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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 19 recordsLinked to original sources
PubMed1992

Assembly of the oligomeric membrane pore formed by Staphylococcal alpha-hemolysin examined by truncation mutagenesis.

The alpha-hemolysin (alpha HL) from Staphylococcus aureus causes the lysis of susceptible cells such as rabbit erythrocytes (rRBCs). Lysis is associated with the formation of a hexameric pore in the plasma membrane. Here we show that truncation mutants of alpha HL missing 2 to 22 N-terminal amino acids form oligomers on the surfaces of rRBCs but fail to lyse the cells. By contrast, mutants missing 3 or 5 amino acids at the C terminus are very inefficient at oligomerization but do lyse rRBCs, albeit extremely slowly. The C-terminal truncation mutants, retarded as monomers on the cell surface, undergo a conformational change in which the protease-sensitive loop located near the midpoint of the polypeptide chain becomes occluded. Judged by this criterion, polypeptides truncated at the N terminus, frozen as nonlytic oligomers, are in a similar conformation. A second proteolytic site near the N terminus of the polypeptide becomes inaccessible in the lytic pore formed by the wild-type polypeptide, supporting the idea that a second conformational change occurs upon pore formation. These findings suggest a pathway for assembly of the lytic pore in which alpha HL first binds to target cells as a monomer, which is converted to a nonlytic oligomeric intermediate before formation of the pore. In keeping with this model, an N-terminal truncation mutant blocks the slow lysis induced by a C-terminal truncation mutant, presumably by diverting the weakly lytic subunits into inactive oligomers.

Amino Acid Sequence↗
PubMed1992

Functional expression of the alpha-hemolysin of Staphylococcus aureus in intact Escherichia coli and in cell lysates. Deletion of five C-terminal amino acids selectively impairs hemolytic activity.

The alpha-hemolysin gene from Staphylococcus aureus, excluding the 5' region encoding the hydrophobic leader sequence, was amplified from genomic DNA. The identity of the disputed C terminus has been confirmed and revisions made to the internal sequence. The hemolysin is expressed at high levels in Escherichia coli and has been purified to homogeneity from this source. In addition, active [35S-Met]alpha-hemolysin of high specific radioactivity can be generated in an E. coli transcription-translation system. By criteria based on protein chemistry, and biological and electrophysiological assays, the recombinant polypeptide is closely similar to the staphylococcal polypeptide ruling out the possibility of functionally important posttranslational modifications in S. aureus. Convenient new assays utilizing the 35S-labeled polypeptide to measure erythrocyte binding, oligomer formation in detergent and on target cells, and hemolysis have been developed. They have been used to demonstrate that a deletion mutant of alpha-hemolysin, in which five C-terminal amino acids are absent, is severely compromised in its ability both to oligomerize and to lyse rabbit erythrocytes. The mutant polypeptide nevertheless binds tightly to erythrocytes as a monomer, strengthening the idea that oligomerization is required for cell lysis.

Animals↗
PubMed1992

The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B.

In this study we report on the synthesis, kinetic characterization and application of a novel biotinylated and active-site-directed inactivator of cathepsin B. Thus the peptidyldiazomethane biotinyl-Phe-Ala-diazomethane has been synthesized by a combination of solid-phase and solution methodologies and has been shown to be a very efficient inactivator of bovine and human cathepsin B. The respective apparent second-order rate constants (k0bs./[I]) for the inactivation of the human and bovine enzymes by this reagent, namely approximately 5.4 x 10(4) M-1.min-1 and approximately 7.8 x 10(4) M-1.min-1, compare very favourably with those values determined for the urethane-protected analogue benzyloxycarbonyl-Phe-Ala-chloromethane first described by Green & Shaw [(1981) J. Biol. Chem. 256, 1923-1928], thus demonstrating that the presence of the biotin moiety at the P3 position is compatible with inhibitor effectiveness. The utilization of this reagent for the detection of cathepsin B in electrophoretic gels, using Western blotting and in combination with a streptavidin/alkaline phosphatase detection system, is also demonstrated. Given that the peptidyldiazomethanes exhibit a pronounced reactivity towards cysteine proteinases, we feel that the present label may well constitute the archetypal example of a wide range of reagents for the selective labelling of this class of proteinase, even in a complex biological milieu containing additional classes of proteinases.

Affinity Labels↗
PubMed1992

The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases.

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (approximately 4.9 x 10(3) M-1.min-1) and reagent 2 (approximately 1.0 x 10(5) M-1.min-1) respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatible with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.

Affinity Labels↗
PubMed1992

The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture.

In this report we demonstrate how the recently developed biotinylated affinity label biotinyl-Phe-Ala-diazomethane (Bio-Phe-Ala-CHN2) [Cullen, McGinty, Walker, Nelson, Halliday, Bailie & Kay (1990) Biochem. Soc. Trans. 18, 315-316; Walker, Cullen, Kay, Halliday, McGinty & Nelson (1992) Biochem. J. 283, 449-453] can be used for the detection of a precursor form of a cathepsin B-like enzyme produced by breast-tumour cells in culture. Thus the cell lines MDA-MB-436, ZR-75-1 and T47-D produce a soluble protein that can be allowed to react with the biotinylated affinity label to yield an SDS-resistant complex; this can be revealed with a streptavidin/alkaline phosphatase label after PAGE and Western blotting. This protein (molecular mass 47 kDa) can also be detected by immunoblotting using sheep anti-(cathepsin B) antibodies in conjunction with a donkey anti-sheep IgG label. None of the cell lines studied produced any mature cathepsin B-like activity, as gauged by the lack of turnover of the fluorogenic substrate benzyloxycarbonyl-Arg-Arg-4-methylcoumarin-7-ylamide (Cbz-Arg-Arg-NH-Mec). However, treatment of medium samples with pepsin resulted in the generation of such activity. When the pepsin-catalysed activation step was analysed by SDS/PAGE, the protein of 47 kDa was completely converted into two species of very similar molecular masses of 30.5 kDa and 29 kDa. Both these proteins can incorporate the biotinylated probe and, in common with the 47 kD species, they can be detected with the streptavidin/alkaline phosphatase label and immunoblotting. We propose that the 47 kD form is the pepsin-activable proform of these lower-molecular-mass species. The release of the proform from the oestrogen-receptor (ER)-positive breast-tumour cell lines ZR-75-1 and T47-D is stimulated 5-10-fold when these cells are grown in medium containing epidermal growth factor (EGF) at a concentration of 10 ng/ml. In contrast, there is no modulation in the amount of proform released by the ER-negative cell line MDA-MB-436, over a range of EGF concentrations from 0 to 100 ng/ml.

Affinity Labels↗
PubMed1992

Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini.

Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons. Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes. Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides. The N2 amino acid sequence is 21 residues longer than the N1 sequence and is homologous to the nonmyristoylated N terminus of the TPK1 gene product, a yeast catalytic subunit homolog. These data support the view that cAMP-dependent protein kinase activity in Aplysia neurons is produced by a complex array of regulatory and catalytic subunits that generate multiple holoenzymes with a spectrum of properties.

Amino Acid Sequence↗
PubMed1992

A nematode hemoglobin gene contains an intron previously thought to be unique to plants.

Hemoglobin genes from plants and animals both have a characteristic chromosomal organization. Plant hemoglobin genes contains a unique intron inserted into the heme-binding domain of exon 2. This intron has not been previously reported in animal globin genes, and its loss was hypothesized to have occurred early in the evolution of hemoglobins. We report here a unique six-intron, seven-exon internally duplicated nematode hemoglobin gene that contains an intron equivalent to the plant central intron in its first repeat. This nematode hemoglobin gene has lost both the central and the normal third intron in its second repeat. The nematode globin also contains a unique intron between its secretory peptide leader sequence and its coding sequence, which is absent in other extracellular invertebrate globin genes. Possible models to explain the head-to-tail duplication of this gene are discussed.

Amino Acid Sequence↗
PubMed1992

Pilot study of the effect of acemannan in cats infected with feline immunodeficiency virus.

Acemannan, a complex carbohydrate shown to stimulate interleukin-1, tumor necrosis factor alpha and prostaglandin E2 production by macrophages, has also demonstrated antiviral activity in vitro against human immunodeficiency virus, Newcastle disease virus and influenza virus. A pilot study was undertaken to determine acemannan's effect in 49 feline immunodeficiency virus (FIV) infected cats with clinical signs of disease (Stage 3, 4 or 5), 23 of which had severe lymphopenia. Cats received acemannan either by intravenous (Group 1) or subcutaneous (Group 2) injection once weekly for 12 weeks, or by daily oral (Group 3) administration for 12 weeks. Upon entry into the study, cats were randomly assigned to one of the three groups. Laboratory analyses were performed at the beginning of the study and at Weeks 6 and 12. Cats were allowed to continue with a predetermined maintenance regimen of acemannan after completing the 12-week study. Thirteen cats died during the course of treatment. Upon necropsy, the most frequent histopathologic findings were neoplastic, kidney and pancreatic disease. Friedman's two-way ANOVA test showed no significant differences in efficacy among groups administered acemannan by the different routes. Therefore, groups were combined and a signed-ranks test was used to determine changes over time. A significant increase was seen in lymphocyte counts (P < 0.001). Neutrophil counts decreased significantly (P = 0.007), as did incidence of sepsis (P = 0.008). When cats entering with lymphopenia were analyzed separately, a much greater increase in lymphocyte counts was noted (235%) compared with non-lymphopenic cats (42%). A survival rate of 75% was found for all three groups. Thirty-six of 49 animals are alive 5-19 months post-entry. These results suggest that acemannan therapy may be of significant benefit in FIV-infected cats exhibiting clinical signs of disease.

Animals↗
PubMed1992

The future of public health.

Significant progress has been made in developing the biological, social, and behavioral science base for public health services. But this knowledge will have limited value unless it is transformed into programs and services for the people who need them. The systems and mechanics for this transmutation may not have reached their full potential. Assuring that they do so offers, in the 1990s, a challenge and an opportunity for the public health community.

Attitude to Health↗
PubMed1992

Violence: a challenge to the public health community.

In the past 2 years, cities have reported skyrocketing rates of violence, crime, and abusive behavior including intrafamilial homicides. Of the 1838 homicide deaths last year among teenagers between the ages of 15 and 19, 71% involved the use of a firearm. Among black males in the same age group, 82% of the deaths were associated with guns. Since 1968, homicide has been among the leading causes of death for black teenagers. These and large volumes of other data clearly indicate that violence and abusive behavior is a compelling public health problem.

Adolescent↗
PubMed1991

Isolation and sequencing of a cDNA for an unusual hemoglobin from the parasitic nematode Pseudoterranova decipiens.

A cDNA clone encoding a 333-amino acid hemoglobin was isolated from the nematode Pseudoterranova decipiens. The protein contains an 18-amino acid hydrophobic signal sequence and has a calculated mass of 37.6 kDa in the mature form. The predicted protein reveals an internal duplication of a 154-amino acid domain (51% identity). Both domains have significant sequence homology to other primitive hemoglobins, in agreement with a duplication event. Hydrophobicity plots reveal identical strongly hydrophobic regions in each domain, which are potential heme binding sites. This confirms previous suggestions that nematode hemoglobins can have two heme groups per molecule. In addition, each domain contains several conserved histidine motifs that may serve as potential copper binding sites. This result provides further evidence that hemoglobins may have evolved from a primitive cytochrome-like molecule.

Amino Acid Sequence↗
PubMed1991

A prospective study of intraoperative pulse oximetry failure.

Since pulse oximetry is now an ASA standard for intraoperative monitoring, we sought to determine the intraoperative failure rate for this device. We prospectively evaluated the intraoperative failure rate of our pulse oximeters at the four University of Washington Hospitals (University of Washington Medical Center, Veterans Affairs Medical Center [VAMC], Children's Hospital and Medical Center, and Harborview Medical Center [HMC]) recorded from April 1989 to August 1989. We defined failure as the inability to obtain any oximetry reading for a cumulative period of more than 30 minutes during any anesthetic procedure after all equipment malfunctions had been eliminated. Our puse oximeters failed in 124 of 11,046 cases studied; this is a failure rate of 1.12%, which ranged from 0.56% at HMC to 4.24% at VAMC. The failure rate at VAMC (4.24%) was significantly higher than the other hospitals (p less than 0.001). Those cases associated with the pulse oximeter failure had the following characteristics: (1) an ASA status of 3 or higher, (2) lengthy operations, and (3) elderly patients. When the device did fail in a patient, it did not function for 32% of the mean anesthesia time. We conclude that the intraoperative use of the pulse oximetry can provide information about blood oxygen saturation in most patients. However, in approximately 1% of the patients we studied in the operating room, mechanically functioning pulse oximeters failed to provide readings of blood oxygen saturations during routine operative use.

Equipment Failure↗
PubMed1991

Wheal and flare responses to muscle relaxants in humans.

Chemically and pharmacologically unrelated molecules release histamine in humans to produce both cutaneous and systemic responses. It has been suggested that molecular changes in the new benzylisoquinoline-derived muscle relaxant, atracurium, make it less likely to cause histamine release. We therefore injected volunteers intradermally with equimolar concentrations of various muscle relaxants, morphine, papaverine (a benzylisoquinoline), and histamine, to evaluate the relative ability of these drugs to cause wheal and flare responses, and mast-cell degranulation. There were no significant differences in wheal and flare responses among the three benzylisoquinoline-derived muscle relaxants, D-tubocurarine, metocurine, and atracurium. The cutaneous effects of morphine were significantly greater than those of the benzylisoquinoline muscle relaxants, suggesting both direct vascular changes and histamine release. Papaverine injection was followed by a significant wheal but no flare. Skin biopsies from vecuronium- and papaverine-induced wheals revealed normal intact mast-cell granules, suggesting a direct cutaneous vascular response rather than histamine release. Skin biopsies after morphine and atracurium injections revealed mast-cell degranulation. All evaluated benzylisoquinoline muscle relaxants are equipotent histamine releasers at equimolar concentrations. A hydrogenated, benzylisoquinoline-nitrogen-containing ring, present in atracurium but not in papaverine, appears to be the molecular conformation responsible for mast-cell degranulation by atracurium.

Atracurium↗