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Biomedical subjects

E Griffiths

Publications and source records attributed to E Griffiths.

At least 73 records · Page 4Linked to original sources
PubMed1989

Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen.

One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.

Antigens, Bacterial↗
PubMed1988

Iron-regulated outer-membrane proteins of Escherichia coli strains associated with enteric or extraintestinal diseases of man and animals.

The SDS-PAGE patterns of the iron-regulated outer-membrane proteins from 70 strains of Escherichia coli isolated from various human and animal infections were analysed and the nature of the siderophores produced was examined. Iron-regulated 81 kDa and 74 kDa protein bands seen in SDS-PAGE gels were characterized further by immunoblotting using anti-81 kDa and anti-74 kDa (Cir) sera. The results showed considerable differences between the patterns of the iron-regulated outer-membrane proteins exhibited by the different strains. Nevertheless, three distinct and characteristic profiles, based on the most prominent bands expressed, could be identified, although not all strains produced patterns which matched with one of these. These results suggest the possibility of using the pattern of iron-regulated outer-membrane proteins expressed, as well as siderophores produced, as a new set of markers to characterize groups of pathogenic E. coli.

Animals↗
PubMed1986

Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron.

Iron restriction was induced in Escherichia coli O 111, E. coli O 164 and E. coli C by growing the organisms in trypticase soy broth containing ovotransferrin, desferal, EDDA (ethylenediamine-dihydroxyphenylacetic acid) or alpha,alpha'-dipyridyl. There were marked qualitative and quantitative differences in the iron regulated outer membrane proteins expressed in the presence of the various iron chelators. Differences in the kinetics of growth were also noted. E. coli C was devoid of a ferric enterobactin iron uptake system.

2,2'-Dipyridyl↗
PubMed1985

Inhibition of uricase by pyrimidine and purine drugs.

A number of barbiturates inhibit uricase from porcine liver competitively, the Ki, values being in the range 1 to 13 X 10(-3) mol/L. In general the Ki increases as the size of the substituents at C-5 increases. Theophylline also inhibits the enzyme competitively (Ki = 1 X 10(-3) mol/L) but theobromine and caffeine have no inhibitory action at 0.01 mol/L. A number of sulphonamides and some other heterocyclic compounds, at 10(-3) mol/L, do not inhibit uricase. The possible effects of such inhibition in clinical assays for plasma/serum urate are discussed but it is concluded that the compounds which were studied are unlikely to cause significant interference in the assay.

Animals↗
PubMed1985

Low-molecular-weight iron complexes and oxygen radical reactions in idiopathic haemochromatosis.

The sera of patients with idiopathic haemochromatosis and iron-overload have been found to contain low-molecular-weight iron complexes detectable in the 'bleomycin assay'. These complexes stimulate both the peroxidation of membrane lipids and the formation of the highly reactive and damaging hydroxyl radical. The iron chelator desferrioxamine interferes with these reactions. We suggest that oxygen radical reactions stimulated by iron salts are important in the pathology of idiopathic haemochromatosis.

Bleomycin↗
PubMed1985

Antigenic alteration of contaminating lipopolysaccharide during extraction of Escherichia coli outer-membrane proteins from polyacrylamide gels.

An antiserum raised to the ferric enterobactin receptor protein of Escherichia coli, isolated from SDS-polyacrylamide gels, contained high-titre antibodies to the lipopolysaccharide (LPS) of E. coli O111. This antiserum was used to show that proteins dissected from polyacrylamide gels can be contaminated with comigrating LPS at levels below those detectable by very sensitive silver staining methods. Using this antiserum it was also shown that the procedures used to extract proteins from polyacrylamide gels can alter the molecular structure and, consequently, the antigenic properties of the contaminating LPS.

Antigens, Bacterial↗
PubMed1985

Antigenic and molecular homology of the ferric enterobactin receptor protein of Escherichia coli.

The ferric enterobactin receptor protein (81 kDal) of Escherichia coli O111 was purified by preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis and used to raise polyclonal antiserum in rabbits. This antiserum was used in conjunction with the immunoblot technique to examine the degree of antigenic homology of the ferric enterobactin receptor protein among 17 pathogenic and laboratory strains of E. coli. Both the molecular weight and the antigenic properties of the enterobactin receptor were highly conserved. However, the laboratory strain C and a pathogenic enteroinvasive strain, E. coli O164, were unusual in not producing the 81 kDal protein. The antiserum also recognized an 81 kDal protein from iron-restricted Salmonella typhimurium and an 83 kDal protein from iron-restricted Klebsiella pneumoniae.

Antibodies, Bacterial↗
PubMed1985

Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coli.

Sera from normal healthy human adults and infants, as well as sera from mice, rabbits, and guinea pigs, were examined by immunoblotting for naturally occurring antibodies reacting with outer membrane proteins of two Escherichia coli strains, O111 and O18. Some individuals had antibodies reacting very strongly with the iron-regulated outer membrane proteins, including the ferric-enterochelin receptor protein (Mr, 81,000), as well as with ompA. However, sera from infants contained predominantly antibodies to ompA; antibodies recognizing the iron-regulated outer membrane proteins were either absent or barely detectable. In human serum the antibodies were mainly of the immunoglobulin G class. No serotype-specific antibodies to the lipopolysaccharide of E. coli O111 or O18 were found in the sera tested.

Antibodies, Bacterial↗
PubMed1985

Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12-Shigella flexneri hybrids and by enteroinvasive strains of Escherichia coli.

One of the chromosomal segments associated with the virulence of Shigella flexneri and transferred to Escherichia coli K-12 by conjugation has been shown to code for the production of aerobactin and for the synthesis of an iron-regulated 76,000-dalton (76K) outer membrane protein. Analysis of various E. coli K-12-S. flexneri transconjugants showed that the genes involved with the synthesis of aerobactin and with the production of the 76K protein were linked to the mtl region of the S. flexneri chromosome. S. flexneri itself synthesized a 76K protein in its outer membrane under iron restriction as well as traces of 81K and 74K proteins. An examination of four enteroinvasive strains of E. coli showed that each produced aerobactin and a 76K outer membrane protein during iron-restricted growth. The profile of the iron-regulated proteins expressed by the enteroinvasive strains of E. coli was virtually identical to that expressed by the laboratory-constructed E. coli K-12-S. flexneri hybrids under the same growth conditions.

Animals↗
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↗