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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 91 records · Page 5Linked to original sources
PubMed1997

Evaluation of some fluorogenic substrates for continuous assay of aminopeptidase P.

Three potential fluorogenic substrates for assay of aminopeptidase P (AP-P) have been prepared and evaluated, using enzyme purified from porcine kidney. They are based on internal quenching of the synthetic, fluorescent amino acid (R, S)-2-amino-3-(7-methoxy4-coumaryl)propanoic acid ((R,S)-Amp) by a 2, 4dinitrophenyl (DNP) group. The compounds are X-Pro-Pro-(R, S)-Amp-NH2, where X is H-Lys(epsilon-DNP), H-Orn(delta-DNP), or L-2-amino-3-(DNP)aminopropionic acid. The first two were found to be excellent substrates for AP-P, with respective Km values of 4.8 and 5.2 microM. An advantageous feature is that under the conditions of assay, using 4-mm2 cells, the substrates are without noticeable quenching effect on the fluorescence of Pro-Pro-(R,S)-Amp-NH2 (the product liberated by the action of AP-P). At concentrations greater than about 30-50 microM, both substrates appear to inhibit the enzyme, but this has little practical consequence since assays can be carried out at substrate concentrations, giving up to approximately 80% of Vmax without this inhibitory effect being noticeable. The Lys derivative was found to be a very useful substrate for a continuous assay for AP-P and equally good in a discontinuous assay of multiple samples using microtiter plates. The racemic center at the Amp residue did not prevent total hydrolysis of the Lys derivative, suggesting that subsite specificity in AP-P does not extend as far as the P3' position.

Aminopeptidases↗
PubMed1997

Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells.

Staphylococcal alpha-toxin is a 293-residue, single-chain polypeptide that spontaneously assembles into a heptameric pore in target cell membranes. To identify the pore-forming domain, substitution mutants have been produced in which single cysteine residues were introduced throughout the toxin molecule. By attaching the environmentally sensitive dye acrylodan to the sulfhydryl groups, the environment of individual amino acid side chains could be probed. In liposomes, a single 23-amino acid sequence (residues 118-140) was found to move from a polar to a nonpolar environment, indicating that this sequence forms the walls of the pore. However, periodicity in side chain environmental polarity could not be detected in the liposomal system. In the present study, the fluorimetric analyses were extended to physiological target cells. With susceptible cells such as rabbit erythrocytes and human lymphocytes, the 23 central amino acids 118-140 were again found to insert into the membrane; in contrast to the previous study with liposomes, the expected periodicity was now detected. Thus, every other residue in the sequence 126-140 entered a nonpolar environment in a striking display of an amphipathic transmembrane beta-barrel. In contrast, human granulocytes were found to bind alpha-toxin to a similar extent as lymphocytes, but the heptamers forming on these cells failed to insert their pore-forming domain into the membrane. As a consequence, nonfunctional heptamers assembled and the cells remained viable. The data resolve the molecular organization of a pore-forming toxin domain in living cells and reveal that resistant cells can prevent insertion of the functional domain into the bilayer.

Adenosine Triphosphate↗
PubMed1997

Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases.

A series of novel synthetic peptides, containing a C-terminal beta-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6x10(6) M-1. s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3x10(7) M-1.s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8x10(-5) s-1 and approx. 4.3x10(-4) s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates.

Catalysis↗
PubMed1997

Evasion of cytotoxic T lymphocyte (CTL) responses by nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells.

Inoculation of macaques with live attenuated SIV strains has been shown to protect against subsequent challenge with wild-type SIV. The protective mechanism(s) remain obscure. To study the effect in more detail, we have investigated the role of virus-specific CTL responses in macaques infected with an attenuated SIV strain (pC8), which has a four-amino acid deletion in the nef gene, as compared with the wild-type SIVmac32H clone (pJ5). Cynomolgus macaques infected with pC8 were protected against subsequent challenge with pJ5 and did not develop any AIDS-like symptoms in the 12 months after infection. The pC8-induced protection was associated with high levels of virus-specific CTL responses to a variety of viral antigens. In contrast, pJ5-infected macaques had little, if any, detectable CTL response to the viral proteins after three months. The latter group of macaques also showed increased Fas expression and apoptotic cell death in both the CD4(+) and CD8(+) populations. In vitro, pJ5 but not pC8 leads to an increase in FasL expression on infected cells. Thus the expression of FasL may protect infected cells from CTL attack, killing viral-specific CTLs in the process, and providing a route for escaping the immune response, leading to the increased pathogenicity of pJ5. pC8, on the other hand does not induce FasL expression, allowing the development of a protective CTL response. Furthermore, interruption of the Fas-FasL interaction allows the regeneration of viral-specific CTL responses in pJ5-infected animals. This observation suggests an additional therapeutic approach to the treatment of AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Adenosine A3 receptor stimulation inhibits migration of human eosinophils.

Activation of adenosine A3 receptors (A3-R) produced a dose-dependent reduction in the chemotaxis of human eosinophils to platelet-activating factor (PAF), RANTES, and leukotriene B4 (LTB4) to a maximum of 58, 48, and 52%, respectively (P < 0.02). This effect was completely reversed by selective A3-R antagonists. In contrast, activation of A1 or A2a-R did not affect PAF-induced eosinophil chemotaxis. PAF up-regulated the expression of CDllb/CD18, down-regulated L-selectin, and also increased F-actin assembly in eosinophils. The expression of these activation markers was not influenced by A3-R, A2a, or A1-R stimulation. Activation of A3-R may play an important role in inflammation by inhibiting eosinophil migration.

Adenosine↗
PubMed1997

Designed protein pores as components for biosensors.

BACKGROUND: There is a pressing need for new sensors that can detect a variety of analytes, ranging from simple ions to complex compounds and even microorganisms. The devices should offer sensitivity, speed, reversibility and selectivity. Given these criteria, protein pores, remodeled so that their transmembrane conductances are modulated by the association of specific analytes, are excellent prospects as components of biosensors. RESULTS: Structure-based design and a separation method that employs targeted chemical modification have been used to obtain a heteromeric form of the bacterial pore-forming protein staphylococcal alpha-hemolysin, in which one of the seven subunits contains a binding site for a divalent metal ion, M(II), which serves as a prototypic analyte. The single-channel current of the heteromer in planar bilayers is modulated by nanomolar Zn(II). Other M(II)s modulate the current and produce characteristic signatures. In addition, heteromers containing more than one mutant subunit exhibit distinct responses to M(II)s Hence, a large collection of responsive pores can be generated through subunit diversity and combinatorial assembly. CONCLUSIONS: Engineered pores have several advantages as potential sensor elements: sensitivity is in the nanomolar range; analyte binding is rapid (diffusion limited in some cases) and reversible; strictly selective binding is not required because single-channel recordings are rich in information; and for a particular analyte, the dissociation rate constant, the extent of channel block and the voltage-dependence of these parameters are distinguishing, while the frequency of partial channel block reflects the analyte concentration. A single sensor element might, therefore, be used to quantitate more than one analyte at once. The approach described here can be generalized for additional analytes.

Bacterial Toxins↗
PubMed1997

Mechanisms of protection induced by attenuated simian immunodeficiency virus. I. Protection cannot be transferred with immune serum.

To evaluate its role in protection, immune serum was collected from four macaques which were chronically infected with live attenuated simian immunodeficiency virus (SIVmacC8) and had resisted challenge with wild-type SIVmacJ5. The immune serum was transferred to two naive cynomolgus macaques by intraperitoneal injection (11 ml/kg). Four control macaques received an intraperitoneal injection of normal saline. One day later, all macaques were challenged with 10 MID50 of the J5M challenge stock of SIV. After challenge, all macaques became infected as determined by virus co-culture and diagnostic PCR. Virus loads in PBMC at 2 weeks post-challenge were indistinguishable between the two groups of macaques. Thus, the failure of passive immunization to transfer protection indicates that serum components alone are not sufficient to mediate the potent protection obtained using live attenuated vaccines. This is the first time that serum has been transferred from animals known to be protected against superinfection.

Animals↗
PubMed1997

Public health in a managed care environment.

As the health care system moves in a new direction, toward managed care, the critical role of public health in society's efforts to mitigate illness and the realization of health become more apparent. Indeed, the public health problems of this era will not yield to simple solutions. They require a multitude of resources, both human and material, and a myriad of services derived from these resources. Public health's role is to serve as the government's presence in assessing health status, developing policy, evaluating the effectiveness of policy implementation, and assuring access to and quality of comprehensive health services. Increasingly, public health must coordinate a wide array of systems in both the private and public sectors to fulfill its purpose.

Community Health Planning↗
PubMed1997

Cancer risk assessment.

Increasing efforts to reduce the burden of cancer have brought into sharp relief the relevance of cancer risk assessment in preventing the occurrence and, when that is not possible, preventing the progression of the disease. Methods for estimating human cancer risk have evolved steadily over the past few decades as more has been learned about molecular, genetic, and biological aspects of cancer. These methods have been applied with increasing frequency in community-based approaches to reduce the risk of environmentally provoked cancers. At the same time, patients are showing increased interest in estimates of their likelihood of developing cancer during the next 10, 20, or 30 years. This interest is involving more physicians in human cancer risk assessment in clinical counseling settings.

Biomarkers↗
PubMed1997

Founders-Sumner lecture.

The revolution in patient care financing and in the structure and changing scope of the health service system calls for leaders in health care to address existing challenges in the health delivery system. Medical leadership cannot ignore, much less resist, the challenges inherent in the flux and dynamic changes going on in the medical care/health sciences environment. Some of these changes are fueled by fundamental advancements in science and technology. Other developments are fueled by political, demographic, economic and social forces. This article highlights some of these challenges and urges the medical leadership to assume the obligation to assure quality of health-care services and not allow it to be over-shadowed by market forces.

Delivery of Health Care↗
PubMed1997

Do prescribing formularies help GPs prescribe from a narrower range of drugs? A controlled trial of the introduction of prescribing formularies for NSAIDs.

BACKGROUND: Previous studies have suggested that prescribing formularies may promote rational prescribing. The range of drugs prescribed may be one aspect of rational prescribing. AIM: To determine whether the introduction of prescribing formularies helps general practitioners (GPs) to prescribe from a narrower range of non-steroidal anti-inflammatory drugs (NSAIDs). METHOD: General practices in Lincolnshire were offered help in developing prescribing formularies. Ten practices decided to develop a formulary for NSAIDs. Level 3 PACT data were used to determine whether changes in prescribing had occurred with the introduction of the formulary. Matched controls were used to determine whether similar changes had occurred in other practices. RESULTS: Between April and June 1992, and during the same period in 1993, practices that introduced a formulary for NSAIDs reduced the mean number of different drugs used (14.3 versus 13.1, P = 0.04) and increased the percentage of NSAID-defined daily doses coming from the three most commonly used drugs (70.1% versus 74.8%, P = 0.02). Similar changes were not seen in control practices. CONCLUSION: Following the development of a formulary for NSAIDs, practices prescribed from a narrower range of drugs and focused a greater proportion of their prescribing on their three most commonly used drugs.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1996

Murine epidermal growth factor peptide (33-42) binds to a YIGSR-specific laminin receptor on both tumor and endothelial cells.

A laminin-antagonist peptide, comprising amino acids 33-42 of murine epidermal growth factor (mEGF-(33-42)), interacts with a breast cancer- and endothelial cell-associated receptor, which is specific for the laminin B1 chain sequence, CDPGYIGSR-NH2 (Lam.B1-(925-933)), and is immunologically similar to a previously described 67-kDa laminin receptor. In whole cell receptor assays, mEGF-(33-42), Lam. B1-(925-933), and laminin all have IC50 values for displacement of 125I-laminin in the range 1-5 nM. Cell attachment to solid-phase laminin is also blocked by all three ligands, but in contrast to the receptor assays, mEGF-(33-42) or Lam.B1-(925-933), while equipotent with each other, were less effective than laminin. The concentrations of the peptides required to produce half-maximal inhibition of attachment were in the range 230-390 nM, but those for laminin were 1000-fold lower, in the range 0.2-0.3 nM. Like laminin, solid-phase mEGF-(33-42) supports cell attachment, and this ability is blocked by anti-67-kDa receptor antibodies. Modeling studies suggest that both peptides present a tyrosyl and an arginyl residue on the same face of a right-handed helical fold with elliptical cross-section.

Amino Acid Sequence↗
PubMed1996

Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin.

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.

2-Naphthylamine↗