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R Wallis

Publications and source records attributed to R Wallis.

At least 37 records · Page 2Linked to original sources
PubMed1997

Asymmetry adjacent to the collagen-like domain in rat liver mannose-binding protein.

Rat liver mannose-binding protein (MBP-C) is the smallest known member of the collectin family of animal lectins, many of which are involved in defence against microbial pathogens. It consists of an N-terminal collagen-like domain linked to C-terminal carbohydrate-recognition domains. MBP-C, overproduced in Chinese-hamster ovary cells, is post-translationally modified and processed in a manner similar to the native lectin. Analytical ultracentrifugation experiments indicate that MBP-C is trimeric, with a weight-averaged molecular mass of approx. 77 kDa. The rate of sedimentation of MBP-C and its mobility on gel filtration suggest a highly elongated molecule. Anomalous behaviour on gel filtration due to this extended conformation may explain previous suggestions that MBP-C forms a higher oligomer. The polypeptide chains of the MBP-C trimer are linked by disulphide bonds between two cysteine residues at the N-terminal junction of the collagen-like domain. Analysis of an N-terminal tryptic fragment reveals that the disulphide bonding in MBP-C is heterogeneous and asymmetrical. These results indicate that assembly of MBP-C oligomers probably proceeds in a C- to N-terminal direction: trimerization at the C-terminus is followed by assembly of the collagenous domain and finally formation of N-terminal disulphide bonds. The relatively simple organization of MBP-C provides a template for understanding larger, more complex collectins.

Amino Acid Sequence↗
PubMed1997

Identification of critical residues in the colicin E9 DNase binding region of the Im9 protein.

1H-15N NMR studies, in conjunction with mutagenesis experiments, have been used to delineate the DNase-binding surface of the colicin E9 inhibitor protein Im9 (where Im stands for immunity protein). Complexes were formed between the 15 kDa unlabelled E9 DNase domain and the 9.5 kDa Im9 protein uniformly labelled with 15N. Approx. 90% of the amide resonances of the bound Im9 were assigned and spectral parameters obtained from 1H-15N heteronuclear single quantum coherence (HSQC) spectra were compared with those for the free Im9 assigned previously. Many of the amide resonances were shifted on complex formation, some by more than 2 p.p.m. in the 15N dimension and more than 0.5 p.p.m. in the 1H dimension. Most of the strongly shifted amides are located on the surfaces of two of the four helices, helix II and helix III. Whereas helix II had already been identified through genetic and biochemical investigations as an important determinant of biological specificity, helix III had not previously been implicated in binding to the DNase. To test the robustness of the NMR-delineated DNase-binding site, a selection of Im9 alanine mutants were constructed and their dissociation rate constants from E9 DNase-immunity protein complexes quantified by radioactive subunit exchange kinetics. Their off-rates correlated well with the NMR perturbation analysis; for example, residues that were highly perturbed in HSQC experiments, such as residues 34 (helix II) and 54 (helix III), had a marked effect on the DNase-immunity protein dissociation rate when replaced by alanine. The NMR and mutagenesis data are consistent with a DNase-binding region on Im9 composed of invariant residues in helix III and variable residues in helix II. The relationship of this binding site model to the wide range of affinities (Kd values in the range 10(-4) to 10(-16)M) that have been measured for cognate and non-cognate colicin DNase-immunity protein interactions is discussed.

Bacterial Proteins↗
PubMed1997

High incidence of Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus in tumor lesions and peripheral blood mononuclear cells from patients with Kaposi's sarcoma in Uganda.

With the advent of AIDS, Kaposi's sarcoma (KS) has become one of the leading malignancies in sub-Saharan Africa. Recently, DNA sequences from a new human herpesvirus called KS-associated herpesvirus (KSHV) or human herpesvirus type 8 have been found in KS tumor lesions in high frequency. Analyses of tumor lesions from 38 Ugandan KS patients indicated a uniform presence of KSHV in KS tumor lesions as revealed by polymerase chain reaction and Southern hybridization. In contrast, only 31% (11/36) of the normal skin biopsies from the same patient population were positive. The frequency of KSHV DNA detection in peripheral blood mononuclear cells (PBMC) of KS patients was also high (84%, 31/37). Similar analyses revealed the presence of cytomegalovirus (21% in KS lesions) to be discordant with KS development. A large number of KS lesions (87%, 33/38) and KS PBMC (70%, 26/37) were, however, positive for Epstein-Barr virus sequences. In addition, KSHV DNA was not found in the PBMC of Ugandans without KS.

Adult↗
PubMed1996

Apolipoprotein E polymorphism does not predict risk of restenosis after coronary angioplasty.

A recent report has suggested that the E4 allele of apolipoprotein (apo) E increases the risk of restenosis after percutaneous transluminal coronary angioplasty (PTCA) and also that it interacts synergistically with the deletion (D) allele of the angiotensin-converting enzyme (ACE) to increase the risk sixteen-fold. To investigate this further, we genotyped 231 subjects with successful PTCA who underwent planned repeat angiography at 4 months to assess the degree of restenosis. Subjects carrying the apo E4 allele (n = 71) were well matched with non-carriers (n = 160) for clinical and pre- and post-PTCA angiographic features. We found no increase in either apo E4 allele frequency (18.4% versus 15.6%, P = 0.42) or apo E4 homozygosity (2/106 versus 5/125, P = 0.30) in those with restenosis compared with those without. The relative risk of restenosis for apo E4 carriers was 1.11 (95% CI = 0.87-1.42). In apo E4 carriers, restenosis frequency was similar in those also carrying the ACE D allele and those without (28/55 (50.9%) versus 9/16 (56.2%), P = 0.71) and there was no significant increase in restenosis risk in carriers of both the apo E4 and ACE D alleles compared to the rest (odds ratio 1.30, 95% CI 0.68-2.50, P = 0.39). We conclude that in our cohort, the apo E4 allele does not either independently or acting synergistically with the ACE D allele increase the risk of restenosis after PTCA, and that apo E genotyping will not be a useful predictor of risk before the procedure.

Alleles↗
PubMed1996

Analysis of quantitative trait loci for blood pressure on rat chromosomes 2 and 13. Age-related differences in effect.

Previous studies have suggested the presence of quantitative trait loci (QTLs) influencing blood pressure on rat chromosomes 2 and 13. In this study, we mapped the QTLs in F2 rats derived from a cross of the spontaneously hypertensive rat and the Wistar-Kyoto rat and analyzed the effect of the QTLs on blood pressures measured longitudinally between 12 and 25 weeks of age. We analyzed 16 polymorphic markers spanning 147.3 cM on chromosome 2 and 13 markers spanning 91.6 cM on chromosome 13. Both chromosomes contained QTLs with highly significant effects on blood pressure (peak logarithm of the odds [LOD] scores, 5.64 and 5.75, respectively). On chromosome 2, the peak was localized to a position at anonymous marker D2Wox7, 2.9 cM away from the gene for the sodium-potassium ATPase alpha 1-subunit. On chromosome 13, the major peak coincided with the marker D13Mit2, 21.7 cM away from the renin gene, but there was a suggestion of multiple peaks. The effect of the QTL on chromosome 2 was seen throughout from 12 to 25 weeks of age, whereas interestingly, the effect for the QTL on chromosome 13 was maximal at 20 weeks of age but disappeared at 25 weeks of age, presumably because of the effect of either epistatic factors or environmental influences. The findings provide important information on QTLs influencing blood pressure on rat chromosomes 2 and 13 that will be useful in localizing and identifying the causative genes and emphasize the importance of age being taken into account when the effects of individual QTLs on a trait that shows significant age-related changes are being analyzed.

Aging↗
PubMed1996

Predictors of survival in human immunodeficiency virus-infected patients with pulmonary tuberculosis. The Makerere University-Case Western Reserve University Research Collaboration.

Infection with the human immunodeficiency virus (HIV) has changed both the epidemiology and natural history of tuberculosis. Despite a generally good response to effective antituberculous therapy, the prognosis remains poor. The objective of this analysis was to determine the independent predictors of survival in HIV-infected Ugandan adults with smear-positive pulmonary tuberculosis. A total of 191 HIV-infected Ugandan adults with smear-positive pulmonary tuberculosis were enrolled into a clinical trial of chemotherapy for tuberculosis. The subjects received either rifampin, isoniazid, and pyrazinamide for two months, followed by rifampin and isoniazid for six months (n = 101) or streptomycin, thiacetazone, and isoniazid for two months followed by thiacetazone and isoniazid for eight months (n = 90). After standard measurements were made at baseline, the group was followed at regular intervals for a mean of 16 months to determine survival. During the course of follow-up, 82 (43%) of the patients died, six within the first month of therapy. The one-year survival proportion was 68% with an estimated median survival of 26 months and did not differ according to treatment regimen. The hazard for death was biphasic, high early in the course of therapy, and then again after about one year. After controlling for the treatment regimen, four independent predictors of survival were found: anergy to purified protein derivative, atypical chest roentgenogram, previous HIV-related condition, and lymphopenia. In this cohort of Ugandan adults, four simple and inexpensive predictors of survival were found. These factors suggest that the degree of immunosuppression was a major determinant of survival.

AIDS-Related Opportunistic Infections↗
PubMed1995

Protein-protein interactions in colicin E9 DNase-immunity protein complexes. 1. Diffusion-controlled association and femtomolar binding for the cognate complex.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a nonspecific DNase housed in the C-terminus of the protein. A kinetic and thermodynamic analysis of complex formation for both the holotoxin and the isolated DNase domain with the cytoplasmic inhibitor of this enzyme, the immunity protein Im9, is presented. The dissociation constant for each complex was calculated from the ratio of the association and dissociation rate constants. Association was monitored by stopped-flow fluorescence and comprises at least two steps for both complexes, an initial fluorescence enhancement followed by a fluorescence quench. The data are consistent with a two-step binding mechanism in which the rate of formation of an encounter complex (k1) is rate determining and essentially diffusion controlled (4.0 x 10(9) M-1 s-1 for colicin E9) in buffer of low ionic strength. This encounter complex then rearranges to the final stable complex. Sequential stopped-flow experiments using 5-hydroxy-L-tryptophan labeled DNase domain support the two-step mechanism and further show that the rate of encounter complex rearrangement is significantly faster than its dissociation. The overall rate of dissociation of the colicin E9-Im9 complex (k(off)) was determined by radioactive subunit exchange to be 3.7 x 10(-7) s-1. Thus, the Kd for the complex (k(off)/k1) is 9.3 x 10(-17) M, which corresponds to a change in free energy on binding of -21.9 kcal mol-1 at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1995

Protein-protein interactions in colicin E9 DNase-immunity protein complexes. 2. Cognate and noncognate interactions that span the millimolar to femtomolar affinity range.

The in vivo and in vitro cross-binding of the colicin endonuclease-specific immunity proteins toward the DNase domain of colicin E9 is described. In vivo cross-protection was tested by toxin plate assays in which bacterial cells overexpressing each immunity (Im2, Im7, Im8, and Im9) were challenged with the ColE9 toxin. Im9, the cognate immunity protein, renders cells completely resistant toward very high concentrations of the toxin (> 1 mg/mL), whereas the noncognate immunities display a spectrum of weaker cross-reactivities (< 0.01 mg/mL). The order of biological protection in this assay was Im9 >> Im2 > Im8, with Im7 providing no colicin E9 resistance. In vitro binding between the immunity proteins and the E9 DNase was analyzed by determining the dissociation constants for E9 DNase-Im protein complexes at pH 7.0 in the presence of 200 mM salt and at 25 degrees C. Stopped-flow fluorescence experiments suggest that both Im2 and Im8 associate with the E9 DNase by a two-step mechanism, in which the rate constants for both the bimolecular association (k1 = approximately 6 x 10(7) M-1 s-1) and the subsequent conformational change (k2 + k-2 = 4-5 s-1) are very similar to Im9 binding under the same conditions. Fluorescence chase experiments defined the dissociation rate constants for Im2 and Im8. The estimated values are 10(6)- and 10(8)-fold, respectively, faster than the off-rate for the Im9 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1994

Sequential assignments and identification of secondary structure elements of the colicin E9 immunity protein in solution by homonuclear and heteronuclear NMR.

1H-1H, 1H-15N, and 1H-1H-15N multidimensional NMR spectroscopic studies of the 86 amino acid protein that provides immunity against the DNase action of colicin E9 are reported. Through a combination of 2D NOESY and TOCSY and 3D TOCSY-HMQC, NOESY-HMQC, and HMQC-NOESY-HMQC experiments, almost complete 1H NMR and backbone 15N NMR assignments have been obtained, and the secondary structure of the protein has been partially elucidated. Approximately 50% of the protein forms three helices. The specificity determining region of the DNase immunity protein, identified from previously reported biochemical studies to include residues 32-40, is helical, indicating that the protein-protein interaction involves residues from at least one helix.

Amino Acid Sequence↗
PubMed1994

Tandem overproduction and characterisation of the nuclease domain of colicin E9 and its cognate inhibitor protein Im9.

We report the overproduction of the non-specific endonuclease domain of the bacterial toxin colicin E9 and its preliminary characterisation in vitro. The enzymatic colicins (61 kDa) are normally released from producing cells in a complex with their cognate inhibitors, known as the immunity proteins (9.5 kDa). Tryptic digestion of the purified ColE9 complex was found to generate two major components, a monomer derived from the N-terminal and central regions of the toxin and a heterodimer comprising the catalytically active C-terminal domain of the colicin bound to its intact immunity protein, Im9. N-terminal amino acid sequencing, in conjunction with electrospray mass spectrometry, shows that preparations of the DNase domain isolated by this method are heterogeneous, thus making subsequent mechanistic and structural analysis difficult. This problem was circumvented by selectively overexpressing the C-terminal 15-kDa nuclease domain of colicin E9 in tandem with its cognate inhibitor in Escherichia coli. This tandem overexpression strategy allowed high-level production of a 25-kDa protein complex comprising the C-terminal DNase domain of colicin E9 tightly bound to its specific inhibitor Im9, thus masking the anticipated toxicity of the nuclease. The DNase domain was then separated from Im9 under denaturing conditions, refolded by removal of the denaturant and the renatured protein shown to possess both endonuclease and Im9 binding activity. These results describe a novel method for the overproduction of a nuclease in bacteria by co-expressing its specific inhibitor and lay the foundations for a full mechanistic, biophysical and structural characterization of the isolated DNase domain of the colicin E9 toxin.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of the protein-protein interaction between the E9 immunity protein and colicin E9.

The specificity-determining region of the colicin E9 immunity protein (Im9) for its interaction with its cognate E colicin has been localized to residues 16-43 of the 86-amino-acid protein by the use of gene fusions. A comparison of the alignment of residues in this region of the Im2, Im8 and Im9 proteins have identified nine candidate specificity-determining residues. Using site-directed mutagenesis, we have changed each of these residues in the Im9 protein to the residue found in the same position in the Im8 protein. The immunity phenotype conferred by the mutant immunity protein was then tested. Of the nine residues, only one (Val34 to Asp) showed any evidence of conferring immunity to colicin E8. Changing other residues in the specificity-determining region to the equivalent Im8 residue did not affect the phenotype conferred by the mutant protein, with the exception of the change of Val37 to Glu, which resulted in low-level E8 immunity. While the substitutions at positions 34 and 37 of the Im9 protein introduced immunity towards ColE8, they did not diminish the immunity towards ColE9, suggesting that the two immunity proteins may have a common specificity framework which can be modified by single mutations. In addition, we have used chemical modification of the unique cysteine residue of Im9 (Cys23) in order to probe further this specificity-determining region. Cys23 in the purified Im9 protein is accessible to modification with the thiol-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) and the stoichiometry of labelling is close to 1:1. This residue, however, cannot be labelled by 5,5'-dithiobis(2-nitrobenzoic acid) when the Im9 protein is complexed to colicin E9. This result is consistent with the Cys23 residue being buried in the complex. However, when the purified Im9 protein modified at Cys23 with a variety of reagents was used in DNase inhibition assays with colicin E9, the modified Im9 proteins still possessed anti-DNase activity but only up to a certain derivative molecular mass. These results are discussed in terms of the proximity of Cys23 to the specificity-determining region.

Amino Acid Sequence↗
PubMed1992

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.

Bacterial Proteins↗
PubMed1992

Evaluation of a rapist as a function of expressed intent and remorse.

Two studies were conducted to examine the effects of expressed versus denied intent and remorse on evaluation of a rapist. In both studies, the rapist was evaluated more negatively and was assigned a longer prison sentence when expressing rather than denying intent. The rapist received more favorable evaluations when expressing rather than denying remorse. Manipulation of the rapist's expressed remorse did not significantly affect the recommended prison sentence. However, the recommended prison sentence did correlate significantly with participants' perceptions of the rapist's remorse. A multiple regression analysis indicated that recommended prison sentence could be best predicted by attributions of cause, intent, and remorse. The results were related to a growing body of research on accounting strategies, responsibility, and excuse making.

Adolescent↗
PubMed1991

A national outbreak of salmonellosis from yeast flavoured products.

A national outbreak of salmonellosis caused by a rare serotype occurred between July and November 1989. A total of 40 cases of Salmonella manchester infection were identified by the PHLS Division of Enteric Pathogens with a further 7 cases reported from Scotland. The median age of those affected was one year. All strains from the outbreak carried a 70mDal plasmid with a distinctive restriction endonuclease. A statistical association was found between infection and consumption of nationally distributed savoury corn snacks. Samples of autolysed yeast powder and flavourings used in the manufacture of many processed foods were also found to be positive for S. manchester.

Adolescent↗
PubMed1990

'DIY dentures!'.

Explore the source record for details and available documents.

Crowns↗
PubMed1986

Is a fat metabolite the major diet dependent accelerator of aging?

Previous work with a high fat/low carbohydrate diet indicated that Drosophila melanogaster aged faster on that diet than on a high carbohydrate/low fat diet. This could be due to one of eight possibilities enumerated. Oxygen consumption of flies on the high fat/low carbohydrate diet is initially the same as that on the control diet. Subsequently the oxygen consumption falls to below control levels at older ages. No increased rate of living is indicated. A starvation resistant strain is described. The male eats less food, and yet maintains a body fat level indistinguishable from the parent strain. The lifespan and most physiological indices of aging used indicate that the process of aging is unchanged. Nutrient dilution produces a nearly compensatory increase in food intake with a reduced body fat and body energy reserves. The indices of aging used indicate that the rate of aging is reduced under these circumstances. These results are consistent with the possibility that fat or a fat metabolite alters the rate of aging. It is argued from previous data that body fat itself is not likely to be the important factor, rather a metabolite is probably involved.

Aging↗
PubMed1979

Pulmonary and systemic vascular responses of perinatal goats to prostaglandins E1 and E2.

Effects of prostaglandins of the E series and their metabolites on pulmonary and systemic circulations of newborn and exteriorized fetal goats (anesthetized with chloralose) were evaluated in situ using an isolated perfused left lung lobe preparation. Prostaglandin E1 (PGE1) and, to a lesser extent, prostaglandin E2 (PGE2) infusions resulted in decreases of pulmonary vascular resistance (PVR) of fetal and neonatal goats. Infusions of PGE1 or PGE2 (less than 2 microgram.kg-1.min-1 for 1 min) directly into left pulmonary arterial blood did not affect systemic arterial pressure (SAP). Infusions of PGEs (greater than 2 microgram.kg-1.min-1 for 1 min) resulted in decreases in SAP and heart rate. The dose-response characteristics of the pulmonary circulation in response to PGE1 and PGE2 were not different in fetal and newborn goats. Fetal asphyxia did not alter the dose-response characteristics of pulmonary circulation in response to PGE1. Metabolites (15-keto) of PGE1 and PGE2 had no effect upon PVR or SAP of perinatal goats. These results demonstrate in perinatal mammals 1) vasodilator action of PGE1 and PGE2 on the pulmonary and systemic circulations, and 2) catabolism by the lungs of these prostaglandins.

Animals↗