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Biomedical subjects

E Griffiths

Publications and source records attributed to E Griffiths.

At least 91 records · Page 5Linked to original sources
PubMed1985

The control of biological medicinal products produced by recombinant DNA technology.

Biological products are considered to be those that cannot be completely characterized by chemical and physical methods. The control of biological medicinal products made by recombinant DNA technology, like those produced by established techniques, is thought to require attention to "in-process' control. This offers an economical and efficient way of assessing the quality of the end product. Evaluation of the starting materials and the manufacturing process may therefore be as relevant to ensuring the safety, purity and potency of biologicals as tests carried out on the final product. Of course, identification and characterization of the product will be essential and should encompass several different physico-chemical properties of the molecule. As yet there is little experience of recombinant DNA technology on a manufacturing scale or of the control problems which may be encountered. Currently proposed requirements should therefore be regarded as flexible and subject to alteration as experience of the production and use of these substances increases.

Animals↗
PubMed1983

Cell killing by lysosomotropic detergents.

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.

Animals↗
PubMed1982

Iron mediated methylthiolation of tRNA as a regulator of operon expression in Escherichia coli.

E. coli growing in the presence of iron-binding proteins produced tRNAtrp and tRNAphe molecules containing i6A instead of ms2i6A adjacent to the anticodon. These undermodified tRNAs functioned less efficiently than the fully modified molecules when translating synthetic polynucleotides containing contiguous codons in an in vitro system, but did not limit the translation of MS2 RNA. We examined the possibility that the altered tRNAs with lowered translational efficiencies could relieve transcription termination at the trp and phe attenuators and lead to increased operon expression under iron restricted conditions. Using trpR mutants we found that there was indeed greater expression of the trp operon during iron restricted growth. This increase was attributable solely to the tRNA alteration induced by iron restriction.

Conalbumin↗
PubMed1981

Iron-related modification of bacterial transfer RNA.

Transfer RNAs isolated from E. coli grown in media where ferric iron is not freely available show well characterized chromatographic changes due to the absence of the methylthio moiety of ms2i6A. The altered tRNA molecules include tRNA trp tRNA tyr, tRNA phe and two minor tRNA ser species. It has been suggested that methylthiolation of tRNA affects its function in regulation. We now show iron-related changes in tRNA trp from S. typhimurium, Ps. aeruginosa and K. pneumoniae. tRNA trp from S. typhimurium contains ms2i6A and it seems probable that the availability of iron affects the synthesis of ms2i6A-tRNA trp from i6A-tRNA trp in this organism. An iron-related methylthiolating system may also be operative in K. pneumoniae. S. marcescens tRNA trp, however was not affected by the availability of iron. Neither ms2i6A nor i6A was found in S. marcescens tRNA, although an, as yet unidentified, hydrophobic nucleoside was present.

Bacteria↗
PubMed1981

Regulation of aromatic amino acid transport by tRNA: role of 2-methylthio-N6-(delta2-isopentenyl)-adenosine.

E. coli growing rapidly in media where ferric iron is not freely available contain a population of specifically undermodified tRNAs. These tRNAs contain isopentenyl adenosine instead of the usual methylthioisopentenyl adenosine adjacent to the 3' end of the anticodon. Iron restricted E. coli also show an enhanced capacity to transport aromatic amino acids into the cell. Our work shows that undermodified tRNAs for phe, tyr and trp can function as positive regulatory elements of the aromatic amino acid transport system in E. coli. This iron related metabolic control, mediated through a specific post-transcriptional modification of the tRNAs, may be an important mechanism for adapting E. coli for growth in an iron restricted environment.

Adenosine↗
PubMed1978

Alterations in tRNAs containing 2-methylthio-N6-(delta2-isopentenyl)-adenosine during growth of enteropathogenic Escherichia coli in the presence of iron-binding proteins.

Escherichia coli grown in chemically produced iron-deficient media have well characterized alterations in the chromatographic properties of tRNAs containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl) adenosine. The present report shows that similar tRNA alterations occur in enteropathogenic E. coli inhibited by human milk and bovine colostrum, the inhibited bacteria containing 10% or less of the normal tRNA species. Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in milk and colostrum reversed these changes which are probably due to a failure to methylthiolate the isopentenyladenosine. Although adding iron led to a rapid replacement of abnormal tRNA by the chromatographically normal species, and to a resumption of multiplication, the tRNA alterations are not directly related to the inhibition of growth. Strains of E. coli which grew normally in milk, colostrum and in defined media containing the iron-binding protein transferrin or ovotransferrin also contained about 90% of the abnormal species. Rapid conversion of abnormal tRNA to normal tRNA occurred on adding iron and in the absence of RNA synthesis. The tRNA changes are discussed in relation to their possible connection with both the adaptation of E. coli to growth under the iron-restricted conditions imposed by iron-binding proteins in tissue fluids and with bacterial pathogenicity.

Adenosine↗
PubMed1978

Alterations in the tRNA's of Escherichia coli recovered from lethally infected animals.

Escherichia coli grown in chemically defined iron-deficient media or in fluids containing the iron-binding proteins transferrin, lactoferrin, or ovotransferrin have well-characterized alterations in the chromatographic properties of tRNA's containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl)-adenosine. The present work shows that similar tRNA alterations occur in E. coli O111 recovered from the peritoneal cavities of lethally infected guinea pigs and rabbits. Adding iron to these in vivo-grown bacteria resulted in the rapid conversion of chromatographically abnormal tRNA's to the normal species. The work strongly suggests that host iron-binding proteins, present in mucosal and other secretions, can affect the metabolism of invading organisms. The idea that the tRNA alterations are connected with the adaptation of E. coli to growth under the iron restricted conditions imposed by iron-binding proteins in tissue fluids, and thus with bacterial pathogenicity, is therefore made particularly attractive.

Animals↗
PubMed1977

Bacteriostatic effect of human milk and bovine colostrum on Escherichia coli: importance of bicarbonate.

At pH 7.4 and in the presence of NaHCO3, human milk and bovine colostrum inhibited the growth of Escherichia coli O111. Adding sufficient iron to saturate the iron-binding capacity of the lactoferrin present in the milk or colostrum prevented bacteriostasis. At pH 6.8 neither molk nor colostrum inhibited E. coli 0111. Adjusting the pH to 7.4 with NaHCO3 resulted in the development of bacteriostatic activity. Adjusting the pH to 7.4 with NaOH was ineffective. Dialyzed colostrum and milk inhibited bacterial growth at pH 6.8 in the absence of added NaHCO3; addition of citrate or iron abolished bacteriostasis. The chromatographic elution profile of tyrosyl-transfer ribonucleic acid (tRNA) from iron-replete E. coli differs significantly from that of tyrosyl-tRNA from iron-deficient organisms. Examination of the elution profile tyrosyl-tRNA from E. coli 0111 growing in colostrum without added NaHCO3 showed that such bacteria were fully replete in iron. The nature of the elution profile of tyrosyl-tRNA also showed that iron was freely available to the bacteria when citrate was added to dialyzed colostrum but not available in its absence, even at pH 6.8. Results support the idea that the bacteriostatic action of milk and colostrum, due to the combined action of antibody and lactoferrin, depends on the addition of bicarbonate to counteract the iron-mobilizing effect of the citrate normally present in these secretions.

Animals↗
PubMed1976

The effect of antipolymorphonuclear leucocyte serum on Pseudomonas aeruginosa infection in rabbits.

Studies were made on the rate of phagocytosis and killing of Pseudomonas aeruginosa by phagocytic cells in the peritoneal cavity of rabbits. In sublethal and lethal infections the phagocytosed bacteria were killed very quickly. In antibody-protected animals, the polymorphs became loaded with liveing bacteria, but this had little effect on the decline in infection. In sublethal infections and in protected animals theproportion of intracellular bacteria labelled with 32O or [14C]uracil was high and antibody greatly enhanced phagocytosis. In lethal infections the rate of phagocytosis was insufficient to prevent the development of a fatal septicaemia. Antipolymorphonuclear leucocyte serum (APS) completely suppressed the normal polymorph response to infection and greatly reduced resistance. The macrophages in the peritoneum, which were not affected by APS, delayed bacterial growth for several hours but were eventually unable to control bacterial mutiplication. The outcome of infection appeared to depend almost entirely on the ratio of bacterial to phagocytes and the presence of antibody. Iron-binding proteins probably make a significant contribution to resistance by reducing the rate of multiplication of extracellular bacteria.

Animals↗
PubMed1975

Effect of pH and haem compounds on the killing of Pasteurella septica by specific antiserum.

The killing of Pasteurella septica by horse antiserum has features not previously associated with serum bactericidal reactions. The present work showed that lowering the pH from 7-4 to 6-8 abolished the action of antiserum. The bactericidal effect and the degradation of RNA seen when antiserum is added to P. septica growing in horse serum, were abolished at pH 6-8 in much the same way as when haem compounds were added to the system. Addition of chloramphenicol, rifampicin or puromycin to P. septica growing apparently normally in antiserum at pH 6-8 or in antiserum containing haem compounds led to rapid killing of the bacteria and to degration of their RNA. Addition of these antibiotics to P. septica growing in normal serum produced only bacteriostasis and did not induce RNA breakdown. In contrast, nalidixic acid, although inhibiting growth, did not induce rapid killing and RNA breakdown under the same conditions. These findings were unexpected and led to a reassessment of ideas concerning the mechanism of action of specific antiserum to P. septica. Although iron compounds clearly abolish the bactericidal based simply on an interference with bacterial iron supply is no longer sufficient. The process is more complex and must involve other factors.

Antibodies, Bacterial↗