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J M Humphreys

Publications and source records attributed to J M Humphreys.

17 recordsLinked to original sources
PubMed2001

Skin microvascular vasodilatory capacity in offspring of two parents with Type 2 diabetes.

AIMS: Microvascular dysfunction occurs in Type 2 diabetes and in subjects with fasting hyperglycaemia. It is unclear whether this dysfunction relates to dysglycaemia. This study investigated in normogylcaemic individuals whether a genetic predisposition to diabetes, or indices of insulin resistance including endothelial markers, were associated with impaired microvascular function. METHODS: Maximum microvascular hyperaemia to local heating of the skin was measured using laser Doppler flowmetry in 21 normoglycaemic subjects with no family history of diabetes (Group 1) and 21 normoglycaemic age, sex and body mass index-matched offspring of two parents with Type 2 diabetes (Group 2). RESULTS: Although Group 2 had normal fasting plasma glucose and glucose tolerance tests, the 120-min glucose values were significantly higher at 6.4 (5.3-6.6) mmol/l (median (25th - 75th centile)) than the control group at 4.9 (4.6-5.9) mmol/l (P = 0.005) and the insulinogenic index was lower at 97.1 (60.9-130.8) vs. 124.0 (97.2-177.7) (P = 0.027). Skin maximum microvascular hyperaemia (Group 1: 1.56 (1.39-1.80) vs. Group 2: 1.53 (1.30-1.98) V, P = 0.99) and minimum microvascular resistance which normalizes the hyperaemia data for blood pressure (Group 1: 52.0 (43.2-67.4) vs. Group 2: 56.0 (43.7-69.6) mmHg/V, P = 0.70) did not differ in the two groups. Significant positive associations occurred between minimum microvascular resistance and indices of the insulin resistance syndrome; plasminogen activator inhibitor type 1 (R(s) = 0.46, P = 0.003), t-PA (R(s) = 0.36, P = 0.03), total cholesterol (R(s) = 0.35, P = 0.02), and triglyceride concentration (R(s) = 0.35, P = 0.02), and an inverse association with insulin sensitivity (R(s) = -0.33, P = 0.03). CONCLUSIONS: In normoglycaemic adults cutaneous microvascular vasodilatory capacity is associated with features of insulin resistance syndrome, particularly with plasminogen activator inhibitor type 1. A strong family history of Type 2 diabetes alone does not result in impairment in the maximum hyperaemic response. Diabet. Med. 18, 541-545 (2001)

Adult↗
PubMed2000

A novel leg-shaking Drosophila mutant defective in a voltage-gated K(+)current and hypersensitive to reactive oxygen species.

1,1'-Dimethyl-4,4'-bipyridinium dichloride (methyl viologen; paraquat), an herbicide that causes depletion of NADPH and generates excessive reactive oxygen species (ROS) in vivo, has been used to screen for ROS-sensitive Drosophila mutants. One mutant so isolated, named quiver(1) (qvr(1)), has a leg-shaking phenotype. Mutants of the Shaker (Sh), Hyperkinetic (Hk), and ether a go-go (eag) genes, which encode different K(+) channel subunits that regulate the A-type K(+) current (I(A)) in different ways, exhibit leg shaking under ether anesthesia and have heightened metabolic rates and shortened life spans. We found that Sh, Hk, and eag mutant flies were all hypersensitive to paraquat. Double-mutant combinations among the three channel mutations and qvr(1) had drastically enhanced sensitivity to paraquat. Synaptic transmission at the larval neuromuscular junction was increased in the qvr(1) mutant to the level of Sh mutants. Similar to eag Sh double mutants, double mutants of eag and qvr(1) showed striking enhancement in synaptic transmission and a wings-down phenotype, the hallmarks of extreme hyperexcitability. Voltage-clamp experiments demonstrated that the qvr(1) mutation specifically disrupted the Sh-dependent I(A) current without altering the other currents [I(K), Ca(2+)-activated fast (I(CF)) and slow (I(CS)) currents, and I(Ca)] in larval muscles. Several deficiency strains of the qvr locus failed to complement qvr(1) and confirmed that ether-induced leg shaking, reduced I(A) current, and paraquat hypersensitivity map to the same locus. Our results suggest that the qvr gene may encode a novel K(+) channel-related polypeptide and indicate a strong link between a voltage-activated K(+) current and vulnerability to ROS.

Animals↗
PubMed1999

New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase.

The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the K(m) values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [(14)C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect.

Arabidopsis↗
PubMed1997

Cloning and characterization of the Drosophila presenilin homologue.

Mutations in two genes, PS1 and PS2, coding for the presenilins, have been linked to the early onset form of familial Alzheimer's disease (AD). Here we report the identification of a Drosophila melanogaster homologue of human PS genes, Dps, which maps to band 77B-C on chromosome 3 and is expressed at multiple developmental stages. The predicted amino acid sequence of the Dps product is 53% identical to human presenilins, with the greatest similarity in the putative transmembrane domains, the hydrophobic domains at the beginning and the end of the cytoplasmic TM6-TM7 loop and the C-terminus. Analysis of Dps in a genetically tractable model system such as Drosophila may provide insight into the mechanisms of Alzheimer's disease (AD) necessary for the development of rational therapeutic approaches.

Alzheimer Disease↗
PubMed1996

Genetic analysis of oxygen defense mechanisms in Drosophila melanogaster and identification of a novel behavioural mutant with a Shaker phenotype.

Mutants of Drosophila melanogaster that lack Cu/Zn superoxide dismutase or urate are hypersensitive to reactive oxygen species (ROS) generated in vivo by the redox-cycling agent paraquat. We have subsequently employed paraquat as a selective agent to identify adult viable mutants potentially defective in other, perhaps unknown, components of ROS metabolism. Paraquat screening of ethyl methanesulfonate-induced second- and third-chromosome mutations yielded 24 paraquat hypersensitive mutants. Two mutants were identified as being new alleles of the previously identified doublesex (dsx) and pink (p) genes. The remainder of the mutations identified previously undescribed genes, including one second chromosome paraquat hypersensitive mutant that was found to exhibit shaking legs, abdomen pulsations, and body shuddering under ether anaesthesia. This recessive mutation was mapped to the polytene chromosome region of 48A5-48B2 and defines a new gene we named quiver (qvr). This mutation is similar in phenotype to the Shaker (Sh), ether-a-gogo (eag), and Hyperkinetic (Hk) mutations, all of which affect potassium channel function in D. melanogaster.

Animals↗
PubMed1993

Effect of cytotoxic drugs on mature neutrophil function in the presence and absence of granulocyte-macrophage colony-stimulating factor.

The effects of the cytotoxic drugs, adriamycin, cyclophosphamide, daunomycin (daunorubicin), prednisolone, actinomycin D, azacytidine and vincristine at concentrations of 1 microM on mature neutrophil function were examined. Up to 5 h incubation with adriamycin, azacytidine, cyclophosphamide, daunomycin and prednisolone had no effect on either luminol chemiluminescence or superoxide secretion. However, after 15 min or 1 h (but not 5 h) incubation vincristine enhanced fMet-Leu-Phe stimulated chemiluminescence, whilst after 5 h incubation with actinomycin D the ability of neutrophils to generate reactive oxidants in response to all stimuli tested was impaired: after 5 h incubation with adriamycin reactive oxidant production was also impaired, but only when fMet-Leu-Phe was used as stimulant. All of the drugs tested except azacytidine inhibited neutrophil oxidant production after 5 h incubation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Actinomycin D and cyclophosphamide also inhibited GM-CSF stimulated protein biosynthesis. These data indicate that cytotoxic drugs may compromise the potentially beneficial effects of CSFs on mature neutrophil function during therapy.

Antineoplastic Agents↗
PubMed1993

Paraquat selection identifies X-linked oxygen defense genes in Drosophila melanogaster.

We have previously shown that homozygous mutants of Drosophila melanogaster deficient in the oxygen radical scavengers, CuZn superoxide dismutase or urate, are adult viable and yet hypersensitive to the oxygen radical-generating agent, paraquat. Thus, paraquat could be used as a selective agent to identify adult-viable mutants potentially defective in other, perhaps unknown, oxygen defense functions. Here we report the successful use of paraquat hypersensitivity in the isolation of X-linked, ethylmethanesulfonate-induced mutations affecting oxygen defense in Drosophila melanogaster. Two paraquat hypersensitive mutants were identified that, by complementation analysis, were shown to be new alleles of the maroon-like gene. In addition to paraquat hypersensitivity, both alleles confer a maternally affected dark brown eye color and a complete lack of enzymatically active xanthine dehydrogenase, both of which are characteristic phenotypes of known maroon-like alleles. We conclude that the lack of xanthine dehydrogenase in these mutants leads to the absence of urate, which is the proximate cause of paraquat sensitivity. Because our search for such mutants on the X chromosome revealed two alleles of only a single selectable gene, we anticipate that the total number of major oxygen defense genes in the complete Drosophila genome may not be large.

Alleles↗
PubMed1991

Effects of recombinant human granulocyte colony-stimulating factor on neutrophil function in vitro and in vivo following chemotherapy and autologous bone marrow transplantation.

Recombinant granulocyte colony-stimulating factor (rG-CSF) primed the ability of human neutrophils to generate increased levels of reactive oxidants in response to fMet-Leu-Phe, and also resulted in an increased rate of protein biosynthesis which was similar to that induced by granulocyte-macrophage colony-stimulating factor. However, rG-CSF reduced the chemotactic activity of neutrophils in response to endotoxin and did not result in an enhanced rate of killing of Staphylococcus aureus. rG-CSF was administered to patients after high dose chemotherapy and autologous bone marrow transplantation for the treatment of either Hodgkin's disease or multiple myeloma. This cytokine decreased the period of neutropenia following such treatment. Neutrophil function in two patients, measured seven days after the final administration of rG-CSF, was severely impaired as indicated by a greatly decreased ability to generate reactive oxidants. However, seven days later (i.e. 14 days post-therapy), the functional activity of the neutrophils from these patients had returned to normal. These data indicate that assays of neutrophil function together with morphological assessment of neutrophil numbers and maturity should be performed in order to evaluate the immune status of patients undergoing such therapy.

Bone Marrow Transplantation↗
PubMed1990

Inhibition of neutrophil superoxide secretion by the preservative, methylhydroxybenzoate: effects mediated by perturbation of intracellular Ca2+?

The preservative, methylhydroxybenzoate inhibited O2- secretion from human neutrophils activated by both the chemotactic peptide fMet-Leu-Phe and phorbol myristate acetate (PMA): the low level of oxidant secretion activated by the ionophore A23187 was similarly reduced in preservative-treated suspensions. Oxidant secretion was similarly reduced in fMet-Leu-Phe and A23187 treated suspensions in which intracellular Ca2+ was buffered by loading with Quin-2, indicating that methylhydroxybenzoate may exert its effects by perturbation of intracellular Ca2(+)-dependent processes. Methylhydroxybenzoate could mimic EGTA in preventing the Ca2+ dependent enhancement of trypsin activity and could also bind this cation in experiments using a Ca2+ electrode, although the preservative bound Ca2+ more slowly and had a lower affinity than EGTA. These data indicate that methylhydroxybenzoate may exert its effects on neutrophils by perturbation of Ca2(+)-dependent activation pathways and this phenomenon may also explain its other known pharmacological effects. Furthermore, these observations provide an insight into the mechanisms by which intracellular Ca2+ may regulate oxidant secretion.

Calcium↗
PubMed1989

Granulocyte-macrophage colony-stimulating factor (GM-CSF) primes the respiratory burst and stimulates protein biosynthesis in human neutrophils.

Pre-treatment of human neutrophils with rGM-CSF resulted in a 3-fold increase in the rate of fMet-Leu-Phe stimulated reactive oxidant generation, as assessed by luminol- and lucigenin-chemiluminescence and O2- secretion. When blood-stream neutrophils were incubated in RPMI 1640 medium supplemented with [35S]methionine, both fMet-Leu-Phe (0.1 microM) and gamma-interferon (100 U/ml) stimulated a 3-4-fold increased incorporation of label into TCA-precipitable material. Similarly, rGM-CSF (50 U/ml) also stimulated protein biosynthesis in bloodstream neutrophils, and newly labelled polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Two classes of polypeptides were visualised on these gels: the relative rate of labelling of one class changed very little upon rGM-CSF treatment whereas the relative rate of labelling of a second group increased 3-12-fold.

Colony-Stimulating Factors↗
PubMed1989

Stimulation of protein synthesis in human neutrophils by gamma-interferon.

Treatment of human, peripheral blood neutrophils with gamma-interferon both "primed" their ability to generate reactive oxidants and increased their rate of protein synthesis. This increased rate of protein synthesis was greatest 60 min after the addition of 100 U/ml gamma-interferon and was not due to an increased intracellular pool of radiolabelled amino acid. Analysis of the newly-synthesized polypeptides by two-dimensional polyacrylamide gel electrophoresis (PAGE) revealed two classes of proteins which were regulated by this agent. The first of these represented proteins whose rate of labelling increased very little (1-2-fold) whereas the rate of biosynthesis of a second group of proteins increased more markedly (10-20-fold). We propose that these newly-synthesized, gamma-interferon regulated proteins play an important role in the function of these cells during an acute inflammatory response.

Blood Proteins↗
PubMed1989

Role of myeloperoxidase in the killing of Staphylococcus aureus by human neutrophils: studies with the myeloperoxidase inhibitor salicylhydroxamic acid.

We have used salicylhydroxamic acid (SHAM) to inhibit intraphagosomal myeloperoxidase activity in order to evaluate the role of this enzyme in the killing of Staphylococcus aureus by human neutrophils. 50 microM-SHAM reduced the luminol-dependent chemiluminescence response stimulated during phagocytosis of unopsonized latex beads and opsonized S. aureus by over 80% and 60%, respectively. When opsonized S. aureus were incubated with neutrophils, 45% were killed within 15 min incubation and 60% by 1 h. However, in neutrophil suspensions incubated with 50 microM-SHAM, only 13% were killed by 15 min whilst 71% still remained viable after 1 h. This inhibitor had no effect upon the number of bacteria phagocytosed or upon degranulation. In a cell-free system, 2.5 microM-H2O2 alone killed 55% of the bacteria, whereas in the presence of myeloperoxidase (i.e. 10 mU myeloperoxidase and 2.5 microM-H2O2) virtually all of the bacteria were killed: the addition of 50 microM-SHAM abolished this myeloperoxidase-enhanced killing but did not affect the H2O2-dependent killing. We therefore conclude that in normal neutrophils whilst H2O2 is required for killing of this pathogen, both myeloperoxidase-dependent and -independent pathways exist.

Cell Degranulation↗
PubMed1988

A shigellosis outbreak traced to commercially distributed shredded lettuce.

In the period August 30-October 7, 1986, 347 persons in adjacent west Texas counties (Ector and Midland) contracted culture-confirmed Shigella sonnei gastroenteritis. A case-control study showed an increased risk of acquiring shigellosis in Ector County with eating at outlets of fast-food Restaurant A, and in Midland County with eating at Restaurant B or C. A second case-control study, of persons who had eaten at Ector County outlets of Restaurant A, showed an increased risk of acquiring shigellosis with eating foods containing shredded lettuce and tomatoes, which were served together (odds ratio = 68.8; 95% confidence interval 8.5-293.1). All implicated restaurants received shredded lettuce produced at one lettuce-shredding plant; two implicated restaurants did not receive tomatoes from the lot delivered to other implicated restaurants. The lettuce-shredding plant distributed shredded lettuce and intact lettuce; restaurants that received only intact lettuce were not involved in the outbreak. Investigation at the lettuce-shredding plant suggested that a food handler might have been the source of contamination and that the method of processing might have allowed cross-contamination to occur. In the laboratory, the outbreak strain of S. sonnei multiplied rapidly on shredded lettuce at 22 C and survived on refrigerated shredded lettuce for at least seven days. This outbreak, one of the largest outbreaks of Shigella infections in the United States in the last decade, indicates that a large, geographically widespread shigellosis outbreak can result from contaminated shredded lettuce that is distributed commercially.

Adolescent↗
PubMed1987

Protein synthesis is activated in primed neutrophils: a possible role in inflammation.

Circulating human neutrophils exhibited low rates of protein biosynthesis, as determined by their ability to incorporate [35S]methionine into TCA-precipitable material. Exposure of cells to the chemotactic peptide (N-formyl-L-methionyl-L-leucyl-L-phenylalanine) increased their rate of protein synthesis, and the maximal stimulation of biosynthesis by this inflammatory agent was observed at 0.1 microM: this concentration of chemotactic peptide "primed" neutrophil activity and only activated the oxidase of these cells by 8% of maximum. The newly-synthesized proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those synthesized in control cells. Two classes of proteins were observed in "primed" cells. The first of these comprised proteins whose rate of biosynthesis changed very little upon "priming" whereas the second class comprised proteins whose rate of synthesis increased greatly after exposure to chemotactic peptide. The fMet-Leu-Phe stimulated protein synthesis was inhibited by actinomycin D and cycloheximide showing that this phenomenon required both transcription and translation. We propose that these fMet-Leu-Phe regulated proteins play an important role in the function of neutrophils during an inflammatory response.

Cycloheximide↗
PubMed1965

Acid phosphatases.

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Acid Phosphatase↗