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C Wilde

Publications and source records attributed to C Wilde.

At least 19 recordsLinked to original sources
PubMed2004

Rho-modifying C3-like ADP-ribosyltransferases.

C3-like exoenzymes comprise a family of seven bacterial ADP-ribosyltransferases, which selectively modify RhoA, B, and C at asparagine-41. Crystal structures of C3 exoenzymes are available, allowing novel insights into the structure-function relationships of these exoenzymes. Because ADP-ribosylation specifically inhibits the biological functions of the low-molecular mass GTPases, C3 exoenzymes are established pharmacological tools to study the cellular functions of Rho GTPases. Recent studies, however, indicate that the functional consequences of C3-induced ADP-ribosylation are more complex than previously suggested. In the present review the basic properties of C3 exoenzymes are briefly summarized and new findings are reviewed.

ADP Ribose Transferases↗
PubMed2003

Requirement of Rac activity for maintenance of capillary endothelial barrier properties.

Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers. In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability. In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min. In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps. To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion. LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not. Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion. We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Actins↗
PubMed2001

Transposition of IS1397 in the family Enterobacteriaceae and first characterization of ISKpn1, a new insertion sequence associated with Klebsiella pneumoniae palindromic units.

IS1397 and ISKpn1 are IS3 family members which are specifically inserted into the loop of palindromic units (PUs). IS1397 is shown to transpose into PUs with sequences close or identical to the Escherichia coli consensus, even in other enterobacteria (Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, and Klebsiella oxytoca). Moreover, we show that homologous intergenic regions containing PUs constitute IS1397 transpositional hot spots, despite bacterial interspersed mosaic element structures that differ among the three species. ISKpn1, described here for the first time, is specific for PUs from K. pneumoniae, in which we discovered it. A sequence comparison between the two insertion sequences allowed us to define a motif possibly accounting for their specificity.

Base Sequence↗
PubMed2000

A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3.

Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C. The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton. We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain. The transferase designated C3(Stau) was cloned from the genomic DNA. At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C. botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S. aureus. In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3. RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA. RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function. The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C. botulinum. The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C. botulinum C2 toxin. The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C. botulinum C3. C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.

ADP Ribose Transferases↗
PubMed2000

Recognition of RhoA by Clostridium botulinum C3 exoenzyme.

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.

ADP Ribose Transferases↗
PubMed2000

Spectrum of abnormal mammographic findings and their predictive value for malignancy in Singaporean women from a population screening trial.

INTRODUCTION: The ability to categorise mammographic features according to their likelihood of malignancy would be valuable in the management of women with abnormal mammograms. The aim of our study was to correlate abnormal mammographic features in a screened population with their histology to identify those features which are predictive of malignancy. The study also examined the spectrum of mammographic features in an Asian population. MATERIALS AND METHOD: This prospective study involved 28,231 women who were randomly selected from a population registry and underwent two-view screening mammography without physical examination. Women with suspicious lesions were recalled for further mammographic views or to a joint assessment clinic prior to biopsy. Mammographic abnormalities and their corresponding histology were assessed. RESULTS: The spectrum of mammographic abnormalities was similar to that in Caucasian populations. The positive predictive value for malignancy was 44.1% of all biopsied cases. Mammographic features could be broadly classified into low-, moderate- and high-risk categories for malignancy. Those features which correspond to high malignancy rates (9.8% to 16.0%) include multiple abnormalities or parenchymal lesions with microcalcifications. The presence of microcalcifications was a good predictor of ductal carcinoma-in-situ (DCIS): 46% of lesions in which the microcalcifications were the sole abnormality were DCIS only. Further, 71% of cancers with any microcalcification on the mammogram had a focus of DCIS on histology. CONCLUSION: Mammographic abnormalities can be segregated into three risk groups for malignancy, and this in turn can improve the selection criteria for breast biopsy, hence reducing unnecessary intervention. Furthermore, the presence of microcalcifications denotes the presence of DCIS, and would be an important determinant of the extent of surgical excision.

Asian People↗
PubMed1999

IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements.

We demonstrate that IS1397, a putative mobile genetic element discovered in natural isolates of Escherichia coli, is active for transposition into the chromosome of E. coli K-12 and inserts specifically into palindromic units, also called repetitive extragenic palindromes, the basic element of bacterial interspersed mosaic elements (BIMEs), which are found in intergenic regions of enterobacteria closely related to E. coli and Salmonella. We could not detect transposition onto a plasmid carrying BIMEs. This unprecedented specificity of insertion into a well-characterized chromosomal intergenic repeated element and its evolutionary implications are discussed.

Amino Acid Sequence↗
PubMed1996

4-1BB is expressed on CD45RAhiROhi transitional T cell in humans.

Murine 4-1BB is an approximately 30-kDa glycoprotein expressed on activated T cells and plays a role in T-cell-mediated proliferative response. To date the majority of work on 4-1BB has been conducted in the mouse. To assess the role of 4-1BB in humans, mAbs were made against the recombinant human (rh) 4-1BB protein. One such mAb 4B4-1 specifically binds SF-21 insect cells expressing rh4-1BB but not irrelevant control protein as measured by flow cytometry (FCM). 4B4-1 mAb stains PMA- and ionomycin-stimulated CEM (human T lymphoma) cells and PHA-stimulated peripheral blood T cells, but not resting cells. 4B4-1 mAb immunoprecipitates both approximately 32 and approximately 80 kDa protein from rh4-1BB expressing SF-21 cells and an approximately 39- and approximately 85-kDa protein from PMA-stimulated CEM cells under reducing conditions by SDS-PAGE. As added proof of its specificity, binding of FITC-labeled 4B4-1 mAb to PHA-stimulated T cells was blocked by rh4-1BB protein. Together these data demonstrate that 4B4-1 is specific for 4-1BB in humans. Unlike in the mouse, 4-1BB is expressed much earlier (within 24 hr) peaking around 2-3 days following PHA stimulation. As in the mouse 4-1BB is induced on both CD4+ and CD8+ T cell subsets. 4-1BB expression is induced upon PHA stimulation in both the naive (CD45RAhi-CD45ROlo/-) and the memory (CD45RAlo/-ROhi) T cell populations. Virtually all CD45RAhiROlo/- cells upon culture in PHA give rise to an intermediate CD45RAhiROhi 4-1BB+ transitional cell and subsequently CD45RAlo/-ROhi 4-1BBlo/- and CD45RAlo/-ROhi 4-1BBhi cells. In contrast, approximately 27% of CD45RAlo/-ROhi 4-1BB- cells when cultured in PHA for 24 hr acquire 4-1BB expression and all remain CD45RAlo/-ROhi.

Animals↗
PubMed1995

Lactation is disrupted by alpha-lactalbumin deficiency and can be restored by human alpha-lactalbumin gene replacement in mice.

Mice carrying either a deletion of the murine alpha-lactalbumin (alpha-lac) gene (null allele) or its replacement by the human alpha-lac gene (humanized allele) have been generated by gene targeting. Homozygous null females are alpha-lac-deficient, produce reduced amounts of thickened milk containing little or no lactose, and cannot sustain their offspring. This provides definitive evidence that alpha-lac is required for lactose synthesis and that lactose is important for milk production. Females homozygous for the humanized allele lactate normally, indicating that human alpha-lac can replace murine alpha-lac. Mouse and human alpha-lac expression was compared in mice heterozygous for the humanized allele. The human gene expressed approximately 15-fold greater mRNA and approximately 14-fold greater protein than the mouse, indicating that the major determinants of human alpha-lac expression are close to, or within, the human gene and that the mouse locus does not exert a negative influence on alpha-lac expression. Variations in alpha-lac expression levels in nondeficient mice did not affect milk lactose concentration, but the volume of milk increased slightly in mice homozygous for the humanized allele. These variations demonstrated that alpha-lac expression in mice is gene dosage dependent.

Alleles↗
PubMed1995

Does an IGF-binding protein (IGFBP) present in involuting rat mammary gland regulate apoptosis?

We examined the effects of GH and prolactin deficiency upon milk production, apoptosis and IGFBP production by the mammary gland. GH deficiency produced a 15% reduction in milk yield, prolactin a 50% reduction and combined prolactin- and GH-deficiency an 85% reduction in milk production. Litter removal led to complete inhibition of milk synthesis within 24 h owing in large part to milk accumulation. GH- and prolactin-deficiency also led to significant loss of mammary cells within 48 h and this was owing at least in part to apoptosis as judged by the appearance of characteristic DNA ladders. Prolactin replacement therapy could prevent all of these changes whilst GH was partially effective. The effects of GH are believed to be mediated via IGF-I, however, we were unable to mimic the effects of GH with IGF-I, IGF-II or a combination of IGF-I, IGF-II and IGFBP-3. We hypothesized that the cell-survival effects of exogenous IGFs might be blocked by an inhibitory IGFBP produced by the gland. Indeed the involuting mammary gland produces large concentrations of an IGFBP which Northern blotting identified as IGFBP-5. There was also a small increase in IGFBP-4 mRNA expression. Both appear to be produced by the secretory epithelial cells as judged by in situ hybridization. Preliminary studies using mouse "mammosphere" cultures suggest that they will be useful for investigating a potential causal relationship between IGFBP-5 synthesis and apoptosis.

Animals↗
PubMed1994

Hypersensitivity reactions to etoposide. A report of three cases and review of the literature.

Etoposide (VP 16) is an antineoplastic agent that has major activity against a number of tumors, including germ cell neoplasms, small cell lung cancer, and malignant lymphoma. It is a derivative of podophyllin, and acts by inhibiting mitosis. Common side effects include bone marrow suppression, alopecia, and gastrointestinal symptoms. Hypersensitivity reactions are uncommon however, and the underlying mechanism is unclear. We report three cases of etoposide hypersensitivity and review the literature.

Adult↗
PubMed1989

Antibiotic proteins of human polymorphonuclear leukocytes.

Nine polypeptide peaks with antibiotic activity were resolved from human polymorphonuclear leukocyte azurophil granule membranes. All but 1 of the 12 constituent polypeptides were identified by N-terminal sequence analysis. Near quantitative recovery of protein and activity permitted an assessment of the contribution of each species to the overall respiratory-burst-independent antimicrobial capacity of the cell. Three uncharacterized polypeptides were discovered, including two broad-spectrum antibiotics. One of these, a defensin that we have designated human neutrophil antimicrobial peptide 4, was more potent than previously described defensins but represented less than 1% of the total protein. The other, named azurocidin, was abundant and comparable to bactericidal permeability-increasing factor in its contribution to the killing of Escherichia coli.

Amino Acid Sequence↗
PubMed1988

Immunologic diversity among serogroup 1 Legionella pneumophila urinary antigens demonstrated by monoclonal antibody enzyme-linked immunosorbent assays.

We tested urine specimens from 222 patients with serogroup 1 Legionella pneumophila pneumonia in two enzyme-linked immunosorbent assays (ELISAs) which used different monoclonal antibodies (A and B) as detector antibodies. Of 171 specimens which contained enough antigen to be detected in the ELISAs, 169 reacted in only one of the two assays. A total of 25 patients whose infections were acquired in any of three Indianapolis hospitals excreted antigen reactive with monoclonal antibody B, but 18 patients who were treated for infections acquired elsewhere reacted with monoclonal antibody A. The urinary antigen ELISA reactivity patterns correlated with the reactivity patterns of L. pneumophila isolates when a separate panel of seven monoclonal antibodies was used. The isolate patterns, in turn, correlated well with environmental isolate patterns from two of the hospitals with nosocomial cases. We conclude that at least two different epitopes exist on the antigen molecules in urine from patients with serogroup 1 L. pneumophila pneumonia and that the subtyping of urinary antigens can be useful epidemiologically.

Animals↗
PubMed1986

Secretion-coupled protein degradation: studies on mammary casein.

Mammary explants from midpregnant rabbits were cultured for 18 h at 37 degrees C with insulin, prolactin and cortisol. Subsequently, explants were labelled for 2 h with inorganic [32P]phosphate, L-[5-3H]proline or L-[4,5-3H]leucine, washed and chased for up to 3 h. The radiolabelling profile of [32P]casein or [3H]casein during the chase period, obtained by isoelectric focussing or immunoprecipitation indicates extensive destruction of neosynthesized casein. The extent of casein destruction in mammary explants in culture (measured after radiolabelling with L-[5-3H]proline), is inversely related to casein secretion. Least casein degradation is observed in explants after 48 h in culture when casein secretion is maximal (observed histochemically). Subsequently, when the extracellular alveolar lumen is filled with secretion products (72 h), rapid intracellular casein destruction is again observed. When the chase was carried out in the presence of drugs which inhibit degradation and/or secretion, the results indicate that secretion-coupled casein degradation is dependent on an intact functional microfilamentous-microtubular network, casein is not degraded by an autophagosome requiring process, degradation is inhibited by leupeptin, amino-acid analogue containing casein does not undergo secretion-coupled degradation and inhibition of N-glycosylation of intracellular vesicular membrane proteins prevents secretion-coupled degradation. Secretion-coupled protein destruction is discussed in relation to the post-translational regulation of the net production of secretory proteins in eukaryotic cells.

Adenine↗