of the T4-analogue tracer used in the Amerlex free T4 radioimmunoassay, in respect of its binding to both T4 binding globulin and prealbumin. The binding constants of the analogue (relative to T4) were approximately 3% for both proteins. These levels were judged unlikely to interfere significantly with the measurement of free T4 in serum samples from patients with very wide variations in concentration of either T4 binding globulin or prealbumin.

Cross Reactions↗
PubMed1982

Methodological background of the Amersham Amerlex free thyroxine RIA.

This contribution describes the essential rules underlying a valid estimation of FT4 and compares the way in which various methods deviate from ideal behaviour. The Amerlex FT4 RIA test has been validated by several approaches. A practical demonstration of the ability of various methods to correct for differences in T4-binding protein concentration has been made by independent studies in a UK hospital on euthyroid non-pregnant, euthyroid contraceptive and euthyroid pregnant women. The Amersham kit has been shown to correspond closely to theoretical expectations for FT4 values in these groups.

Female↗
PubMed1976

Turnover as a control of ribonucleic acid accumulation in bacteria undergoing stepdown.

The synthesis of ribosomes was compared in rel+ and rel- strains of Escherichia coli undergoing "stepdown" in growth from glucose medium to one with lactate as principal carbon source. Two strains (CP78 and CP79), isogenic except for rel, showed similar behaviour with respect to (1) the kinetics of labelling total RNA and ribosomes with exogenous uracil, (2) the proportion of newly formed protein that could be bound with nascent rRNA in mature ribosomes, and (3) the rate of induction of enzymically active beta-galactosidase (relative to the rate of ribosome synthesis). It was concluded that, as there was no net accumulation of RNA during stepdown in either strain, rRNA turnover must be occurring at a high rate. The general features of ribosome maturation in rel+ and rel- cells were almost identical with those found in auxotrophic rel+ organisms starved of required amino acids. In both cases, there was a considerable delay in the maturation of new ribosomal particles, owing to a relative shortfall in the rate of synthesis of ribosome-associated proteins. Only about 4-5% of the total protein labelled during stepdown was capable of binding with newly formed rRNA. This compared with 3.5% for rel+ and 0.5% for rel- auxotrophs during amino acid starvation. The turnover rate for newly formed mRNA and rRNA was virtually the same in "stepped-down" rel+ and rel- strains and was similar to that of the same fraction in amino acid-starved rel+ cells. The functional lifetime of mRNA was also identical. It seems that in the rel- strain many of the characteristics typical of the isogenic rel+ strain are displayed under these conditions, at least as regards the speed of ribosome maturation and the induction of beta-galactosidase. Studies on the thermolability of the latter enzyme induced during stepdown indicate that inaccurate translation, which occurs in rel- strains starved for only a few amino acids, is less evident in this situation than in straightforward amino acid deprivation.

Bacterial Proteins↗
PubMed1975

RNA synthesis and the accumulation of guanine nucleotides during growth shift down in the blue-green alga Anacystis nidulans.

Shift down of growth rate in the blue-green alga Anacystis nidulans by reduction of the incident light intensity produced a reduction in the rate of stable RNA accumulation which was correlated with increased concentrations of three phosphorylated compounds, two of which were identified as guanosine 5'-diphosphate-3'-diphosphate (ppGpp) and guanosine 5'-triphosphate3'-diphosphate (pppGpp). The step also causes a large but transient increase in the concentration of GTP. Stable RNA fails to accumulate for a considerable length of time after the concentrations of ppGpp and pppGpp have fallen, suggesting the involvement of another mechanism in the control of stable RNA accumulation.

Cyanobacteria↗
PubMed1974

The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation.

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.

Amino Acids↗
PubMed1973

The effect of trimethoprim on macromolecular synthesis in Escherichia coli.

In trimethoprim-inhibited RC(str) strains of Escherichia coli, the expression of the RC control of stable RNA synthesis arose primarily from a decrease in the intracellular concentrations of glycine and methionine, and not from inhibition of the initiation of new protein chains. In non-supplemented cultures, experiments with rifampicin showed that the immediate response to the addition of trimethoprim was a rapid decrease in the rate of initiation of RNA chains. This was followed after a few minutes by a sufficiently large fall in the rate of endogenous synthesis of nucleotide bases to cause a decrease in the rate of RNA chain polymerization. Inhibition of RNA chain initiation was thus overridden by an accumulation of DNA-dependent RNA polymerases upon the cistrons. RC(rel) strains also accumulated polymerases upon the DNA in similar circumstances, but did not suffer the initial effects on chain initiation. If purines were supplied before adding trimethoprim, RC(str) strains polymerized RNA chains at normal rates, but initiation rates were permanently decreased. In either situation, an increased% of the RNA formed was mRNA. However, in RC(rel) strains supplemented with bases, trimethoprim did not affect either the rate of initiation of new chains or their rates of polymerization or the relative rates of synthesis of stable RNA and mRNA. Protein synthesis was also severely inhibited by trimethoprim. Though the addition of glycine and methionine to base-supplemented, trimethoprim-inhibited RC(str) strains did not apparently affect the decreased rate of protein synthesis, RNA accumulation resumed at its normal rate. Thus, the inhibition of protein chain initiation had no effect on the rate of RNA accumulation in either RC(str) or RC(rel) bacteria. The RC control does not express itself through inhibitions of protein synthesis at this level.

Adenine↗
PubMed1973

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Ribosome maturation in RCstr and RCrel strains.

When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RC(str) and RC(rel) strains continued to accumulate RNA at rates very close to those in growing controls. The effects of trimethoprim on protein synthesis were studied by using as an experimental basis the rate of maturation of ribosomal particles from RNA-rich precursors. 1. In RC(str) cultures given nucleotide bases but no amino acids, RNA accumulation was inhibited because of amino acid lack. However, maturation of ribosomes from their precursors was more severely inhibited than was the synthesis of rRNA. The restraints on protein synthesis were more severe at the level of translation than the transcription of operons specific for the formation of ribosomal proteins. The kinetic delay time in the passage of rRNA from RNA-rich intermediates to the final ribosome products was therefore increased some three- to four-fold. 2. In RC(rel) cultures in the same conditions, trimethoprim inhibition stopped ribosomal particle synthesis, but rRNA-rich precursors accumulated. 3. If glycine+methionine were also added to inhibited RC(str) cultures, RNA accumulation resumed at a high rate. However, ribosomal maturation was still considerably disturbed because of a disproportionate response of the cells in the formation of protein and RNA. 4. With RC(rel) cultures, addition of the amino acids caused a large increase in the rate of ribosome maturation, though the degree of disproportionation between the rates of rRNA and ribosomal protein synthesis was now identical with that found in RC(str) strains. 5. When inhibited RC(rel) cultures were supplemented, there was still a severe inhibition of protein synthesis at the level of chain initiation, but inaccuracies in the process of polypeptide chain elongation were greatly decreased. This suggests that the effects of the RC(rel) mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.

Bacterial Proteins↗
PubMed1973

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Regulation of ribonucleic acid synthesis by 'Magic Spot' nucleotides.

During the inhibition of RC(str), but not RC(rel) mutants of Escherichia coli by trimethoprim the unusual nucleotides MSI (guanosine tetraphosphate, ppGpp) and MSII rapidly accumulated. The production of these nucleotides was not dependent on the addition of nucleotide base supplements to RC(str) cultures before trimethoprim, and the MSI nucleotide concentrations in non-supplemented or purine-supplemented cultures were comparable with the concentrations obtained when the cells were inhibited with l-valine (1g/l). Rifampicin rapidly decreased MSI and MSII nucleotide concentrations in trimethoprim-inhibited cultures to the basal values. Several situations were noted, in which MS nucleotide concentrations in trimethoprim-inhibited RC(str) cells could be drastically lowered without giving rise to an immediate resumption of stable RNA accumulation. If RC(str) mutants were first inhibited with trimethoprim and then given purines 15min later, MS nucleotide concentrations fell rapidly, because of a temporarily enhanced rate of accumulation of stable RNA. However, after a further 5min, RNA accumulation stopped, though MS nucleotide concentrations remained low. Also, if either glycine or methionine were added to trimethoprim-inhibited cultures supplemented with purines, RNA accumulation did not resume, though MS nucleotide concentrations rapidly declined. With both amino acids present, there was both a decline in MS nucleotide concentration and a resumption in stable RNA synthesis. These findings suggest that MSI nucleotide concentrations in trimethoprim-inhibited bacteria are not the sole factors in the control of stable RNA synthesis. It is possible that, during the period when the RC(str) cells contained high concentrations of MS nucleotides, some factor important in the MSI-mediated control of stable RNA synthesis was irreversibly inactivated. However, as antibiotics (e.g. chloramphenicol) both abolished high MS nucleotide concentrations and permitted a rapid resumption of stable RNA accumulation in the same conditions, it is more likely that an additional control mechanism has come into play.

Centrifugation, Density Gradient↗
PubMed1972

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acids in relaxed and stringent amino acid auxotrophs of Escherichia coli.

The biosynthesis and stability of various RNA fractions was studied in RC(str) and RC(rel) multiple amino acid auxotrophs of Escherichia coli. In conditions of amino acid deprivation, RC(str) mutants were labelled with exogenous nucleotide bases at less than 1% of the rate found in cultures growing normally in supplemented media. Studies by DNA-RNA hybridization and by other methods showed that, during a period of amino acid withdrawal, not more than 60-70% of the labelled RNA formed in RC(str) mutants had the characteristics of mRNA. Evidence was obtained for some degradation of newly formed 16S and 23S rRNA species to heterogeneous material of lower molecular weight. This led to overestimations of the mRNA content of rapidly labelled RNA from such methods as simple examination of sucrose-density-gradient profiles. In RC(rel) strains the absolute and relative rates of synthesis of the various RNA fractions were not greatly affected. However, the stability of about half of the mRNA fraction was increased in RC(rel) strains during amino acid starvation, giving kinetics of mRNA labelling and turnover that were identical with those found in either RC(str) or RC(rel) strains inhibited by high concentrations of chloramphenicol. Coincidence hybridization techniques showed that the mRNA content of amino acid-starved RC(str) auxotrophs was unchanged from that found in normally growing cells. In contrast, RC(rel) strains deprived of amino acids increased their mRNA content about threefold. In such cultures the mRNA content of accumulating newly formed RNA was a constant 16% by wt.

Amino Acids↗
PubMed1972

The control of ribonucleic acid synthesis in bacteria. Polymerization rates for ribonucleic acids in amino acid-starved relaxed and stringent auxotrophs of Escherichia coli.

Polymerization rates of newly formed chains of various RNA fractions were measured in Escherichia coli CP78 (RC(str)) and CP79 (RC(rel)) multiple amino acid auxotrophs, deprived of four amino acids essential for growth. Immediately after the onset of severe amino acid deprivation, in RC(str) strains the rate of labelling of RNA by exogenous nucleotide bases was greatly diminished. At first, the initiation of new RNA chains declined faster than the rate of polymerization in RC(str) organisms, but as starvation proceeded the rate of polymerization was eventually lowered to about 10% of that found during normal growth. In strain CP79 (RC(rel)), on the other hand, chain-polymerization rates were unaffected by amino acid withdrawal. Artificial depletion of the intracellular purine nucleotide pools in RC(str) or RC(rel) strains by trimethoprim, before the onset of amino acid deprivation, showed that in the RC(str), but not the RC(rel) strain, amino acid withdrawal gave rise to an inability of the cells to utilize exogenously supplied purine or pyrimidine bases for RNA synthesis. During a prolonged starvation, the observed 100-fold decrease in the total rate of incorporation of exogenous nucleotide bases into the RNA of RC(str) organisms was ascribed to a combination of a tenfold decrease in the overall rate of RNA chain polymerization, at least a fivefold decrease in the ability of the cells to utilize exogenous bases and a preferential inhibition of initiation of stable RNA chains. None of these changes occurred in the corresponding RC(rel) strain.

Adenine↗