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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 145 records · Page 8Linked to original sources
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

Editorial.

Explore the source record for details and available documents.

Demography↗
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↗
PubMed1994

Synthesis of a cyclic analogue of the C-loop region of epidermal growth factor, containing 1-aminocyclopropane-1-carboxylic acid.

We report the synthesis of a cyclic analogue of epidermal growth factor sequence 33-42 with substitution of 1-aminocyclopropane-1-carboxylic acid for glycine at position 39 (N-acetyl-Cys-Val-Ile-Gly-Tyr-Ser-ACPCA-Asp-Arg-Cys-NH2). The analogue was synthesised by solid-phase methods, using t-Boc chemistry and acid-labile side-chain protecting groups. The use of the 4-methoxybenzyl protecting group for C- and N-terminal cysteine residues resulted in the spontaneous formation of the desired intramolecular disulfide bond after HF deprotection.

Amino Acid Sequence↗
PubMed1994

Interdisciplinary evaluation of cerebral hemodynamics in the treatment of arteriovenous fistulae associated with giant varices.

The techniques for the treatment of intracranial arteriovenous fistulae include angiographic balloon occlusion of the fistula as well as direct surgical attack. Regardless of the method, the occurrence of severe hyperemia caused by a lack of autoregulation after obliteration of the fistula remains a significant concern. We report the use of single photon emission computed tomography and transcranial Doppler studies to assess the occurrence of hyperemia during trial balloon occlusion of such fistulae in two patients. Single photon emission computed tomography and transcranial Doppler studies confirmed the lack of hyperemia during the test occlusion, allowing consideration of treatment plans involving acute fistula occlusion without the difficulty imposed by gradual occlusion and permitting a more accurate evaluation of risk. The purpose of this report is to illustrate how clinical evaluation of intracranial hemodynamics can contribute significantly to treatment decisions.

Adult↗
PubMed1994

The detection, quantification and partial characterisation of cathepsin B-like activity in human pathological synovial fluids.

In this study, the levels of the cysteine proteinase--cathepsin B were measured in diseased synovial fluids using a steady state fluorometric assay. Cathepsin B-like activity was shown to be present in all the samples analysed, with the rheumatoid arthritic synovial fluids possessing significantly higher concentrations (mean value ca. 416 mg/l) than the osteoarthritic fluids (mean value ca. 142.4 mg/l). In addition, upon treatment with pepsin, all of the rheumatoid arthritis samples were shown to possess additional cathepsin B-like activity, suggesting the presence of a reservoir of latent precursor molecules. By utilising a recently developed biotinylated affinity label for cathepsin B-like proteinases and sheep anti-(human cathepsin B) antibodies, used in combination with SDS-PAGE and Western blotting, the rheumatoid arthritic synovial cathepsin B was shown to exist in two forms with apparent molecular masses of M(r) 29,000 and 42,000. We propose that the former is a functionally active proteinase, whereas the latter is a pepsin activatable proform which, when cleaved by this aspartyl proteinase, is converted into a catalytically competent species of M(r) 20,000.

Affinity Labels↗
PubMed1994

Psychosocial needs of family caregivers of terminally ill patients.

The primary caregivers (mean age = 63 yr.) of 28 terminally ill veterans (mean age = 67 yr.) who were living at home were surveyed for their concrete and psychosocial needs. The caregivers also completed the Beck Depression Inventory, the Life Satisfaction Index, the Provision of Social Relations scale, and the Burden Inventory. The rank order of concrete and psychosocial needs is reported for these caregivers. Increased scores on depression were associated with decreased life satisfaction as was low social support. Not surprisingly, higher numbers of concrete and psychosocial needs were associated with increases in depression.

Adult↗
PubMed1994

Prediction of tolerance to carotid artery occlusion using transcranial Doppler ultrasound.

Surgical sacrifice of the carotid artery is a frequently anticipated event during the treatment of certain aneurysms and tumors. The ability to predict tolerance to carotid artery occlusion is therefore of benefit when planning procedures in which the carotid artery is at risk. A trial of carotid artery occlusion using an angiographic balloon during concurrent neurological examination or blood flow studies is an accepted method for testing tolerance, but it carries the risks of an angiogram and cannot be performed at the bedside. Transcranial Doppler ultrasound (TCD) is a noninvasive modality that permits measurement of blood velocity in cerebral vessels. The immediate effects of carotid artery occlusion on middle cerebral artery (MCA) perfusion can be obtained by insonating this artery during manual carotid artery compression. To compare the TCD response to carotid artery compression with the data obtained with more formal testing, the MCA of 22 patients was insonated during manual carotid artery compression and the results compared with the clinical tolerance to balloon occlusion in all patients and to blood flow studied by single photon emission computerized tomography before or during balloon occlusion in 14 of the 22 patients. Surgery was planned to treat giant unruptured aneurysms in 17 cases, intracranial tumors in three, a carotid-cavernous fistula in one, and a carotid artery injury in one. Fifteen patients showed a reduction in TCD flow velocities by no more than 65%; of these, 14 (93%) clinically tolerated the balloon occlusion test. Of the seven patients showing a TCD flow velocity decrease of more than 65%, six (86%) developed a transient focal deficit during the occlusion. It is concluded that the change in MCA velocity measured with TCD studies during manual carotid artery occlusion is a useful predictor of the clinical and blood flow responses to a trial of carotid artery occlusion with an angiographic balloon.

Adult↗
PubMed1994

Impediments to the implementation of environmental policy.

During the past two decades Congress has enacted numerous pieces of legislation for the prevention of environmentally-provoked disease and for reducing ecological risk. The transmutation of these policies into action has been plagued by problems of delay, interagency disagreement, and inadequate resources. As the executive and legislative branches of the federal government try within the next four years to settle and move beyond a number of large environmental health and ecological issues, they must be constantly alerted to the limitations inherent in the implementation phase of the policy process, and not raise public expectations beyond what is realistically achievable in terms of policy impact (i.e. changes in the environment that are associated with measures of government activities).

Environmental Health↗
PubMed1994

Single lung transplantation: a temporal look at rejection, infection, and survival.

Single lung transplantation (SLT) has emerged as routine therapy for selected patients with end-stage lung disease. This study examines the incidence of rejection, infection, and survival during the first posttransplant year. Twenty-one patients (12 male, 9 female; mean age 46 +/- 13 years) underwent 23 SLT procedures (12 left, 11 right lung). Indications were pulmonary fibrosis in six, emphysema in seven, primary pulmonary hypertension in three, Eisenmenger's syndrome in one, pulmonary veno-occlusive disease in one, a-1 antitrypsin deficiency in two, CREST syndrome in one, and retransplantation (graft failure and bronchiolitis obliterans) in two. All were maintained on triple immunotherapy. Survival at 1 year was 100%. The five patients with preoperative pulmonary hypertension had normal hemodynamics at follow up. Freedom from event at 1 year was rejection 23 per cent, all infections 6 per cent, viral 40 per cent, bacterial 55 per cent, fungal 74 per cent. At 1 year, cumulative incidence (events/patient-year) was rejection 1.61, all infections 2.18, viral 0.78, bacterial 1.12, fungal 0.28. Two of 21 patients have developed bronchiolitis obliterans at 1 and 2 years posttransplant. SLT provided safe, effective treatment for a wide variety of end-stage lung diseases. Rejection and infection, although common, may be safely treated with resolution.

Actuarial Analysis↗
PubMed1993

Peptide glyoxals: a novel class of inhibitor for serine and cysteine proteinases.

A series of novel synthetic dipeptides, containing a C-terminal glyoxal grouping (-COCHO), have been tested as inhibitors against typical members of the serine- and cysteine-proteinase families. For example, the sequences benzyloxycarbonyl (Cbz)-Pro-Phe-CHO (I) and Cbz-Phe-Ala-CHO (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and cathepsin B respectively, have been found to be potent reversible inhibitors of their respective target proteinase. Thus I was found to inhibit chymotrypsin with a Ki of approximately 0.8 microM, whereas II exhibits a Ki of approximately 80 nm against cathepsin B. These Ki values are some 10-fold and 3-fold lower than those reported for the corresponding peptide-aldehyde inhibitors of chymotrypsin and cathepsin B upon which the peptidyl-glyoxals were fashioned. Unexpectedly, the sequence Cbz-Pro-Ala-CHO, which was designed to inhibit elastase-like proteinases, exhibited no inhibitory activity towards porcine pancreatic elastase, even when used at concentrations as high as 200 microM.

Animals↗
PubMed1993

Functional complementation of staphylococcal alpha-hemolysin fragments. Overlaps, nicks, and gaps in the glycine-rich loop.

The final steps in assembly of the lytic pore formed by staphylococcal alpha-hemolysin (alpha HL) involve the formation of a nonlytic oligomeric pore precursor, followed by the formation of a transmembrane channel. In this study, truncation mutants of alpha HL encompassing the NH2-terminal or COOH-terminal half of the polypeptide chain and all, part, or none of the central glycine-rich loop were obtained by in vitro, coupled transcription and translation of mutant plasmid DNAs. These polypeptides were unable to oligomerize upon or cause lysis of rabbit erythrocytes (rRBCs). Twenty-one pairs of the same truncation mutants constituting discontinuous alpha HL chains with overlaps, nicks, and gaps in the central loop were obtained by cotranslation. When incubated with rRBCs, many of the pairs were able to form hetero-oligomers with wild-type alpha-hemolysin (s-alpha HL) and most of these formed homo-oligomers in the absence of s-alpha HL. However, only members of a subset of these pairs were able to lyse the cells. The lytic combinations contained overlaps, nicks, or gaps, but only two pairs, with nicks between amino acid residues 128 and 129 and between 131 and 132 had hemolytic activities approaching that of the wild-type polypeptide. Active combinations could also be obtained by separately translating NH2- and COOH-terminal truncation mutants and then combining them. These findings suggest that the integrity of the central loop is of little significance for oligomer formation but that it is more important for the final step in pore assembly or alternatively for determining the correct structure of the conductive channel. Our findings disagree with previous reports of NH2- and COOH-terminal fragments with hemolytic activity and of the prevention of hemolysis by proteolytic cleavage in the central loop. This discord is attributed to experimental and interpretative ambiguities in the earlier protein chemistry. For example, we show that loss of hemolytic activity after treatment with trypsin is not due to cleavage after Lys-131, as previously proposed, but to the removal of a small NH2-terminal peptide through cleavage after Lys-8.

Amino Acid Sequence↗
PubMed1993

Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels.

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala-CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25-26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25-26 kDa. The major 25-26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

Affinity Labels↗
PubMed1993

1H-nuclear magnetic resonance studies of human synovial fluid in arthritic disease states as an aid to confirming metabolic activity in the synovial cavity.

1. A 1H-n.m.r. method was used to measure concentrations of valine, alanine, lactate, acetate, hyaluronan and lipids in synovial fluid obtained, during the normal course of examination from the knee joints of patients attending rheumatology and orthopaedic clinics. Fluid was available from 16 patients with osteoarthritis, 18 patients with rheumatoid arthritis, four patients with meniscal tear and one patient each with systemic lupus erythematosis, mono-arthritis, synovitis and loose bodies. Four normal specimens were obtained for comparison. 2. Valine, alanine and acetate levels all showed a normal Gaussian distribution, reflecting the distributions within the serum of the sample population. 3. Lactate concentrations divided into two distinct patterns. At concentrations below 2.5 mmol/l the lactate levels showed a Gaussian distribution, reflecting the distribution in normal serum. The normal synovial fluid specimens belong to this distribution. Above 2.5-3.0 mmol/l, lactate levels were asymmetric in distribution with a long tail at higher concentrations. These high levels of lactate can be explained by the generation of lactate through anaerobic metabolism within the synovial cavity. This metabolic process is triggered by a general inflammatory condition such as in rheumatoid arthritis. 4. The distribution of n.m.r.-observable lipid concentrations in rheumatoid arthritis and osteoarthritis each shows a normal distribution and the mean concentration is significantly higher in rheumatoid arthritis. 5. An increased n.m.r.-observable hyaluronan concentration is associated with an inflammatory situation. 6. It is concluded that raised levels of lactate and n.m.r.-observable hyaluronan and lipids are useful markers to aid the clinical distinction between rheumatoid arthritis and osteoarthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗