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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 55 records · Page 3Linked to original sources
PubMed1995

An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch.

BACKGROUND: Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel. RESULTS: Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II). CONCLUSIONS: The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.

Bacterial Toxins↗
PubMed1995

Restoration of pore-forming activity in staphylococcal alpha-hemolysin by targeted covalent modification.

In previous studies, the replacement of His35 in the pore-forming protein alpha-hemolysin (alpha HL) with Leu, Ile, Pro, Arg, Ser, Thr or Cys yielded inactive polypeptides. Here, we show that modification of the inactive single-cysteine mutant alpha HL-H35C with iodoacetamide, to form H35CamC, generates significant pore-forming activity. The closely related polypeptides H35N and H35Q have, respectively, essentially no activity and greatly reduced activity. The modified residue in H35CamC, S-carboxamidomethyl-cysteine, mimicks histidine in volume, polarity and hydrogen bonding potential, but is unable to ionize. Unmodified H35C is defective in the final step of pore formation: the conversion of an inactive heptameric membrane-bound assembly intermediate to a structure containing open channels. It is this step in assembly that is ameliorated in H35CamC.

Animals↗
PubMed1995

Rate of growth of Pseudomonas fluorescens in donated blood.

AIMS: To examine how delayed refrigeration of blood affects the growth of Pseudomonas fluorescens, one of the two most important causes of sepsis resulting from transfusion of contaminated blood. METHODS: Two donations of whole blood were each divided into three aliquots and inoculated with 5-10 cfu/ml of a P fluorescens strain from a case of transfusion associated sepsis. From each donation, one aliquot was placed at 4 degrees C, one was held at 20 degrees C for six hours prior to refrigeration and the third was held at 20 degrees C for 24 hours prior to refrigeration. Samples were aseptically withdrawn over 17 days and bacterial counts were determined using a pour plate technique. RESULTS: The rate of growth of P fluorescens in blood at 20 degrees C was increased compared with blood at 4 degrees C. At 24 hours the aliquots held at 20 degrees C for six and 24 hours had, respectively, 174 and 29,000 cfu/ml compared with 15 cfu/ml in aliquots held at 4 degrees C. There was no evidence of increased killing of P fluorescens at the higher temperature. CONCLUSIONS: These results suggest that blood for transfusion should be refrigerated as soon as possible after collection.

Blood↗
PubMed1995

Substance P (NK1)- and neurokinin A (NK2)-receptor gene expression in inflammatory airway diseases.

The tachykinin neuropeptides substance P and neurokinin (NK) A have been postulated to participate in the inflammatory reaction in airways of smokers and asthmatics. We have examined the hypothesis that the expression of one or more of the three cloned tachykinin receptors (NK1, NK2, and NK3) is increased in inflammatory airway disorders, which could result in augmentation of the effect of released tachykinin neuropeptides. NK1 receptor and NK2 receptor but not NK3-receptor mRNA were detected by ribonuclease protection assay in RNA from both cartilaginous and membranous bronchi and subpleural lung. In lung samples containing membranous airways, NK2-receptor mRNA expression was increased fourfold in asthmatics compared with nonsmoking controls, whereas NK1-receptor mRNA levels were similar in the two groups. NK1- and NK2-receptor mRNA expression was increased twofold in smokers without airflow obstruction compared with nonsmokers, whereas NK1-receptor mRNA expression was significantly lower in patients with chronic obstructive pulmonary disease compared with smoking controls. In situ hybridization indicated NK1-receptor mRNA was expressed in submucosal glands and airway epithelial cells, whereas NK2-receptor and NK3-receptor mRNA were not detected. These observations have implications for the pathophysiology and treatment of both asthma and tobacco smoke-induced airway inflammation.

Adult↗
PubMed1995

Differences in cancer incidence, mortality, and survival between African Americans and whites.

This report highlights selected evidence of different cancer patterns among African Americans and whites and considers potential risk factors associated with these cancers. During the years 1987 to 1991, African Americans experienced higher incidence and mortality rates than whites for multiple myeloma and for cancers of the oropharynx, colorectum, lung and bronchus, cervix, and prostate. African Americans had lower incidence and mortality for cancer of the urinary bladder. The incidence of breast cancer was higher among white women, but mortality was higher among African American women. Five-year relative survival for the period 1983 to 1990 was generally lower among African Americans than whites for cancers of the oropharynx, colorectum, cervix, prostate, and female breast but slightly higher for multiple myeloma. From 1973 to 1991, there were significant declines in cervical cancer incidence among women of both races, oropharyngeal cancer mortality among whites, and bladder cancer mortality for whites and African Americans. Risk factors for the more prominent cancers suggest that efforts aimed at changing lifestyles, achieving socioeconomic parity, and insuring environmental equity are likely to relieve African Americans of much of their disproportionate cancer burden.

Black or African American↗
PubMed1995

Cytokine profile of circulating T cells of leprosy patients reflects both indiscriminate and polarized T-helper subsets: T-helper phenotype is stable and uninfluenced by related antigens of Mycobacterium leprae.

Cytokine profiles of circulating mononuclear cells were studied with the aim of delineating T-cell subsets in leprosy patients with active disease. Using reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA and enzyme-linked immunoassay (ELISA) for the secreted products, interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were studied. Three antigens, native Mycobacterium leprae, a recombinant antigen LSR/A15 of M. leprae and peptide 624 spanning 58-77 amino acids of the latter, were used to induce cytokine expression and release. Half of the subjects, irrespective of the clinical type or antigen used, showed a mixed T-helper type 0 (Th0)-like cytokine pattern, with evidence of the concomitant presence of IFN-gamma and IL-4. The remainder showed a polarized pattern based on the type of leprosy. Lepromatous patients with disseminated disease had Th2-type cytokines, with IL-4 but not IFN-gamma. In contrast, tuberculoid leprosy patients with localized disease showed a Th1-like profile, with the presence of IFN-gamma but not IL-4. Of interest was the stability of the Th phenotype for M. leprae-related antigens. Both the recombinant and the peptide antigens induced the same phenotype as the natural M. leprae bacillus in all except four of 45 leprosy patients.

Antigens, Bacterial↗
PubMed1995

Environmental health and African Americans: challenges and opportunities.

Demographic shifts and other social and economic forces as well as the increasing concerns about environmental determinants of disease and dysfunction have focused more attention on the disproportionate burden of environmental risk on blacks and other minorities. This article reviews these developments and identifies related challenges and opportunities confronting the medical community in the prevention of environmentally provoked disease among black Americans.

Black or African American↗
PubMed1994

Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal alpha-hemolysin.

Structural changes in staphylococcal alpha-hemolysin (alpha HL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel-shift assay to determine the rate of modification of key single-cysteine mutants with the hydrophilic sulfhydryl reagent, 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). The central glycine-rich loop of alpha HL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of approximately 1000 Da. By contrast, residues near the N-terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C-terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.

Animals↗
PubMed1994

Identification and characterization of the cysteine and serine proteinases of the trematode, Haplometra cylindracea and determination of their haemoglobinase activity.

The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22-23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-Argp(OPh)2 or biotin-Lysp(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

Affinity Labels↗
PubMed1994

Modeling the growth of Yersinia enterocolitica in donated blood.

BACKGROUND: Sepsis and death subsequent to the transfusion of blood containing Yersinia enterocolitica is an increasing problem. The organisms probably originate from bacteremia in the donor and can subsequently multiply at low temperature. STUDY DESIGN AND METHODS: Reported here are experiments with a strain of Y. enterocolitica associated with a case of transfusion-associated bacteremia. RESULTS: It was found that the rapid early killing of Y. enterocolitica injected into donated blood does not require viable phagocytes and can be explained by complement-mediated killing. Complement resistance in Y. enterocolitica is known to be plasmid-coded. It is expressed at 37 degrees C, but not at 20 degrees C, and is favored by calcium-deficient culture media. Y. enterocolitica organisms induced to express complement resistance were still killed in donated blood, though the initial rate was slower. Such organisms multiplied in plasma at 37 degrees C, but were killed after 6 hours of incubation at 20 degrees C, presumably because complement resistance genes are switched off at this temperature. CONCLUSION: This experiment is thought to reflect the natural history of Y. enterocolitica contamination of blood, in which complement-resistant organisms in the donor blood encounter lower temperatures after donation. These observations suggest that the practice of plasma depletion may have contributed to the increased incidence of mortality due to Y. enterocolitica contamination of donated blood.

Blood↗
PubMed1994

A pore-forming protein with a protease-activated trigger.

alpha-Hemolysin (alpha HL) is a 293 amino acid pore-forming toxin, which is secreted as a water-soluble monomer by Staphylococcus aureus. By forming a hexameric pore, alpha HL damages the plasma membranes of target cells. Previous studies established that alpha HL proteins with nicks near the midpoint of a central glycine-rich loop are held together by a domain-domain interaction and are hemolytically active. In contrast, alpha HL proteins comprising two alpha HL truncation mutants that overlap in the central loop have no or greatly reduced pore-forming activity, even though the two chains again form a tight complex. Based on these findings, overlap mutants have now been designed that are activated when redundant amino acids in the loop are removed by proteases. Further, the identity of the activating enzyme can be specified by additional mutagenesis of the protease recognition site in the overlap sequence. Mutants of alpha HL that are activated by tumor-associated proteases might be useful components of immunotoxins.

Animals↗
PubMed1994

A pore-forming protein with a metal-actuated switch.

Staphylococcal alpha-hemolysin, a pore-forming exotoxin, is a polypeptide of 293 amino acids that is secreted by Staphylococcus aureus as a water-soluble monomer. It assembles to form hexameric pores in lipid bilayers. Previous studies of pore formation have established the involvement of a central glycine-rich loop. Here, we show that when five consecutive histidine residues replace amino acids 130-134 at the midpoint of the loop, they provide a switch with which pore activity can be (i) turned off by micromolar concentrations of divalent zinc ions and (ii) turned back on with the chelating agent EDTA. Planar bilayer recordings show that Zn2+ and EDTA can act on open channels from either side of the bilayer and thus demonstrate that the central loop lines part of the conductive pathway. Our results suggest that genetically-engineered pore-forming proteins might make useful components of metal ion sensors.

Bacterial Toxins↗