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Biomedical subjects

B Walker

Publications and source records attributed to B Walker.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1996

MMPI-2 patterns in African-American females.

Researchers have reported conflicting conclusions about the relation of the MMPI (MMPI-2) clinical scale elevations and race. Consequently, this study examined the concurrent validity of the MMPI-2 in evaluating African-American females. Seventy-eight (78) African American college student volunteers were administered the MMPI-2, along with other measures of personality, achievement, and coping style. Scores revealed 76% of the sample had elevated profiles. Subjects were divided into three groups based on frequency of clinical scale code type. Subjects with the 5/9 profile elevations functioned as well as normals on measures of coping skills and mood disorders. Discussion emphasizes the importance of clinical interpretation of MMPI data in research programs and the relationship of the data to coping style.

Adaptation, Psychological↗
PubMed1996

Assessment of urinary gonadotropin in solid carcinomas other than gynecological tumors.

To the already long list of existing tumor markers, a new marker has been recently added, the urinary gonadotropin peptide (UGP). This marker is determined in the urine of cancer patients and is considered to be particularly specific for ovarian carcinomas. The purpose of our study was to assess the specificity of UGP in a variety of malignancies other than ovarian carcinomas, e.g., breast, colonic, lung, and urogenital tumors (n = 50 each). The tumors were compared with benign lesions of the same organs. Urine samples of 50 healthy donors served as controls. The 450 urine samples were tested in duplicate using the UGP EIA-kit from Ciba Corning Diagnostics. All tumors were staged and histologically classified. For normalization in all samples, creatinine levels were determined. UGP was found in all tested tumors, however, with very low sensitivity of 20% in urogenital tumors, 46% in lung, and 30% or 27% in colon and breast carcinomas, respectively. The specificity of UGP was comprised between 100% (breast) and 88%. Clearly elevated UGP-concentrations were seen in postmenopausal women. A comparison of UGP with the optimal markers for each tumor system showed that UGP is not superior to these markers. However, we can confirm UGP as being an optimal marker for gynecological carcinomas.

Adolescent↗
PubMed1996

IL-4 and TNF-alpha-mediated proliferation of the human megakaryocytic line M-O7E is regulated by induced autocrine production of GM-CSF.

In this study, the authors examined the effects of recombinant human interleukin 4 (rhIL-4) and recombinant human tumour necrosis factor alpha (rhTNF-alpha) alone or in combination on proliferation of the human cytokine dependent myeloid cell line, M-O7e. While rhIL-4 or rhTNF-alpha alone induced only a weak proliferative response, a synergistic proliferative signal was clearly evident on stimulation of cells with a combination of both cytokines. The stimulatory effect of rhTNF-alpha is mediated predominantly by the 55-kDa TNF receptor because the agonistic monoclonal antibody htr-9 and the Trp32 Thr86 TNF-alpha mutant protein specific for this receptor type produced similar results to rhTNF-alpha. In contrast, the Asn143 Arg145 TNF-alpha mutant protein specific for the 75-kDa TNF receptor produced only minimal proliferation of M-O7e cells. Using RT-PCR, we found that rhTNF-alpha rapidly and strongly induced granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA production, while rhIL-4 was a slow and less efficient inducer of GM-CSF mRNA. However, there was little evidence of the TNF-alpha/IL-4 combination acting synergistically on GM-CSF mRNA production as the levels of GM-CSF mRNA increased only marginally compared with IL-4 or TNF-alpha alone. Thus, the observed synergistic effect of TNF-alpha/IL-4 costimulation of M-O7e cells seems to be mediated via induction of GM-CSF secretion rather than an enhanced production of GM-CSF mRNA. Higher levels of GM-CSF were detectable in supernatants of cells treated with both rhIL-4 and rhTNF-alpha than in cells stimulated with either cytokine alone. Furthermore, addition of a neutralising antibody against GM-CSF abrogated the observed synergistic effect of rhIL-4 and rhTNF-alpha treatment, indicating that the rhIL-4/TNF-alpha combination acts to significantly increase GM-CSF release which then acts in an autocrine manner to enhance the proliferation of M-O7e cells.

Antibodies, Monoclonal↗
PubMed1996

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of pore-forming bacterial cytolysins.

Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin are well-studied prototypes of pore-forming bacterial cytotoxins. Each is produced as a water-soluble single-chain polypeptide that inserts into target membranes to form aqueous transmembrane pores. This review will compare properties of the three toxin prototypes, highlighting the similarities and also the differences in their structure, mode of binding, mechanism of pore formation, and the responses they elicit in target cells. Pore-forming toxins represent the most potent and versatile weapons with which invading microbes damage the host macroorganism.

Amino Acid Sequence↗
PubMed1996

Distal vessel atherosclerosis as a cause for false-positive renal scintigraphy.

While captopril-enhanced renal scintigraphy is acknowledged to be a useful screening technique to detect clinically silent obstructive lesions of the main renal arteries, the presence of significant atherosclerosis of distal, smaller renal vessels as a cause of positive scintigraphy scans has not been reported extensively. In a retrospective 2-year analysis of 31 consecutive captopril-enhanced renal scintigrams, we found a total of 13 studies in 11 patients that were classified as "positive" for renal artery stenosis. Of these 11 patients with positive scintigraphic studies, 4 patients underwent 5 renal arteriography procedures; only 1 of these renal arteriograms showed significant stenosis of the main renal artery. In the other 4 cases, an angiographic pattern of diffuse intrarenal distal arterial disease correlated with scintigram lateralization. Angiography was also performed in 4 patients with negative captopril renal scintiscans. In each of these cases the arteriogram was also negative for significant renal artery stenosis, and only 1 patient had diffuse bilateral intrarenal arterial disease. We conclude that distal renal arterial narrowing should be considered in the differential diagnosis of lateralized renal scintigrams. A negative renal scintigraphic study may be more reliable for excluding significant main renal artery obstructive disease.

Adult↗
PubMed1995

Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification.

The alpha-hemolysin (alpha HL) polypeptide is secreted by Staphylococcus aureus as a water-soluble monomer that assembles into lipid bilayers to form cylindrical heptameric pores 1-2 nm in effective internal diameter. We have individually replaced each charged residue (79 of 293 amino acids) and four neutral residues in alpha HL with cysteine, which is not found in the wild-type protein. The properties of these mutants have been examined before and after modification with the 450-Da dianionic sulfhydryl reagent 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonate (IASD). This modification was highly informative as 28 of 83 modified polypeptides showed substantially reduced pore forming activity on rabbit erythrocytes (rRBC), while only five of the unmodified cysteine mutants were markedly affected. Through detailed examination of the phenotypes of the mutant and modified hemolysins, we have pinpointed residues and regions in the alpha HL polypeptide chain that are important for binding to rRBC, oligomer formation and pore activity. Residues in both the N-terminal (Arg-66 and Glu-70) and C-terminal (Arg-200, Asp-254, Asp-255, and Asp-276) thirds of the protein are implicated in binding to cells. The His-35 replacement mutant modified with IASD was the only polypeptide in this study that failed to form SDS-resistant oligomers on rRBC. Altered hemolysins that formed oligomers but failed to lyse rRBC represented the most common defect. These alterations were clustered in the central glycine-rich loop, which has previously been implicated as a component of the lumen of the membrane-spanning channel, and in the regions flanking the loop. Alterations in mutant and modified hemolysins with the same defect were also scattered between the N terminus and His-48, in keeping with previous suggestions that an N-terminal segment and the central loop cooperate in the final step of pore assembly.

Amino Acid Sequence↗
PubMed1995

Murine epidermal growth factor (EGF) fragment (33-42) inhibits both EGF- and laminin-dependent endothelial cell motility and angiogenesis.

Laminin, murine epidermal growth factor (mEGF), and the synthetic laminin peptide Lam.B1(925-933) (a linear peptide from the B1 chain of murine laminin, CDPGY1GSR-amide) all stimulate endothelial cell motility above basal rates, whereas a synthetic mEGF fragment, mEGF33-42 (a linear peptide from the C-loop of mEGF, acetyl-C-[S-Acm]-VIGYSGDR-C-[S-Acm]-amide), inhibits motility. In both human SK HEP-1 and embryonic chick endothelial cells, mEGF33-42 blocks both EGF- and laminin-stimulated locomotion of endothelial cells. In vivo, mEGF33-42 also blocks both laminin- and mEGF-induced angiogenesis in the chick. In the human cell line. Lam.B1(925-933) has an additive effect in coincubation with either laminin or mEGF, but it blocks their effects in the chick cells. Lam.B1(925-933) alone stimulates angiogenesis in the chick but blocks laminin-induced angiogenesis. Thus, mEGF33-42 acts as a general laminin antagonist, whereas Lam.B1(925-933) acts as a laminin agonist in human cells, but in chick cells it acts as a partial antagonist. We propose that the presence of an anionic group at the eighth residue of mEGF33-42 may be the source of the antagonistic effects seen with this peptide as compared with the laminin fragment. These findings have important implications in the design of human antiangiogenic agents, and also in the use of chick models in the study of human disease.

Amino Acid Sequence↗
PubMed1995

Early first-trimester ultrasound provides a window through which the chorionicity of twins can be diagnosed in an in vitro fertilization (IVF) population.

PURPOSE: Early and accurate diagnosis of placentation alerts the obstetrician to potential clinical sequelae. The reproductive endocrinologist has a unique opportunity to sonographically evaluate the very early intrauterine pregnancy. We undertook this study to determine whether chorionicity could accurately be predicted using early first-trimester transvaginal ultrasound. RESULTS: Of 47 sets of twins conceived through our IVF-ET program, all underwent detailed transvaginal sonography. These findings were then compared with results of placental pathology examination, after birth. Transvaginal sonography was performed 41 days following embryo transfer. All 3 monochorionic placentas were correctly predicted by ultrasound, while the remaining 44 placentas were dichorionic. CONCLUSIONS: The significance of our findings lies both in our 100% accuracy in diagnosis and in the extremely early gestational age at which we were able to establish correctly the diagnosis of chorionicity. The errors in diagnosis made by previous investigators were often the result of not recognizing single placentae which were later histologically shown to be the result of placental fusion. The infertility specialist has a unique window of opportunity to evaluate placentation and should provide useful information regarding chorionicity to the obstetrical team.

Chorion↗
PubMed1995

Carboxyfluorescein and biotin neuromedin C analogues: synthesis and applications.

Two neuromedin C (NC) analogues were constructed by Fmoc synthesis and in situ coupling of 4(5)-carboxyfluorescein or biotin to the N-terminus. Both displayed full agonism in an amylase release assay and cross-reacted fully with a NC-specific antiserum. Biotin NC functioned in a streptavidin-capture ELISA. Carboxyfluorescein NC was used to probe receptor localization in rat stomach. Specific NC binding sites, which did not interact with substance P, angiotensin I, or neurokinin A, were labeled in the antrum. Identity of NC binding sites was confirmed by microautoradiography. The specifically labeled cells were all found in the lamina propria and at least some of cells were identified as eosinophils.

Amylases↗
PubMed1995

A photogenerated pore-forming protein.

BACKGROUND: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells. RESULTS: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. CONCLUSIONS: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.

Animals↗
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↗