Search PubMed⌕ Search

Biomedical subjects

K L Manchester

Publications and source records attributed to K L Manchester.

At least 37 records · Page 2Linked to original sources
PubMed1994

The 9-kDa calbindin gene of Rousettus aegyptiacus: its identification and isolation from a genomic library.

A genomic library of the fruit bat (Rousettus aegyptiacus) was constructed in lambda phage gt11. The titre of the library was determined to be 2 x 10(5) pfu/ml. The genomic library was amplified and the titre of the amplified library increased 300-fold to 7 x 10(7) pfu/ml. The library was screened by in situ hybridization techniques using a fragment of the mouse 9-kDa calbindin cDNA as a probe. Screening of 10(5) plaques yielded a positive clone. Three additional rounds of screening were performed to purify the positive. Lambda phage DNA was isolated from the positive clone and restriction digest analysis, followed by hybridization studies, was performed on these digests in order to determine the location of the bat 9-kDa calbindin gene in the insert of the lambda phage vector. Restriction maps so derived were interpreted from the published sequence for the rat 9-kDa calbindin gene and indicate the successful isolation of the 9-kDa calbindin gene of Rousettus aegyptiacus.

Animals↗
PubMed1994

Is there a need for channelling in the functioning of the protein synthesis initiation factors eIF-2 and eIF-2B?

The possibility that channelling is necessary in the initiation of eukaryotic protein synthesis is evaluated. Calculations suggest that despite the very high association rate constant found for the interaction of eIF-2 and eIF-2B under in vitro conditions, it is unlikely that rates in vivo can be high enough to permit measured rates of protein synthesis. Channelling therefore seems a priori a requirement.

Animals↗
PubMed1993

The transport of aluminum and water across the rat small intestine.

Aluminum transport across the epithelium of the rat small intestine has been investigated to determine factors affecting its absorption and its effect on the transport of other substances across the membrane. The intestines were attached to a perfusion apparatus and perfused with Krebs-Ringer-bicarbonate buffer containing aluminum. The transport of aluminum and buffer ions across the small intestine were measured. Phosphate transport was found to be the most satisfactory marker for viability. It is impossible to accurately measure the aluminum transport across a biological membrane unless the aluminum concentration of the solution is stable over the period of measurement. Hence, the solutions were stabilized with citrate ions which made them stable over a period of at least two hours. The velocity of transport of aluminum across the epithelium increased steadily and only became constant after about one hour. The steady state value of 0.12 micrograms atom of Al/hr/mg dry tissue compares well with that reported in the literature for stable aluminum solutions. Aluminum inhibited the transport of water across the membrane, but the inhibition took about two hours to reach a steady state of about 50% of the control value. This indicates that aluminum-containing medications and foods are able to interfere with the absorption of nutrients from the gut. Aluminum salts may therefore be useful to prevent rapid dehydration in the treatment of certain diseases such as cholera.

Aluminum↗
PubMed1993

Detection of the 9-kDa vitamin D-dependent calbindin gene in a fruit bat (Rousettus aegyptiacus) fibroblast cell line.

1. A fibroblast cell line was established from skin and lung tissue of the fruit bat (Rousettus aegyptiacus). 2. Genomic DNA, isolated from the fibroblast cells, was restriction digested and probed for the 9-kDa calbindin gene with a 180 base-pair fragment of mouse calbindin-D9K cDNA. Genomic DNA from rats and humans was similarly probed. 3. Hybridization and thus homology was observed between the mouse probe and DNA from the rat and the fruit bat, but not the human. 4. The results are discussed in relation to the detection of calbindin-D9K in the fruit bat despite very low circulating levels of its inducer 1,25(OH)2D3.

Animals↗
PubMed1992

Adenine nucleotide concentrations and energy charge in muscle of chronic haemodialysis patients.

Adenine nucleotide concentrations and energy charge ratios were measured in muscle samples collected during transplant surgery of 7 patients suffering from chronic renal failure and undergoing haemodialysis. The energy charge ratio of 0.75 in the muscle of transplant patients was significantly lower (p less than 0.01) than that of 0.89 found in muscle from controls. The lower energy charge ratio and increased concentrations of adenosine diphosphate and adenosine monophosphate may contribute to the patients' reduced exercise ability and their poor metabolic state.

Adenine Nucleotides↗
PubMed1992

Rate laws for the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The rate laws governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts are set out and the information that is available from various studies is analysed in terms of the laws. In particular the consequences of k-1 being equal to k-4 in the reaction sequence as set out below are examined in relation to results obtained by Hwang and Miller (J. Biol. Chem. 260, 11498-11502) and the contradiction these pose in relation to other published results. Measurement of on and off rate constants for binding of GTP to EF-Tu.EF-Ts and of the affinity of EF-Ts for EF-Tu.GTP should prove of importance in resolving discrepancies.

Binding Sites↗
PubMed1991

Structural studies of amyloglucan and a soluble glucan produced from starch by Streptococcus sanguis 1 MC 204.

Two alpha-D-glucans, produced from amylopectin by an oral isolate Streptococcus sanguis 1 MC 204, were shown to contain both (1----4) and (1----6) linkages. The first alpha-D-glucan (amyloglucan) was adherent and highly insoluble, and methylation analysis, i.r. spectroscopy, and enzymic analyses showed it to be similar to amylopectin but less branched with longer interior and exterior chains. The second polymer was a non-adherent soluble alpha-D-glucan that was similar to amyloglucan but with long exterior chains. These alpha-D-glucans were not synthesised de novo, but were the products of the modification of amylopectin.

Amylopectin↗
PubMed1991

Separation of ribosomal subunits on Trisacryl GF 2000.

The separation of rat liver and E. coli ribosomal subunits was attempted on Trisacryl GF 2000. Contrary to experiments with Sepharose 4B and Bio-Gel A-15 the 60S mammalian subunit did not bind to the resin at 4 degrees C but eluted within the column volume ahead of the 40S subunit. Puromycin, however, used to prepare the subunits, which on the agarose gels had eluted at the total column volume, exhibited anomalous retardation on the Trisacryl resin. Trisacryl therefore behaves as the more non-polar resin, and the binding of 60S subunits to agarose gels is a result of hydrophilic interaction.

Animals↗
PubMed1991

Control of initiation of eukaryotic protein synthesis by guanine nucleotides and methionyl-tRNAi.

The influence of changing concentrations of GDP, methionyl-tRNAi, eIF-2 and eIF-2B on possible rates of initiation of protein synthesis have been explored in calculations based on previously derived rate constants for interaction of the components involved in formation of ternary or quaternary complexes of eIF-2B, eIF-2, GTP and Met-tRNAi. When allowance is made for the limitation of diffusional coefficients imposed on macromolecules by the intracellular milieu it is apparent that recent estimates by Rowlands et al. (Eur. J. Biochem. 175, 93:1988) of higher concentrations of eIF-2 and eIF-2B in cells than hitherto proposed become necessary to support known rates of initiation. Under these conditions changing concentrations of met-tRNAi as proposed by Cooper and Braverman (J. Biol. Chem. 256, 7461:1981) are likely to have an important regulating influence.

Animals↗
PubMed1991

Re-evaluation of rate constants involved in the action of the initiation and elongation factors eIF-2B and EF-Ts.

Rate constants calculated previously by the author (Biochem. Int. 22, 523-533:1990) for the reactions catalysed by eIF-2B (GEF) in which free GDP exchanges with GDP bound to eIF-2 have been re-evaluated using the computational procedures developed by Chau et al. (J. Biol. Chem. 256, 5591-5596:1981) for the analogous reactions catalysed by EF-Ts. Modification of the equations used by Chau et al. emphasises the interrelationships of the rate constants for the binding of GDP and of EF-Ts (eIF-2B) to the ternary complex EF-Ts.EF-Tu.GDP (eIF-2B.eIF-2.GDP). The modification leads to some revision of the previously published values for the rate constants involved in the action of EF-Ts as put forward by Chau et al. as well as for those involved in the action of eIF-2B.

Guanine Nucleotide Exchange Factors↗
PubMed1991

Analysis of rate constants governing the exchange of guanine nucleotides bound to EF-Tu catalysed by EF-Ts.

The kinetics of the heterologous exchange of GDP bound to EF-Tu by free GTP catalysed by EF-Ts have been analysed with a view to correlating results obtainable with different computational procedures. The affinity of EF-Ts for EF-Tu.GTP was found to be somewhat less than previously proposed by Romero et al. (Biochemistry 260, 6167:1985) though still greater than for EF-Tu.GDP. There is a close interrelationship between the constants for the binding of GTP to EF-Tu.EF-Ts and of EF-Ts to EF-Tu.GTP. The declining fractional rate of exchange observed by Romero et al. during displacement of GDP by GTP appears to be dependent on the ratio of the rate constants (k-1 + k-2)k4/k1k-2 as defined in the text, not on that of K4/K1 as they proposed.

Guanine Nucleotides↗
PubMed1990

Kinetic modelling of the effect of alpha subunit phosphorylation on the activity of the protein synthesis initiation factor eIF-2.

The ability of eIF-2.GDP in which the alpha subunit of eIF-2 is phosphorylated (eIF-2(alpha P).GDP) to act as a competitive inhibitor of eIF-2B-catalysed exchange of eIF-2-bound GDP has been investigated by modelling data provided by Rowlands et al. (J. Biol. Chem. 263, 5526-5533:1988). Some revision of previously determined dissociation and rate constants proved to be necessary. Under the conditions employed it was not possible to demonstrate significant inhibition of GDP exchange by eIF-2 (alpha P).GDP without substantial increase in its affinity for eIF-2B over that of eIF-2.GDP. Classic double reciprocal plots for competitive inhibition were found only when [eIF-2B] was low in relation to [eIF-2 (alpha P).GDP]. Relatively high cellular [eIF-2B] lessens the inhibitory effect of eIF-2(alpha P).GDP and suggests the possibility of other potential controls of initiation.

Eukaryotic Initiation Factor-2↗
PubMed1990

Kinetic parameters governing the formation of eIF-2.methionyl-tRNAi complexes in protein synthesis.

Published data have been analysed to determine the rate constants governing the exchange of GDP in the complex of the eukaryotic protein synthesis initiation factor eIF-2 with GDP, catalysed by eIF-2B. The interaction of eIF-2B with eIF-2.GDP appears to include a very high 'on' rate constant of up to 4 x 10(8) M-1 sec-1 - a value very similar to that found by others for the interaction of the bacterial elongation factors Tu and Ts. Assuming a substituted enzyme mechanism that leads to displacement of GDP and ultimately to formation of a quaternary complex eIF-2B.eIF-2.GTP.methionyl-tRNA, minimum rate constants have been estimated for the additional reactions assuming in vivo rates of protein synthesis. Rate constants for the other reactions are unexceptional.

Chemical Phenomena↗
PubMed1989

Transfer-RNA, magnesium and the formation of ternary complexes by eukaryotic initiation factor eIF-2.

The likely concentrations of free magnesium ions in assay systems measuring ternary complex formation with the eukaryotic initiation factor eIF-2 and the exchange of bound GDP have been calculated. Contrary to the suggestion of Roy et al. (Biochem. Biophys. Res. Commun. 146, 114-120) amounts of added tRNA are unlikely to sequester sufficient magnesium ions to affect significantly their role in the assays. There seems little correlation between methionyl-tRNA added and the extent of ternary complex formation in published data other than that expected from mass action. In vivo the concentration of methionyl-tRNA is probably greater than or equal to that of eIF-2 which is shown to be necessary for efficient functioning of the initiation factor.

Chemical Phenomena↗
PubMed1988

Role of polyamines and intracellular pH change in the hypertrophy of the denervated rat hemidiaphragm.

The transient hypertrophy of the denervated rat hemidiaphragm is associated with a rise in concentrations of putrescine and spermidine. Hypertrophy still occurs following denervation in rats injected with difluoromethylornithine which reduces the increase in spermidine and particularly putrescine levels. Administration of isobutylmethylxanthine tended to raise putrescine concentrations in the denervated but not innervated tissue. No evidence could be found that the enhanced protein synthesis rate in the denervated tissue is associated with an increase in intracellular pH.

1-Methyl-3-isobutylxanthine↗
PubMed1987

eIF-2B and the exchange of guanine nucleotides bound to eIF-2.

Available data for the formation of the ternary complex eIF-2 X GTP X methionyl-tRNAi involved in eukaryotic initiation and of the inhibition of ternary complex formation by GDP have been examined with a view to determining the mechanism by which eIF-2B facilitates nucleotide exchange. Two mechanisms have been considered--first a displacement reaction in which eIF-2B displaces GDP and GTP in a manner analogous to a "ping-pong" enzyme mechanism, and secondly the possibility that binding of eIF-2B to eIF-2 nucleotide complexes enhances the rate of nucleotide exchange without itself inducing nucleotide displacement. Comparison has been made between the properties of eIF-2 and eIF-2B and of the bacterial elongation factors Tu and Ts. It seems most probable that, as previously suggested by others for Ts, eIF-2B effectively catalyses an exchange reaction through a "ping-pong" type mechanism. Possible explanations of data suggesting otherwise are put forward. Both eIF-2 and bacterial Tu are complex allosteric proteins subject to a variety of influences which in the case of eIF-2 include phosphorylation of the alpha subunit. This phosphorylation appears to change the equilibria in the reaction mechanism such that the transferred entity (eIF-2) becomes firmly bound to the catalyst (eIF-2B). Minimum rate constants for the formation of eIF-2 X eIF-2B from eIF-2 X GDP and eIF-2 X GTP and reverse reactions are derived. These values suggest that the initiation factors are likely to have to operate in a restricted environment if rates of protein synthesis seen in vivo are to be sustained.

Eukaryotic Initiation Factor-2↗