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Biomedical subjects

G Payne

Publications and source records attributed to G Payne.

At least 37 records · Page 2Linked to original sources
PubMed1995

Functional implications of the expression of PilC proteins in meningococci.

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.

Bacterial Adhesion↗
PubMed1994

The phosphopeptide-binding specificity of Src family SH2 domains.

BACKGROUND: Src homology 2 (SH2) domains mediate protein/protein interactions by binding phosphotyrosyl proteins with high specificity. The protein Lck, a Src-like lymphocyte-specific tyrosine kinase which is important in signals involved in T-cell development, contains one such domain. The crystal structure of a complex of the Lck SH2 domain with a high-affinity ligand, pY324, is known. This ligand has the sequence EPQpYEEIPIYL. RESULTS: We designed and synthesized a series of phosphopeptides with single amino-acid changes in the four residues C-terminal to the phosphotyrosine (pTyr) in pY324. Surprisingly, the Glu one residue C-terminal to the phosphotyrosine (at position pY + 1) is sensitive to substitution, whereas the Ile at position pY + 3 is much less sensitive, accommodating a Glu with only modest loss of binding affinity. Replacement of the Glu and Pro on either side of the Ile had little effect, as predicted. Truncated phosphopeptides that end at position pY + 5 and have only an acetyl group N-terminal to the pTyr bound with only slightly lower affinity than pY324. In addition, naturally occurring phosphopeptide sequences that span a 1,000-fold range in binding affinity for the Lck SH2 domain have been identified. CONCLUSIONS: The Lck SH2 domain is highly selective for phosphotyrosyl-peptide binding; its specificity is dictated by the first and third residues C-terminal to the pTyr. The unexpected effects of some amino-acid substitutions indicate that the interactions seen between SH2 domains and ligand in the crystal structure may not be identical to those that occur in solution.

Crystallography, X-Ray↗
PubMed1994

Thoracic impedance and pulmonary atrial natriuretic peptide during head-up tilt induced hypovolaemic shock in humans.

Head up and down tilts were used for manipulating the central blood volume in eight volunteers. During head-up tilt thoracic electrical impedance (TI) increased from 36.7 (33.9-52.1) ohm (mean and range) to 41.9 (36.9-59.2) ohm, heart rate from 60 (49-72) to 80 (65-90) beats min-1 (P < 0.05) and decreased again to 57 (48-67) beats min-1 accompanying a fall in mean arterial pressure from 86 (76-97) to 54 (41-79) mmHg and in cardiac output from 9.2 (5.9-12.1) to 6.9 (3.4-8.8) 1 min-1 (n = 7, P < 0.07). Central venous pressure did not change significantly. Pulmonary arterial mean, 6 (3-12) mmHg, and wedge pressures, 4 (1-9) mmHg, decreased to 4 (1-11) and 1 (0-7) mmHg, respectively, and mixed, 78 (77-79%), and central venous oxygen saturations, 72 (71-73)%, fell to 62 (46-75) and 54 (44-58)%, respectively (P < 0.05). Atrial natriuretic peptide (ANP) was determined from blood of the superior vena cava and pulmonary and brachial arteries. Pulmonary artery ANP, 18.4 (7.5-30.7) pmol l-1, was higher than in vena cava, 13.3 (5.2-20.9) pmol l-1 (P < 0.05). At the time of presyncope, pulmonary artery ANP decreased from 20.8 (37.4-10.1) to 13.7 (19.7-5.7) pmol l-1, in vena cava from 13.8 (23.1-7.1) to 10.2 (17.9-6.7) pmol l-1 and in the brachial artery from 16.9 (34.1-5.2) to 11.3 (18.5-5.1) pmol l-1 (P < 0.05). Head-down tilt did not affect the recorded variables significantly. Thoracic electrical impedance, pulmonary artery pressure and venous oxygen saturations were sensitive indices of the central blood volume as reflected in the release of atrial natriuretic peptide from the right side of the heart.

Adult↗
PubMed1994

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Amino Acid Sequence↗
PubMed1994

Mother-daughter correlations of obesity and cardiovascular disease risk factors in black and white households: the NHLBI Growth and Health Study.

OBJECTIVES: This study sought to evaluate obesity as a potential explanatory factor for the increased relative risk for cardiovascular disease in Black compared with White women. METHODS: Familial associations for obesity and cardiovascular disease risk factors were assessed in 720 White and 580 Black mother-daughter pairs from the National Heart, Lung, and Blood Institute's Growth and Health Study by using Pearson's chi square, Spearman's correlations, and partial correlations. RESULTS: Black girls and mothers were significantly heavier and had higher body mass indices than their White counterparts. In each racial group, significant, positive mother-daughter correlations existed for weight, body mass index, and triceps skinfolds, and for all cardiovascular disease risk factors. Obesity measures correlated positively with systolic blood pressure and triglycerides and inversely with high-density lipoprotein cholesterol in girls and mothers of both races. Correlations between mothers and daughters for exercise and ideal body shape were weak and did not explain obesity associations. CONCLUSIONS: Intrafamilial associations of obesity, cardiovascular disease risk factors, and the obesity-cardiovascular disease risk factor relationship support the position that increased cardiovascular disease morbidity and mortality rates in Black women may be linked to excess obesity in Black women compared with White ones.

Black or African American↗
PubMed1993

Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.

Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.

Amino Acid Sequence↗
PubMed1993

An indifference point for electrical impedance in humans.

Regional electrical impedance was used over eight sections of the human body (two thoracic; one abdominal; two thigh; two around the knee; and one lower leg) to determine the volume indifference point during passive head-up tilt in eight subjects. Head-up tilt was performed in 10 degrees increments from 0 degree to 60 degrees over 6 min. Electrical impedance increased over the thorax in proportion to the head-up tilt angle, while abdominal impedance did not change significantly, and over the thigh and the lower leg it decreased with increasing head-up tilt angle. No change in electrical impedance was noted just above the knee, and electrical impedance just below the knee decreased only marginally. Results demonstrate minimal fluid accumulation around the knee during head-up tilt. Furthermore, in humans the electrical impedance and therefore probably the volume indifference point is positioned between the umbilicus and crista iliaca.

Adult↗
PubMed1993

Effect of limb anesthesia on middle cerebral response to handgrip.

Transcranial Doppler ultrasound was used to measure middle cerebral arterial mean flow velocity (Vmean) on both sides of the brain in 12 subjects during hand contractions before and during regional anesthesia of the left arm. At rest Vmean was 49 (36-104) cm/s (median and range) and was unaffected by regional anesthesia. During right-hand contractions Vmean remained unchanged on the right side of the brain, whereas it increased 20 (4-37)% (P < 0.01) on the left side of the brain. Similarly, during left-hand contractions Vmean increased 24 (2-42)% (P < 0.01) on the right side of the brain, while it remained unchanged on the left side of the brain. Regional anesthesia did not quantitatively affect Vmean during right-hand contractions. In contrast, during left-hand contractions, both right and left Vmean tended to decrease. Increases in Vmean appeared despite a decrease in arterial carbon dioxide tension (P < 0.01). Heart rate and blood pressure responses to hand contractions were enhanced during regional anesthesia (P < 0.01), but left-hand contractions resulted in a less pronounced increase in blood pressure (P < 0.01). These data demonstrate a contralateral increase in cerebral perfusion during hand contractions that is dependent on intact afferent input from the working limb.

Adult↗
PubMed1992

Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco.

Complementary DNA clones encoding acidic and basic isoforms of the class III chitinase were isolated from Nicotiana tabacum. The clones share ca. 65% identity, are equally homologous to the class III chitinases from cucumber and Arabidopsis, and are members of small gene families in tobacco. An acidic class III chitinase was purified from the intercellular fluid of tobacco leaves infected with tobacco mosaic virus (TMV). Partial amino acid sequencing of the protein confirmed that it was encoded by one of the cDNA clones. The mRNAs of the class III chitinases are coordinately expressed in response to TMV infection, both in infected and uninfected tissue. The acidic and basic class III chitinases constitute previously undescribed pathogenesis-related proteins in tobacco.

Amino Acid Sequence↗
PubMed1992

Effects of axillary blockade on regional cerebral blood flow during dynamic hand contractions.

Regional cerebral blood flow (rCBF) was measured at orbitomeatal (OM) plane +5.0 and +9.0 cm in 10 subjects at rest and during dynamic hand contractions before and after axillary blockade. Handgrip strength was significantly reduced, and rating of perceived exertion increased after blockade. During hand contractions before blockade, contralateral hemispheric cerebral blood flow (CBF) at OM +9.0 increased from a resting value of 58 (49-75) to 63 (52-82) ml.100 g-1.min-1; contralateral motor sensory rCBF at OM +9 from 58 (50-77) to 71 (64-84); motor sensory rCBF at OM +5 from 67 (54-76) to 77 (64-87) and 70 (62-84) contralaterally and ipsilaterally, respectively; and supplementary motor area (SM) rCBF from 64 (53-69) to 75 (67-88) ml.100 g-1.min-1. During dynamic hand contractions after axillary blockade, CBF did not increase at OM +5 or in the SM. Furthermore, contralateral motor sensory rCBF at OM +9 increased much less. Axillary blockade had no effect on resting CBF, rCBF, or increases in the two during hand contractions of the opposite hand. Thus neural feedback from the contracting muscle is necessary for the increases in SM bilateral OM +5 motor sensory rCBF and the maximal increase in contralateral OM +9 motor sensory rCBF during dynamic hand contractions.

Adult↗
PubMed1991

A phase II clinical and pharmacokinetic study of Lonidamine in patients with advanced breast cancer.

Lonidamine is a substituted indazole carboxylic acid with a unique mechanism of action and early clinical studies have reported anti-tumour activity. In a phase II study 32 patients with previously treated advanced breast cancer were given Lonidamine in a daily divided oral dose of 600 mg. Of 28 patients evaluable for response, three (11%) achieved a partial response (4-24+ months) and three (11%) a minor response. Two patients have stable disease (greater than 3 months) and 20 progressed. Toxicity was very mild. Sixteen (53%) of 31 patients had myalgia which lasted a median of 2 weeks. This was investigated with nuclear magnetic resonance spectroscopy in four patients but the changes were unrelated to the degree of myalgia. No other major side-effect was seen, and no dose reduction was required. Lonidamine pharmacokinetics have been investigated in 17 patients 1 month after the start of therapy. Lonidamine was detected in the plasma of all patients, but there was no clear relationship between Lonidamine levels and clinical response or toxicity. Lonidamine appears to be active against advanced breast cancer and its low toxicity would allow combination studies with chemotherapy.

Adenosine Triphosphate↗
PubMed1990

Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase.

Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA. The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF). To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer. We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69. The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59. From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69. The chemical nature of the chromophores did not change after several catalytic cycles. The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency. Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield.

Base Sequence↗
PubMed1990

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues.

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.

Apoenzymes↗
PubMed1990

Evidence for a third structural class of beta-1,3-glucanase in tobacco.

Glucan endo-1,3-beta-glucosidases (beta-1,3-glucanases) have been implicated in several developmental processes and they may also play a direct role in the plant's defense against fungal pathogens. In an effort to characterize the glucanase gene family, complementary DNA clones encoding an acidic form of beta-1,3-glucanase have been isolated from tobacco. The cDNA was expressed in E. coli and shown to encode a beta-1,3-glucanase activity. The protein sequence encoded by the cDNA was found to match the partial protein sequence of PR-35, a previously characterized beta-1,3-glucanase. The protein encoded by the cDNA was purified from the extracellular fluid of TMV-infected tobacco leaves and found by immunological methods to correspond to glucanase PR-Q'. From a detailed analysis of the cDNA it is clear that this glucanase represents a third structural class of enzyme which differs substantially from both the basic, vacuolar glucanase and the acidic, extracellular forms (PR-2, PR-N and PR-O). It has previously been demonstrated that the basic form of beta-1,3-glucanase is synthesized as a pre-pro-enzyme and upon maturation the 21 amino acid signal peptide and a 22 amino acid carboxy-terminal peptide are removed. This processing event has been proposed to be involved with the vacuolar localization of the enzyme. By comparing the deduced protein structure of PR-Q' to that of the basic form it is evident that this extracellular enzyme is missing the carboxy-terminal 22 amino acids. The role of a conserved phenylalanine-glycine dipeptide in the processing of glucanases and other pathogenesis-related proteins from tobacco is discussed.

Amino Acid Sequence↗
PubMed1990

Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco.

Complementary DNA clones encoding two isoforms of the acidic endochitinase (chitinase, EC 3.2.1.14) from tobacco were isolated. Comparison of amino acid sequences deduced from the cDNA clones and the sequence of peptides derived from purified proteins show that these clones encode the pathogenesis-related proteins PR-P and PR-Q. The cDNA inserts were not homologous to either the bacterial form of chitinase or the form from cucumber but shared significant homology to the basic form of chitinase from tobacco and bean. The acidic isoforms of tobacco chitinase did not contain the amino-terminal, cysteine-rich "hevein" domain found in the basic isoforms, indicating that this domain, which binds chitin, is not essential for chitinolytic activity. The accumulation of mRNA for the pathogenesis-related proteins PR-1, PR-R, PR-P, and PR-Q in Xanthi.nc tobacco leaves following infection with tobacco mosaic virus was measured by primer extension. The results indicate that the induction of these proteins during the local necrotic lesion response to the virus is coordinated at the mRNA level.

Amino Acid Sequence↗
PubMed1989

Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase.

An extracellular, acidic chitinase was purified to homogeneity from tobacco necrosis virus-infected leaves of Cucumis sativis. The amino acid sequences of the intact protein and of peptides isolated following endoproteinase Lys-C digestion, cyanogen bromide cleavage, and trypsin digestion were determined. Oligonucleotide probes derived from this sequence were used to isolate a cDNA clone encoding this protein. No significant homology was found between this chitinase and either the basic chitinase isolated from bean or tobacco or the chitinase isolated from Serratia marcescens; however, strong homology was found between the cucumber chitinase and a lysozyme/chitinase from Parthenocissus quinquifolia. The induction of the protein by tobacco necrosis virus infection or salicylate was found to be at the level of RNA accumulation. Genomic Southern analysis indicates that a single gene in the cucumber genome encodes this protein.

Amino Acid Sequence↗