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Biomedical subjects

B F Clark

Publications and source records attributed to B F Clark.

At least 73 records · Page 4Linked to original sources
PubMed1994

Kinetin delays the onset of ageing characteristics in human fibroblasts.

Kinetin (Kn) is a synthetic cytokinin plant growth hormone having some senescence-retarding effects in plants. Its effects on animal cells have been, until now, best considered as growth inhibitory and anti-tumorigenic. However, we have observed that an addition of 40-200 microM Kn in the culture medium of human diploid fibroblasts can both delay the onset and decrease the extent of many of the ageing characteristics that appear during serial passaging of normal cells in culture. Age-related changes that are affected by Kn include morphological alterations, growth rates, cell size, cytoskeletal organisation, macromolecular synthesis and the intensity of autofluorescence due to the oxidative damage product lipofuscin. These anti-ageing effects of Kn are achieved without any increase in the cell culture lifespan in terms of maximum proliferative capacity in vitro.

Adenine↗
PubMed1994

Dual hydrolytic role for Pb(II) ions.

RNA phosphodiester bonds can be cleaved by metal ions, of which Pb2+ is one of the most effective. It can cleave both generally and site-specifically, depending on the substrate and the conditions. In addition, metal ions are also known to cleave ester bonds between amino acid and the 3'-end of transfer RNA. Here we report that in aminoacylated transfer RNA, Pb2+ ions cleave internucleotide bonds in the 3'-end of tRNA and also cleaves the bond between tRNA and its amino-acid, attached at the 3'-end via an ester bond to the terminal ribose in aminoacyl tRNA. The two reactions proceed at different rates. The rate of deacylation is significantly faster than the rate of cleavage of phosphodiester bonds, with a pH-optimum of 7. This dual hydrolytic role is not seen for other metal ions examined, namely Zn(II), Cd(II) and Mn(II). The rate of the two kinds of hydrolyses by Pb2+ ions is compared with that of other metal-ions. The mechanism of cleavage is investigated further by modification of the 3'-end of tRNA.

Acylation↗
PubMed1994

Towards an understanding of structure-function relationships of elongation factor Tu.

In light of the recently determined structure of elongation factor Tu, and taking into account chemical studies mapping functional sites, a number of residues have been selected for site-directed mutagenesis studies. Gly94, Gly126, His66, His118, Lys89 and Asp90 have each been point-mutated. Preliminary in vitro characterization data are presented.

Amino Acids↗
PubMed1993

Tissue-dependent variation in the expression of elongation factor-1 alpha isoforms: isolation and characterisation of a cDNA encoding a novel variant of human elongation-factor 1 alpha.

A novel isoform of human elongation factor-1 alpha (EF-1 alpha 2) has been characterised. It shows a high similarity to other EF-1 alpha proteins, especially to a rat EF-1 alpha variant and it has all the characteristics of a functional EF-1 alpha protein. The pattern of expression of both EF-1 alpha 2 and EF-1 alpha was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF-1 alpha 2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF-1 alpha was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.

Amino Acid Sequence↗
PubMed1993

Elongation factor 2-specific calcium and calmodulin dependent protein kinase III activity in rat livers varies with age and calorie restriction.

Elongation factors involved in polypeptide chain elongation are considered to be rate limiting for the slowing down of total protein synthesis during ageing. The activities of elongation factors are themselves regulated by various means, including phosphorylation. Here we have compared the activity of a protein kinase, called calcium- and calmodulin-dependent protein kinase III (CaM PK III), specific for the phosphorylation of elongation factor eEF-2, in cell-free extracts prepared from livers isolated from young and old male Fischer 344 rats maintained under freely fed or calorie-restricted dietary regimes. There was a significant increase of more than 70% in the activity of CaM PK III in 24 month old freely fed rat livers as compared with young animals. This age-related increase was found but to a lower extent (46%) in calorie-restricted rats of the same age. Therefore, slowing down of ageing in calorie-restricted animals is also reflected at the level of the regulation of the activity of protein elongation factor eEF-2 by CaM PK III.

Aging↗
PubMed1993

Reduction in the amount of 8-hydroxy-2'-deoxyguanosine in the DNA of SV40-transformed human fibroblasts as compared with normal cells in culture.

DNA damage due to oxidative free radicals is considered to be a major cause of ageing and age-related diseases including cancer. Of more than 20 modifications formed in DNA by the action of hydroxyl radicals, 8-hydroxy-2'-deoxyguanosine (oh8dG) is potentially highly mutagenic and is known to occur most frequently. Using HPLC combined with electrochemical (HPLC/EC) detection of oh8dG, fivefold higher levels of oh8dG are detected in the DNA of cultured normal human skin fibroblasts as compared with SV40-transformed human fibroblasts MRC-5V2. In comparison, the levels of oh8dG were similar in the growth medium of both types of cells. Applications of this method range from studies on the genomic stability and instability of normal and cancerous cells to the clinical and laboratory testing of toxic substances and drugs.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Phorbol ester PMA stimulates protein synthesis and increases the levels of active elongation factors EF-1 alpha and EF-2 in ageing human fibroblasts.

Phorbol esters modulate gene expression, reorganize the cytoskeleton and stimulate bulk protein synthesis and the steps of initiation and elongation. We have observed that a phorbol ester PMA stimulates protein synthesis and increases the amounts of active elongation factors, EF-1 alpha and EF-2 in cultured human fibroblasts MRC-5 undergoing ageing. Although bulk protein synthesis slows down during ageing, the cellular response to the stimulatory effects of PMA is higher in senescent cells. Similarly, despite the age-related decline in the amounts of active EF-1 alpha and EF-2, senescent cells exhibit a higher response to PMA. The results indicate an age-dependent increase of cellular responsiveness to PMA and provide evidence about both the integrity of the translational apparatus and the effectiveness of the signal transduction pathways during cellular ageing. In comparison, the effects of PMA on SV40-transformed MRC-5V2 cells were minimal.

Aging↗
PubMed1993

Altered lead(II)-cleavage pattern of free Phe-tRNAPhe and Phe-tRNAPhe in ternary complex with EF-Tu:GTP.

Pb2+ ions in sub-millimolar concentrations are known to cleave internucleotide bonds of phenylalanine-specific transfer RNA (tRNAPhe) from Saccharomyces. cerevisiae specifically between nucleotides D17 and G18 in the D-loop, with additional minor cleavages after D16 and G15. This makes lead(II) a sensitive structural probe for correct folding of tRNAPhe. In the present paper we use Pb2+ ions as a functional probe to determine whether this part of tRNA is protected by the Escherichia coli elongation factor EF-Tu in the ternary complex formed between Phe-tRNAPhe and EF-Tu.GTP. Our results show that for tRNA in complex with EF-Tu:GTP, the phosphodiester bond after D17 is cleaved, yet the phosphodiester bonds after D16 and G15 are not. To our knowledge, this is the first time that Pb2+ ions, bound at a specific site in tRNA, have been used both to investigate the correct folding of tRNA in complex, and to footprint a functional complex with components whose individual structures are known.

Autoradiography↗
PubMed1992

Protein synthesis, posttranslational modifications, and aging.

Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.

Adenosine Diphosphate Ribose↗
PubMed1992

Substitution of aspartic acid-80, a residue involved in coordination of magnesium, weakens the GTP binding and strongly enhances the GTPase of the G domain of elongation factor Tu.

The functional role of Asp80, a residue involved in the coordination of the Mg(2+).guanine nucleotide complex in elongation factor Tu (EF-Tu), has been investigated by its substitution with Asn in the isolated N-terminal domain (G domain). The G domain D80N is characterized by a strong decrease in binding affinity for GTP and magnesium, whereas the affinity for GDP is unchanged. This effect can be mimicked in wild-type G domain by the addition of EDTA. In contrast to this, EDTA does not essentially influence the selective effects of the mutation on the GTP and GDP binding of G domain D80N, indicating that the action of Asp80 is mainly mediated by the GTP-coordinated magnesium ion. The GTPase activity of the G domain D80N is very unstable, but can be markedly stabilized by the addition of glycerol without essentially modifying the specific effects of the mutation. In the absence of glycerol G domain D80N can express a short-lived GTPase activity. The presence of glycerol transforms this evanescent activity into a linear multiple-round activity that under optimal conditions can be almost 2 orders of magnitude higher than the GTPase of wild-type G domain. This enhanced catalytic activity represents the most striking consequence of the mutation and stresses the key role of Asp80 in the GTPase of EF-Tu.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Phorbol ester-induced reorganization of the cytoskeleton in human fibroblasts during ageing in vitro.

Phorbol esters induce drastic morphological alterations in cells of different origin by altering the conformation and the interrelationship of the elements of the cytoskeletal system. Treatment of early passage (young) and late passage (senescent) human fibroblasts MRC-5 with phorbol-12-myristate-13-acetate (PMA) results in the rearrangement of actin and tubulin filaments. PMA brings about the disorientation and diffusion of the heavily criss-crossed network of actin and microtubulin fibres characteristic of senescent cells suggesting thereby an increased sensitivity of senescent cells to phorbol esters. Since phorbol esters are known to be specific activators of protein kinase C (PKC), the PMA-induced modulation of the cytoskeleton patterns in ageing fibroblasts provides further support for the view that the effectiveness of the signalling mechanisms is retained during cellular ageing.

Actins↗
PubMed1992

Specific incorporation of kinetin into eukaryotic and prokaryotic transfer ribonucleic acid molecules.

We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase. We also show that kinetin is specifically incorporated into E. coli tRNA(Tyr) and most probably at position 37. To our knowledge, this is the first report of a nucleic acid base exchange reaction occurring at this position.

Adenine↗
PubMed1992

Senescent human fibroblasts are more sensitive to the effects of a phorbol ester on macromolecular synthesis and growth characteristics.

4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular growth properties by modulating gene expression in a wide variety of cell types. Human diploid fibroblasts MRC-5 were treated with PMA at different phases of their lifespan in vitro and the alterations of their short-term growth characteristics and macromolecular synthesis in response to PMA were analysed. PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III (senescent) MRC-5 cells. Treatment of senescent cells with various PMA concentrations results in a greater stimulation of DNA and RNA synthesis than that in young cells. Senescent cells are also more sensitive to the PMA-induced alterations of growth characteristics and higher concentrations of PMA become toxic for them. The age-related alterations of cellular responsiveness are also apparent in the gradual loss of responsiveness to serum, observed in parallel with the increased sensitivity to PMA. Furthermore, serum-induced stimulation of macromolecular synthesis is inhibited by PMA. Since it is known that serum and PMA elicit their effects via different signal transduction pathways, our results point to suggest the differential regulation of the signalling mechanisms during cellular ageing.

Cell Division↗
PubMed1992

One-step purification of E. coli elongation factor Tu.

The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one-step procedure. The construct was designed in order to make possible the release of authentic EF-Tu by cleaving the fusion protein with the protease factor Xa.

Amino Acid Sequence↗
PubMed1991

Dietary calorie restriction does not affect the levels of protein elongation factors in rat livers during ageing.

Dietary calorie restriction of rats has been previously shown to increase protein synthetic rates in liver and kidney cells during ageing. Here we have compared the activity and amounts of active elongation factors EF-1 alpha and EF-2 in cell-free extracts prepared from livers isolated from male Fischer 344 rats of different ages. Although there is some age-related decline in the catalytic activity and amounts of active EF-1 alpha during ageing, no differences between freely-fed and calorie-restricted animals were observed. In the case of EF-2, the amounts of ADP-ribosylatable EF-2 neither declined during ageing nor differed between freely-fed and calorie-restricted animals. Thus differences in the protein synthetic rates in calorie-restricted and freely-fed rats are not reflected at the level of protein elongation factors, and may involve some other mechanisms of regulation.

Aging↗
PubMed1991

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA.

Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

42Sp48 in previtellogenic Xenopus oocytes is structurally homologous to EF-1 alpha and may be a stage-specific elongation factor.

We have isolated the cDNA for 42Sp48 and EF-1 alpha from mixed stage oocytes and tailbud (stage 22) Xenopus laevis cDNA libraries by use of the cDNA for human elongation factor-1 alpha (EF-1 alpha) as probe. The nucleotide and deduced amino acid sequences of the entire coding region of 42Sp48 and EF-1 alpha cDNA were established. The proposed functional homology of the proteins is reflected in highly conserved amino acid sequences (91% identity), while the large number of silent mutations at the gene level may serve to prevent recombination at their loci. 42Sp48 is apparently encoded by two genes in Xenopus, while no sequences corresponding to 42Sp48 could be found in murine or human genomic DNA. 42Sp48 has been proposed to act as a stage-specific elongation factor in Xenopus. Comparison of the deduced amino acid sequences of 42Sp48 and EF-1 alpha with that of elongation factor Tu from E. coli, for which the three-dimensional structure including that of the GTP binding sites have been determined, supports this hypothesis.

Amino Acid Sequence↗