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Biomedical subjects

R H Burdon

Publications and source records attributed to R H Burdon.

At least 37 records · Page 2Linked to original sources
PubMed1989

Cell proliferation and oxidative stress.

Antioxidants such as mannitol, butylated hydroxytoluene and alpha-tocopherol enhance the growth of polyoma virus transformed and non-transformed BHK-21 cells. In the case of mannitol this is observed even in the absence of added calf serum. In part these effects may operate to protect cellular growth control mechanisms. On the other hand oxidants such as H2O2 and t-butyl hydroperoxide can inhibit growth and overall cellular protein synthesis, through mechanisms that are likely to involve radicals. In the case of H2O2 the inhibitory effects can nevertheless be reduced by 'prestressing' the cells with mild heat or with H2O2 itself. Paradoxically very low concentrations (10(-8) M) of H2O2 or t-butyl hydroperoxide can actually stimulate cell growth, even in the absence of serum. These stimulatory effects however do not appear to involve radicals as they are enhanced by inclusion of mannitol or DMSO in the medium.

Amino Acids↗
PubMed1988

Hyperthermic toxicity and the modulation of heat damage to cell protein synthesis in HeLa cells.

That some of the effects of hyperthermia on HeLa cell protein synthesis may involve free radical activity is suggested by experiments with free radical scavengers. A possible source of damaging free radicals induced by heat may lie in peroxidative events within lipid-containing membranes. These can be blocked by EGTA. Other experiments with t-butylhydroperoxide indicate that cycloheximide can also neutralise peroxidative effects. These effects are discussed in the light of observations that calcium depletion (or EGTA treatment) can induce thermotolerance and that cycloheximide does not prevent the development of thermotolerance.

Benzaldehydes↗
PubMed1987

Oxidative stress and heat shock protein induction in human cells.

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis. Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.

Ditiocarb↗
PubMed1987

Temperature and animal cell protein synthesis.

A predominant feature of brief heat stress to animal cells is the vigorous but transient activation of a small number of specific genes, previously either silent, or active at low levels. New mRNAs are actively transcribed from these genes and are translated into proteins, known collectively as the heat shock proteins, or hsps. The number of different types of hsp varies considerably in different organisms and cell types but in all cases proteins of approximately 84 and 70 kDa are amongst the most prominent. A dramatic feature that emerges from a study of their genes is that these proteins have been highly conserved during evolution. Gene sequence data reveal specific nucleotide sequences upstream of the transcription start sites that are essential for induction. These are known as 'heat shock elements' and are believed to be the region to which activated 'heat shock transcription factors' bind to facilitate hsp gene transcription. A recent model suggests that transcriptional regulation is based on competition between abnormal intracellular proteins and such a labile regulatory factor. Other experiments suggest such hsp inducers as heat, ethanol, arsenite, or oxygenation after anoxia, may cause protein damage through oxygen-derived free radical action. Although heat shock can cause very considerable changes in transcriptional patterns, effects specifically on translational control are no less dramatic. Different organisms achieve a rapid change in different ways. In Drosophila, heat shock promotes the translation of hsp mRNAs. In yeast there is no mechanism for sequestering pre-existing mRNAs from translation. Instead these mRNAs simply disappear rapidly from the cell. Mammalian cells are different in yet another way. There is neither sequestration of pre-existing mRNAs nor their removal from the cell. Superimposed upon the transient heat induced activation of hsp genes there are now clear indications of developmental regulation. The mechanism for the specific developmental control of hsp expression is not yet known. However, as oncogene products are known to induce hsp synthesis it may be that hsps are involved in eukaryotic growth control. Hypothermia causes loss of protein synthetic activity in cultured animal cells. No specific proteins are however induced and recovery at 37 degrees C is rapid.

Animals↗
PubMed1987

Thermotolerance and the heat shock proteins.

Mammalian cells can dramatically increase their tolerance to thermal damage after prior heat conditioning. The thermal history, the heat fractionation interval and the recovery conditions, all modify significantly the degree of thermotolerance exhibited. Several lines of evidence have suggested that perhaps that shock proteins (hsps) provide the protective mechanism. For example by following the synthesis and degradation of heat shock proteins during development and decay of thermotolerance, strong circumstantial evidence has been obtained in certain cases that hsps are involved in the acquisition, maintenance and decay of thermotolerance. The levels of certain heat shock proteins, particularly the class at 68-70 kDa, can also correlate with thermotolerance. On the other hand these correlations do not always hold. Moreover cycloheximide treatment during the heat shock does not appear to block the development of thermotolerance. In addition depletion of medium Ca2+ which also inhibits hsp induction can produce thermotolerance. However it should be emphasized that whilst hsp synthesis is elevated after heat shock, it is clear that there are low level of hsps, always present in unheated cells, which may be sufficient to confer tolerance. Other data now show that thermotolerance measured in terms of cells survival is closely parallel by thermal resistance of total protein synthesis. Moreover it is possible to demonstrate the development of thermotolerance in the total protein synthetic activity of Hela cells, either held continuously at 42 degrees C or treated briefly at 45 degrees C and returned to normal growth temperature. This development of tolerance in the total protein synthetic activity is nonetheless reduced by actinomycin D and a role for the nucleolus is suggested. The properties of hsps that might be of significance in the generation of thermotolerance are examined. For example there appears to be a specific role of hsp 70 in aiding repair of heat damaged nucleoli. Another heat shock protein appears to be ubiquitin which is likely to have a role in the degradation of the abnormal proteins which are postulated to be involved in the transcriptional activation of the hsp genes themselves.

Adaptation, Physiological↗
PubMed1986

Mouse ascites DNA methylase: characterisation of size, proteolytic breakdown and nucleotide recognition.

We have purified the DNA methylase from mouse ascites tumour cells to a specific activity of 11,500 units per mg protein using denatured Micrococcus luteus DNA as methyl acceptor. Methyl groups are transferred to cytosines almost exclusively in CpG dinucleotides. The purified enzyme contains two polypeptides of molecular mass 185 and 160 kDa, and an antiserum raised in a rabbit to the purified enzyme specifically reacts with these two proteins in crude extracts. The two proteins can be partially separated by affinity chromatography when activity is associated with the 185 kDa protein which can be proteolytically degraded to give polypeptides of 170 and later 100 and 50 kDa. Only the 185 kDa methylase is lost when cells are treated with azadeoxycytidine and this is the predominant form firmly bound in the nucleus of dividing cells. Antibody bound to the 185 kDa band in protein blots will itself bind native DNA methylase, which can be detected by its binding 14C-labelled, azacytosine-containing DNA.

Animals↗
PubMed1986

Characterization of a human orphon 28 S ribosomal DNA.

We have isolated clones in which two regions of the human genome are represented, each containing an orphon: a dispersed copy of 28S rDNA. Nucleotide (nt) sequence analysis established that one of these, H28S-O1, corresponds to nt 3627-4105 of human 28S rDNA, but in a mutated form. The orphon was flanked on one side by a portion of the L1Hs long interspersed repeat family of the human genome. Although H25S-O1 is not flanked by the terminal direct repeats characteristic of transposed DNA, it is possible that it is a processed pseudogene.

Base Sequence↗
PubMed1985

Nuclear matrix-associated DNA methylase.

Previous procedures for the extraction of DNA methylase (EC 2.1.1.37) from nuclei of mouse ascites cells have involved the use of buffers containing 0.2M NaCl. Whilst such 'soluble' methylase accounts for the bulk (70-80%) of DNA methylase activity a further portion of activity is detectable in a 'bound' form firmly associated with 2 M NaCl-resistant nuclear matrix-like structures. This association, which in part requires continuing DNA replication and protein synthesis, can, however, be disrupted in vitro with high concentrations of ammonium sulphate, and the enzymic properties of the 'bound' form of DNA methylase are similar to those described for the 'soluble' form.

Ammonium Sulfate↗
PubMed1985

Delayed methylation and the matrix bound DNA methylase.

It is shown that the methylation of DNA that occurs in isolated nuclei is "delayed methylation". This methylation is not reduced in nuclei which have been pretreated with 0.2M NaCl to extract the soluble methylase suggesting that this methylation is the product of a firmly bound matrix associated DNA methylase. Evidence is provided that, like the methylase, the DNA substrate is associated with the nuclear matrix.

Animals↗
PubMed1984

Heat shock causes diverse changes in the phosphorylation of the ribosomal proteins of mammalian cells.

When HeLa cells or BHK cells were subjected to heat shock at 42 degrees C (for 2 h) or 45 degrees C (for 10 min) there was extensive dephosphorylation of ribosomal protein S6. Concomitantly ribosomal protein L14, which is not significantly phosphorylated in normal cells, became phosphorylated, as did a non-structural protein of Mr = 27000, associated with the ribosomes. The latter effects were not prevented by cycloheximide or actinomycin D. When cells shocked at 45 degrees C for 10 min were returned to 37 degrees C for 2 h there was rephosphorylation of ribosomal protein S6 and dephosphorylation of the 27 kDa protein, but not of ribosomal protein L14.

Animals↗
PubMed1984

Hyperthermia, Na+K+ATPase and lactic acid production in some human tumour cells.

When HeLa cells are exposed to brief heat shock at 45 degrees C there is a reduction in the cellular level of Na+K+ATPase. Return of the cells to the normal growth temperature of 37 degrees C leads to a partial restoration of enzyme activity. The pattern of this recovery of activity suggests that it may be associated with the induction of heat shock proteins. Indeed other means of heat shock protein induction such as continuous heat treatment at 42 degrees C, or treatment of cells at 37 degrees C with sodium arsenite, leads to elevated levels of Na+K+ATPase activity and alterations in the kinetic properties of the enzyme. Continuous hyperthermia at 42 degrees C led to increased lactate production which could be blocked with ouabain suggesting that effects on Na+K+ATPase activity could partly influence glycolysis. A number of other human and hamster cells also showed increased lactate production at 42 degrees C and also an inhibition of lactate production by ouabain. Whilst incubation of HeLa cells with cyanide had little effect on glycolysis at 37 degrees C elevation of the temperature to 42 degrees C (or 45 degrees C), in the presence of cyanide, impaired glycolysis. The possible role in this phenomenon, of an unusual oxygen-sensitive isoenzyme of lactate dehydrogenase, expressed in human cancers, is discussed.

Animals↗
PubMed1983

Stimulation of de novo methylation following limited proteolysis of mouse ascites DNA methylase.

The ability of mouse Krebs II ascites cell DNA methylase to add methyl groups to native, unmethylated DNA (de novo activity) is stimulated by limited proteolysis. The affinity of the enzyme for DNA is not altered by this treatment but the rate of reaction is increased so that 40% or more of methylatable sites are methylated within 4.5 h. The activation is associated with a decrease in size of the enzyme to 6.2 S.

Animals↗
PubMed1983

Methylation of satellite DNA.

The lower amount of 5 methylcytosine in DNA from bull sperm relative to DNA of other bovine tissues is a result of the absence of this minor base from several of the satellite DNAs in sperm. This applies particularly to the 1.715, 1.711b and 1.709 satellites and less so to the 1.706 and 1.711a satellites. Mouse sperm DNA is also partially undermethylated.

Animals↗
PubMed1982

HeLa cell messenger RNAs of widely differing sizes encode certain heat shock proteins in the Mr region of 72 000-74 000.

Cloned cDNA sequences coding for specific 72 000-74 000 Mr HeLa cell heat shock proteins (HSPs) have been used as probes to examine cytoplasmic and nuclear RNA of heat shocked HeLa cells. The mRNAs for these HSPs vary widely in size. Whilst the beta-, delta- and epsilon-HSPs are encoded in mRNAs of 1.9 kb, the gamma-HSP arises from an mRNA three times that size. A search for putative nuclear precursors to these mRNAs revealed high Mr candidates at 15.8 kb which accumulate 1-2 h after the initial heat shock, thus supporting the notion that transcriptional control is important in the initial heat shock response.

Centrifugation, Density Gradient↗