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J Mowbray

Publications and source records attributed to J Mowbray.

At least 19 recordsLinked to original sources
PubMed2006

Survival of a five-strain cocktail of Escherichia coli O157:H7 during the 60-day aging period of cheddar cheese made from unpasteurized milk.

The U.S. Food and Drug Administration Standard of Identity for Cheddar cheeses requires pasteurization of the milk, or as an alternative treatment, a minimum 60-day aging at > or =2 degrees C for cheeses made from unpasteurized milk, to reduce the number of viable pathogens that may be present to an acceptable risk. The objective of this study was to investigate the adequacy of the 60-day minimum aging to reduce the numbers of viable pathogens and evaluate milk subpasteurization heat treatment as a process to improve the safety of Cheddar cheeses made from unpasteurized milk. Cheddar cheese was made from unpasteurized milk inoculated with 10(1) to 10(5) CFU/ml of a five-strain cocktail of acid-tolerant Escherichia coli O157:H7. Samples were collected during the cheese manufacturing process. After pressing, the cheese blocks were packaged into plastic bags, vacuum sealed, and aged at 7 degrees C. After 1 week, the cheese blocks were cut into smaller-size uniform pieces and then vacuum sealed in clear plastic pouches. Samples were plated and enumerated for E. coli O157:H7. Populations of E. coli O157:H7 increased during the cheese-making operations. Population of E. coli O157:H7 in cheese aged for 60 and 120 days at 7 degrees C decreased less than 1 and 2 log, respectively. These studies confirm previous reports that show 60-day aging is inadequate to eliminate E. coli O157:H7 during cheese ripening. Subpasteurization heat-treatment runs were conducted at 148 degrees F (64.4 degrees C) for 17.5 s on milk inoculated with E. coli O157:H7 at 10(5) CFU/ml. These heat-treatment runs resulted in a 5-log E. coli O157: H7 reduction.

Animals↗
PubMed2000

The comparative content of the mitochondrial phosphoglyceroyl-ATP-containing polymer, purinogen, in rat tissues.

Here, we describe an assay for the tissue content of purinogen, a highly phosphorylated labile polymer containing ATP and phosphoglycerate found in the mitochondrial intermembrane space. We report the purinogen content (as adenine nucleotide equivalents) of rat heart and, for the first time, of rat liver, kidney, brain and mixed skeletal muscle. The findings show that purinogen contains very significant proportions of cell adenine nucleotides ranging from 25% of the free pool in brain and skeletal muscle to 135% of it in kidney. The evidence that purinogen may form a controlled intracellular reservoir of inorganic phosphate is briefly discussed.

Adenine Nucleotides↗
PubMed1998

The mitochondrial phosphoglyceroyl-ATP-containing polymer, purinogen, is unchanged by cardiac ischaemia and reperfusion but may function in the regulation of free intracellular inorganic phosphate concentrations.

Previous work in our laboratory demonstrating large unexplained systematic variations in the heart contents of free adenine nucleotides led us to propose the existence of some unrecognised sequestered form and thence to the purification of very labile acid-insoluble oligomers which we characterised as oligo[3-phospho-glyceroyl-gamma-triphospho(5')adenosine(3')] , abbreviated to (PG-ATP)n. More recently, we provided evidence that these oligomers appear to be the end chains of a complex polymer located in the mitochondrial intermembrane space of a number of rat tissues. We called this polymer purinogen and devised a means of assaying it quantitively [Patel, B., Sarcina, M. & Mowbray, J. (1994) Eur. J. Biochem. 220, 663-669]. Here we report measurements of purinogen in perfused hearts subjected to moderate and severe global ischaemia and reperfusion. Measurements of tissue and perfusate nucleotides, nucleosides and purine degradation products demonstrate that ischaemia led to the augmentation of the free nucleotide content by up to 30% and its re-sequestration on reperfusion in reversible but not in irreversible ischaemia. The purinogen content was unchanged by ischaemia or reperfusion implying the existence of some other unidentified storage pool. By contrast, glucose addition to glycolytically deprived hearts or removal of Pi from perfusion medium, conditions which might be expected to alter demand for intracellular Pi, led to the quantitative transfer of nucleotides between phosphate-rich purinogen and free nucleotides. The possibility that purinogen may act as a rapidly accessible reservoir of intracellular inorganic phosphate is discussed.

Adenosine Triphosphate↗
PubMed1997

Maternal response to paternal trophoblast antigens.

PROBLEM: What is the function of the immunoglobulin (Ig) G antibody bound to trophoblast in normal pregnancy, and what is the antigen? METHOD: IgG was acid eluted from term human placental microvesicles and reacted with the antigen, R80K, left on the vesicles. The eluted antibody was used to detect the antigen on monocytes, lymphocytes, and lymphoblastoid cell lines. The eluted antibody is highly polymorphic, but monoclonal antibodies (mAbs) were made against conserved regions of the molecule. These also reacted with the murine equivalent of the human R80K and were used in inhibition studies of natural killer (NK) cell killing and the mouse abortion models, CBA x DBA2 F1 resorption in CBA females, the endotoxin-induced resorption model, and a sonic stress-induced murine resorption model. RESULTS: All 600 syncytiotrophoblast microvesicle preparations of human term placenta had IgG antibody bound, elutable at pH 3.0. The eluted antibody reacted with about 15% of unrelated human placentae. In horses mares make detectable antibody early in pregnancy, at about the time of implantation. The IgG antibody was bound to an 80-kDa protein (R80K) also detected on B lymphocytes and monocytes. In HLA homozygous lymphoblastoid B cell lines, which reacted with one or more eluted antibodies, had a pattern of cytotoxicity independent of HLA Class I; and as a single 80-kDa peptide chain, R80K did not resemble HLA molecules. Genetic studies in horses show that of the two paternal allotypes of R80K detectable by placental alloantibodies, only one, usually the grandpaternal one, is present in all the placentae of a sibship. Two of 26 eluted human antibodies had affinity for K562 and inhibited killing by human peripheral blood NK cells. One mAb, BA11, against a conserved site on R80K inhibited killing of K562, and also reacted with the murine R80K homologue. BA11 inhibited murine NK cell killing and virtually completely inhibited three NK cell-dependent mouse resorption models. CONCLUSION: R80K protein is a target molecule for NK cell activity expressed on all placentae. It has a polymorphic alloantigenic determinant completely covered with maternal antibody in all successful term pregnancies. In murine NK cell-dependent models of abortion, a mAb against a monomorphic determinant present in human and murine R80K prevents abortion very effectively. It seems that the R80K molecule must be covered with antibody to prevent NK attacks on trophoblast.

Animals↗
PubMed1996

Direct thyroid hormone signalling via ADP-ribosylation controls mitochondrial nucleotide transport and membrane leakiness by changing the conformation of the adenine nucleotide transporter.

Addition of triiodothyronine at 10 pM in vitro to hypothyroid rat liver mitochondria doubles the rate of the adenine nucleotide transporter at low ADP concentrations. Nicotinamide abolishes this effect in parallel with its inhibition of the ADP-ribosylation of an inner membrane protein identical in size to the transporter. Nicotinamide also renders euthyroid preparations indistinguishable from hypothyroid ones. A mechanism is offered to explain these findings in which it is proposed that the adenine nucleotide transporter is a true allosteric protein and that its covalent modification by ADP-ribosylation increases the stability of the less favoured externally-facing C-conformation and thus increases the proportion of transporters in this orientation: although the C-conformation is significantly more leaky to cations than the tight matrix-facing M-conformation, this enhances ADP import. This model is shown to offer an explanation not only for the transport effects of T3 but also for those of oxidative stress and ADP-ribosylation inhibitors on Ca2+, H+ and K+ transfer across the mitochondrial inner membrane. Ca2+ at 30 nM appears to stabilize the M-conformation of the transporter by a mechanism other than ADP-ribosylation.

Adenine Nucleotides↗
PubMed1995

Novel pathways involved in recovery of guanine nucleotides in reperfused ischaemic rat hearts and reoxygenated myocytes.

During global ischaemia for various lengths of time followed by reperfusion, the adenine and guanine nucleotide concentrations altered in parallel. Measurements of purine precursors and breakdown products suggested that GTP was resynthesised from inosine, adenosine or the adenine nucleotides in reversible ischaemia: however, the use of three different inhibitors of the key enzyme in this pathway. IMP-dehydrogenase, was entirely without effect. To eliminate the endothelial cell barrier, the experiments were repeated with anoxic/reoxygenated cardiac myocytes. The lower rate of recovery of purine nucleotide triphosphates in myocytes compared with perfused hearts would tend to confirm the important role suggested for endothelial cells in nucleoside salvage. GTP resynthesis took place after reoxygenation by some unknown pathway unaffected by the presence of large concentrations of an IMP-dehydrogenase inhibitor. The possible relationship of these findings to purinogen, the recently discovered major source of adenine nucleotides in heart, is discussed.

Animals↗
PubMed1994

Evidence that the recently discovered high-energy nucleotide derivative, oligo(phosphoglyceroyl-ATP) forms the end chains of purinogen, a complex polymeric major storage form of adenine nucleotide and phosphate in heart.

Previous work in our laboratory has shown that free adenine nucleotides in heart, liver and kidney exchange rapidly with a major acid-insoluble species which we have characterised as oligo[3-phospho-glyceroyl-gamma- triphospho(5')adenosine(3')], abbreviated to (PG-ATP)n. This has been found in liver and in heart to be complexed to a specific 3'-phosphodiesterase which releases PG-ATP monomers and is located in mitochondrial inter-membrane space. In [14C]adenosine-perfused hearts, (PG-ATP)n has been shown to reach specific radioactivity equilibrium with free ATP within 30 min. Attempts to estimate the quantities of nucleotide in (PG-ATP)n and free nucleotides in response to a variety of stimuli using previously labelled hearts showed by contrast that the free and sequestered nucleotide pools were not at equilibrium. Re-examination of the rapidly labelled acid-insoluble species led to the recognition of radioactive inhomogeneity and of three additional nucleotide components. Perfusion of hearts with phosphate-free medium increased the proportion of 14C incorporated into the sequestered from by 70% and caused the net transfer of 0.9 mumol.g-1 of purine from free adenine nucleotides to the sequestered form. The findings point to the existence of a more complex polymer whose rapidly exchanging end chains we had previously isolated and characterised as (PG-ATP)n; we suggest the name purinogen for this polymer and show that it can contain between 25% and 55% of the tissue nucleotide pool in rat heart.

Adenine Nucleotides↗
PubMed1993

The control of cellular adenine nucleotides: the discovery and metabolism of an oligomeric derivative of ATP and phosphoglyceric acid in mitochondrial intermembrane space.

The observation of large systematic variations in the purine nucleotides in perfused rat hearts inexplicable by known metabolic transformations led us to propose the existence of some unknown major form of nucleotide. Radiolabelling studies located a rapidly metabolized TCA/methanol-insoluble species in heart, kidney and liver which selective digestion and chromatography allied to 31P-nmr and mass spectrometry suggested to be a tetraphosphate derivative of adenosine and phosphoglyceric acid of the form p3Gri1p[pp5'p3Gri1p]npp5'A, oligo-phosphoglyceroyl-ATP. Partial chemical synthesis has confirmed this basic structure. We have shown that substantial amounts of oPG-ATP are located in liver mitochondrial intermembrane space tightly bound to a specific 3' phosphodiesterase which releases the monomer p3Gri1ppp5'A. Here we show that oPG-ATP is remarkably stabilized by Mg2+ ions and describe the 300-fold purification of the stable substrate-free form of the 3' phosphodiesterase; it has an apparent M(r) = 165k with possibly up to 4 subunits of M(r) = 42.5k, 41.9k, 40.5k and 38.9k. The potential function of oPG-ATP is briefly discussed.

Adenosine Triphosphate↗
PubMed1992

The rapid response of isolated mitochondrial particles to 0.1 nM-tri-iodothyronine correlates with the ADP-ribosylation of a single inner-membrane protein.

Under defined conditions liver mitochondria from hypothyroid rats show an apparent lowering of the ADP/O ratio, which can be corrected by addition in vitro of 0.1 nM-tri-iodothyronine (T3). Nicotinamide prevents this restoration by hormone, lowers the ADP/O ratio of euthyroid-rat mitochondria to hypothyroid-rat values and induces T3-sensitivity in euthyroid-rat mitoplasts indistinguishable from that found with hypothyroid-rat preparations. Incorporation into the trichloroacetic-acid insoluble fraction of mitoplasts and hypothyroid-rat mitochondria of radiolabel from [adenine-14C]-NAD+ was stimulated by T3: this stimulation was abolished by nicotinamide. The findings strongly suggest that this incorporation occurs external to the matrix. Confirming the work of others, PAGE of radiolabelled mitoplasts shows alkali-labile modification of a major species of approx. 30 kDa: both nicotinamide and T3 abolish this modification. By contrast, T3 promotes incorporation of label into a single major 11 kDa species: this incorporated label is somewhat acid-labile, and the incorporation is abolished by nicotinamide. Comparative electrophoresis of purified sub-mitoplast fractions show that the 11 kDa species is in the inner membrane and absent from the matrix. The findings are consistent with a receptor-mediated ADP-ribosylation mechanism for the rapid action of T3 on mitochondria.

Adenosine Diphosphate↗
PubMed1991

Substantial quantities of the high energy derivative oligophosphoglyceroyl-ATP are located in mitochondria in rat liver.

Perfusion with [8-14C]adenosine demonstrated the likely existence in rat liver of oligophosphoglyceroyl-ATP (OPG-ATP). Purification followed by assay with a new specific 3' phosphodiesterase confirmed this. The quantities present were 5-10-fold those found previously and comparable to total soluble nucleotides. OPG-ATP was also purified from the mitochondrial fraction, shown to co-distribute with succinate dehydrogenase and can be co-purified with an enzyme confined to intermembrane space.

Adenosine Triphosphate↗
PubMed1991

The characterization of a new enzyme from rat liver mitochondria, oligophosphoglyceroyl-ATP 3'-phosphodiesterase.

By using an assay based on the precipitation of intact 14C-labelled substrate, an activity has been located in the mitochondrial fraction of rat liver which selectively hydrolyses the 3' ester link in the fairly recently discovered oligomeric tetraphosphate derivative of ATP and glyceric acid for which the structure 3-phospho[glyceroyl-gamma-triphospho-5'-adenosine-3'-3-phospho]n-glyceroyl- gamma-triphospho-5'-adenosine has been proposed [Hutchinson, Morris & Mowbray (1986) Biochem. J. 234, 623-627]. This enzyme activity (Mr 85,000) has been purified approx. 30-fold from washed mitochondria by (NH4)2SO4 precipitation and f.p.l.c. The apparent Km for substrate (adenosine equivalents) is around 35 microM. The recovery of total activity is about 20%, and this, allied to the relatively low Vmax. found in contrast with the rapid turnover of oligomer seen in post-ischaemic tissues, suggests that some activating factors have been lost in purification. Percoll-gradient studies confirm that the activity is mitochondrial and not lysosomal or endoplasmic-reticular. The activity is latent in intact mitochondria; it is not, however, associated with intact inner-membrane vesicles but released during their preparation, implying an intermembrane-space location. The product of the enzyme is proposed to be the monomeric unit 3-phosphoglyceroyl-gamma-triphospho-5'-adenosine, from which digestion with snake-venom phosphodiesterase releases ADP.

Animals↗
PubMed1989

Epidemiology of human T-cell leukaemia/lymphoma virus type 1 (HTLV-1) infections in a subpopulation of Afro-Caribbean origin in England.

Epidemiological studies on neurological diseases in residents of Afro-Caribbean origin in the West Midlands region of England have identified eight patients with tropical spastic paraparesis (TSP), all of whom were found to be infected with human T-cell leukaemia/lymphoma virus type 1 (HTLV-1). The husband of one of the patients with TSP was also infected with HTLV-1 and had a T-cell lymphoma. In addition, six asymptomatic HTLV-1-infected first-degree relatives of the TSP patients have been found. By anonymous testing of over 700 sera obtained from individuals of Afro-Caribbean, African, or Asian ethnic origin, seven HTLV-1-infected individuals were detected, who were all immigrants from the Caribbean. Overall, these numbers yielded a seroprevalence of HTLV-1 infections of 3.4% among the immigrant population of Afro-Caribbean origin, which is comparable with the prevalence of HTLV-1 in Jamaica in an equivalent age and sex cohort. Sera were tested for HTLV-1 antibody by means of three different procedures: passive particle agglutination test (Serodia), indirect enzyme-labeled immunosorbent assay (ELISA; Dupont), and indirect immunofluorescence test (in-house, using HTLV-1-infected MT2 cells). The results of all three tests correlated very well with each other. HTLV-1 antibody titres in TSP patients were on the whole significantly higher than those of asymptomatic carriers, but some of the apparently healthy first-degree relatives and one anonymously tested individual had titres as high as most of the TSP patients.

Black or African American↗
PubMed1987

Evidence for ADP-ribosylation in the mechanism of rapid thyroid hormone control of mitochondria.

Triiodothyronine in vitro at concentrations between 10(-13) and 10(-11) M very rapidly activates oxidative phosphorylation in hypothyroid rat liver mitochondria. Comparing the concentrations of hormone with estimates of the amounts of respiratory chain components present suggests that this activation may involve an amplification mechanism. Here we present evidence that while no changes in phosphorylation were detected following hormone administration, nicotinamide, an inhibitor of mono ADP-ribosylation reported to occur rapidly and reversibly in mitochondria, prevented activation by hormone. Moreover incubation with nicotinamide of euthyroid mitochondria and derived intact inner membrane vesicles revealed lowered ADP/O ratios under the same conditions as shown by hypothyroid preparations. While this lesion could be reversed simply by washing the intact mitochondria, the membrane vesicles required triiodothyronine addition.

Adenosine Diphosphate Ribose↗
PubMed1987

The influence of nanomolar calcium ions and physiological levels of thyroid hormone on oxidative phosphorylation in rat liver mitochondria. A possible signal amplification control mechanism.

Using different conditions mitochondria from hypothyroid rats can show both unchanged ADP/O ratios and lowered ADP/O ratios without evidence of uncoupling when compared with euthyroid controls. Raising the free Ca2+ concentration to around 25 nM progressively lowered the ADP/O ratio in hypothyroid but not in euthyroid mitochondria. Ruthenium Red did not alter this behaviour and further increasing the Ca2+ concentration to levels below those which stimulate State 3 respiration had no additional effect. Measurements of the free Ca2+ concentration in the mitochondrial suspending medium using a Quin 2 fluorescence assay showed that the mitochondria did not buffer the free Ca2+ at these low concentrations. At 25 nM-free Ca2+, addition of 10-13) M-T3 to hypothyroid mitochondria produced an immediate and significant increase in the ADP/O ratio without altering the free Ca2+ concentration. The hormone effect was maximal by 10(-11) M. The concentration of ATP synthetase can be estimated to lie at about 10 nM in these experiments. Hence it appears possible that a substantial amplification of the hormone signal may have taken place. Comparison with binding studies suggests that T3 may have been maximally stimulating when somewhat less than half its receptor sites had been filled. The possible mechanisms by which this receptor mediated alteration of the ADP/O ratio might be achieved are discussed.

Adenosine Diphosphate↗
PubMed1987

A comparison of rapid adenine nucleotide incorporation into phosphoglyceroyl-ATP and into RNA-like species in perfused rat heart.

Four main species of rapidly synthesised trichloroacetic acid-insoluble derivatives of adenylate can be separated in extracts from rat hearts. The major species, accounting for more than 70% of the total, is phosphoglyceroyl-ATP; two others (16% of the total) are closely related to it. Around 10% of incorporated radioactivity is in a high-molecular-weight form with a phosphate/purine ratio of 0.8; comparison of [14C]uridylate and [14C]adenylate incorporation supports the suggestion that this is a rapidly synthesised species of RNA which represents about 4% of total RNA in heart. Values are given for the contents of UTP, UDP and UDPglucose in adult rat hearts.

Adenosine Triphosphate↗
PubMed1987

The rapid alteration by tri-iodo-L-thyronine in vivo of both the ADP/O ratio and the apparent H+/O ratio in hypothyroid-rat liver mitochondria.

Mitochondria from the livers of thyroidectomized rats have a lowered ADP/O ratio, which can be restored to normal within 15 min after intravenous injection of a near-physiological dose of tri-iodothyronine. Thyroidectomy lowered the measured delta pH, which appears to be compensated by a rise (not statistically significant) in the membrane potential, so that the protonmotive force is unaltered. A simple simulation technique is described for use in estimating H+/O ratios by the oxygen-pulse technique, which circumvents the problem that this ratio can be seriously underestimated because of re-uptake of protons from the bulk phase by the mitochondria before their expulsion is complete. By this procedure the H+/O ratio of hypothyroid mitochondria is shown to be lowered by the same factor as the ADP/O ratio, and both these ratios are very rapidly restored in parallel by hormone administration. Although these findings could be consistent with a proposal that tri-iodothyronine rapidly modulates by some mechanism the efficiency of the respiratory-chain-linked proton pumps, the kinetic properties of the proton exchange suggest that the bulk-phase protons measured may not reflect faithfully those that drive the ATP synthetase.

Adenosine Diphosphate↗