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R A Welch

Publications and source records attributed to R A Welch.

At least 37 records · Page 2Linked to original sources
PubMed1995

Binding of antibodies to functional epitopes on the pore formed by Escherichia coli hemolysin in cells and model membranes.

Escherichia coli hemolysin (HlyA) inserts into target membranes producing a cation-selective pore. We approached the problem of determining which portions of this protein remain exposed on the side of attack by applying specific antibodies. Results obtained with resealed erythrocyte ghosts and planar phospholipid membranes were compared. The effects of one polyclonal and four monoclonal anti-hemolysin antibodies (mAbs) were studied. Using ghosts we found one mAb which strongly reduced the ion-permeability through the preinserted HlyA channels and one which clearly increased it. Experiments with planar bilayers corroborated these results by showing that the former mAb effectively promoted the closed state of the channel whereas the latter forced the HlyA channel into an open configuration. Anti-hemolysin polyclonal antibodies initially stimulated but then prevented channel opening, indicating they contained clones able to act on both these channel determinants. They were effective only when applied on the same side as the hemolysin indicating that the epitopes were exposed to that side. Finally, the antigenic epitopes of three of the mAbs were localised on the HlyA molecule by using different mutants (amber and frame shift mutants and hemolysin gene hybrids).

Animals↗
PubMed1995

Construction of Actinobacillus pleuropneumoniae-Escherichia coli shuttle vectors: expression of antibiotic-resistance genes.

We constructed several cloning vectors, designated pGZRS-18/19 and pGZRS-38/39, which were based on an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3-kb plasmid. They carry the lacZ alpha-complementation fragment and MCS from pUC18/19, and either the bla gene under the control of a putative Apl promoter or the KmR gene from Tn903. These vectors replicate in representative strains of Apl serotypes 1 and 7, Escherichia coli, Pasteurella haemolytica (Ph) and Haemophilus (Actinobacillus) actinomycetemcomitans. We also found that Apl and Ph did not express genes under the control of the lacZ or bla promoters, suggesting that their RNA polymerases may not utilize these promoters.

Actinobacillus pleuropneumoniae↗
PubMed1995

Biological effects of RTX toxins: the possible role of lipopolysaccharide.

RTX toxins are a family of related exotoxins with hemolytic, leukotoxi c and leukocyte-stimulating activities that are produced by a diverse array of Gram-negative bacteria. Lipopolysaccharide might be required for the maximal production of some RTX toxins and might be a cofactor in some of the biological effects of RTX toxins.

Animals↗
PubMed1995

Battling against host phagocytes: the wherefore of the RTX family of toxins?

The RTX family of bacterial exotoxins is a group of related cytolytic proteins produced by a wide variety of gram-negative human and animal pathogens. While diverse in their associated diseases and in their target cell specificities, there remain several themes common to RTX toxins, including genetic organization, structural and functional features, and effects on target cells. In this review, we summarize and discuss the genetics, regulation, epidemiology, structure/function relationships, and in vivo and in vitro activities of the best characterized RTX toxins, and speculate on their roles in pathogenesis and their use in immunotherapy.

Antitoxins↗
PubMed1994

Assays of hemolytic toxins.

The ability to produce a cytolytic toxin contributes to the success of many organisms in a particular niche by such diverse means as lysis of a phagolysosomal membrane of the macrophage by hemolysin from the intracellular parasite Trypanosoma cruzi, disruption of leukocyte activity by the Escherichia coli hemolysin, and destruction of invading bacteria by hemolysin from the annelid Glycera dibranchiata. The relative contribution of erythrocyte lysis to survival of the cytolysin producer is still under investigation. Nevertheless, the hemolytic phenotype is both a powerful tool for identifying novel cytolysins and a convenient marker for studying cytolytic activity in established toxins.

Animals↗
PubMed1994

Laparoscopically assisted vaginal hysterectomy appears to be an alternative to total abdominal hysterectomy.

Seventy percent of hysterectomies in the United States are performed as total abdominal hysterectomies (TAH). Laparoscopic assisted vaginal hysterectomy (LAVH) has been promoted by some to be a low morbidity substitute for TAH. In this study, we compared the outcomes of 61 recent LAVH cases to 65 TAH cases performed during an earlier time interval. Data were abstracted from the charts of 65 TAH cases performed between August 1988 and December 1990 and 61 LAVH cases performed between June 1991 and September 1992 by the same gynecologist (E.D.R.). Patient characteristics and perioperative morbidities were compared. Patient characteristics were similar between the two groups except that LAVH procedures were performed on thinner women (148 +/- 3.4 vs 162 +/- 4.3 lbs, p < or = 0.01, LAVH vs TAH) and operating time was longer (137 +/- 4.1 vs 66 +/- 1.7 min, p < or = 0.0001). Uterine weights were comparable (152 +/- 12 vs 194 +/- 31 g, p = 0.2). There was less intraoperative blood loss (337 +/- 21 vs 417 +/- 16 ml, p < or = 0.003), febrile morbidity (9.8 vs 66%, p < or = 0.0001), respiratory complications (6.6 vs 22%, p < or = 0.05), and urinary morbidity (6.6 vs 24.4%, p < or = 0.02) and shorter postoperative hospitalization (22 +/- 1.2 vs 99 +/- 2.3 h, p < or = 0.0001) in the LAVH group. Thinner patients were selected for the LAVH procedure, and operating room time was twice that of those undergoing TAH. Conversely, perioperative morbidity and postoperative hospitalization time were significantly less with LAVH. LAVH appears to be an alternative to TAH.

Adult↗
PubMed1994

Effects of temperature, time, and toxin concentration on lesion formation by the Escherichia coli hemolysin.

We performed osmotic protection experiments to test the hypothesis that the Escherichia coli hemolysin forms a discrete-size pore in erythrocyte membranes. The effects of toxin concentration, assay time, temperature, and protectant concentrations were examined. The results we present here raise doubts about the existing model of pore formation by hemolysin. We demonstrate that osmotic protection by various sugars of different sizes is a function of hemolysin concentration and assay time. The data indicate that under various conditions, lesion sizes with a diameter ranging from < 0.6 to > 1.2 nm can be inferred. Quantification of hemolysin permitted the estimation of the number of HlyA structural protein molecules required per erythrocyte for lysis in the presence of each protectant. It appears that hemolysin induces heterogeneous erythrocyte lesions which increase in size over time. Influx experiments utilizing radioactive sugar markers indicated that time-dependent osmotic protection patterns are independent of the diffusion rates of individual protectants. We demonstrate that the rate of the putative growth in the size of hemolysin-mediated lesions is temperature dependent. The erythrocyte membrane lesions formed at 37 degrees C can be stabilized in size when shifted to 4 degrees C. On the basis of these data, new models for the nature of the hemolysin-mediated erythrocyte membrane lesions are presented.

Animals↗
PubMed1994

Analysis of toxinogenic functions associated with the RTX repeat region and monoclonal antibody D12 epitope of Escherichia coli hemolysin.

Amino acids (aa) 550 through 850 of the Escherichia coli hemolysin (HlyA) contain sequences important for several steps in cytolysis. These include the Ca(2+)-binding glycine-rich tandem repeats recognized by the monoclonal antibody A10, the putative HlyC-dependent acylation site that corresponds to the monoclonal antibody D12 epitope, and the erythrocyte specificity domain which confers erythrolytic activity to the Pasteurella haemolytica leukotoxin. To further investigate the toxinogenic functions associated with this region of HlyA, we constructed mutants in the hlyA sequences coding for the repeat region and the D12 epitope. Mutants were analyzed for anti-HlyA antibody reactivity, cytolytic activities, target cell binding, Ca2+ requirements, and virulence. The D12 epitope was mapped to aa 673 through 726, with portions of the epitope both amino terminal and carboxy terminal to aa 700. This region was necessary, but not sufficient, for toxin binding to erythrocytes. A substitution at aa 684 resulted in loss of the D12 epitope, while cytolytic activity was retained. The nature of the D12 epitope and its associated functions are discussed. The A10 epitope mapped to residues 745 through 829, corresponding to repeats 4 through 11. Insertions within the glycine-rich repeats resulted in mutant forms of HlyA which retained A10 reactivity but required increased Ca2+ for lytic activity. These in vitro effects on cytolysis corresponded to a significant decrease in HlyA-mediated virulence in mice. HlyA from one insertion mutant was able to associate with leukocyte membranes under conditions that were Ca2+ deficient for cytolysis. The role of the glycine-rich repeats and Ca2+ in HlyA activity are discussed.

Animals↗
PubMed1994

Use of two Greenfield caval filters to prevent recurrent pulmonary embolism in a heparin-allergic gravida.

BACKGROUND: Maternal mortality may be reduced by prompt diagnosis and treatment of pulmonary embolism. CASE: A 25-year-old pregnant woman required a second Greenfield filter after developing a heparin allergy and recurrent pulmonary embolism. CONCLUSION: Heparin allergy has not been reported previously in pregnancy. A Greenfield filter may be used in this circumstance. Extension of the thrombus cephalad to the filter can cause recurrent emboli, so deployment of a second caval filter may be an effective remedy.

Adult↗
PubMed1993

Low periconceptional maternal serum thymosin alpha 1 levels are associated with blighted pregnancies.

OBJECTIVE: The thymus-derived peptides, thymosin alpha 1 and thymosin beta 4, are believed to contribute to the maintenance of immune homeostasis. They are also associated with the hypothalamic-pituitary-adrenal-gonadal axis and may play a role in reproduction. STUDY DESIGN: Patients were recruited from a university hospital setting. Eligible candidates were 24 to 38 years old who were being seen in an ovulation induction and in vitro fertilization program. Serial maternal serum thymosin alpha 1 and beta 4 levels were assayed preconceptual and then twice in the first trimester by ELISA in 28 women with known ovulation dates who successfully conceived as demonstrated by positive serum beta human chorionic gonadotropin (hCG). Thymosin alpha 1 and beta 4 serum levels for viable pregnancies (group I; N = 19) were compared to pregnancies that aborted (group II; N = 9) using repeated measures of multivariate analysis of variance (MANOVA). Periconceptional (preovulatory and early pregnancy) thymosin alpha 1 and beta 4 values between groups I and II were compared using repeated measure one-way ANOVA. RESULTS: Thymosin alpha 1 levels from pregnancies that remained viable were significantly higher than those from pregnancies that spontaneously aborted. Preovulation thymosin alpha 1 levels also tended to be lower in pregnancies that subsequently aborted. Thymosin beta 4 levels were similar between the two groups. CONCLUSION: Decreased maternal serum thymosin alpha 1 levels may be associated with periconceptional endocrine and/or immune disturbances preceding miscarriage.

Abortion, Spontaneous↗
PubMed1992

Markers for endothelial injury, clotting and platelet activation in preeclampsia.

The etiology of disseminated intravascular coagulation (DIC) in preeclampsia is not well understood. We measured plasma levels of fibronectin (FN), which may reflect endothelial cell injury, fibrinopeptide A (FPA), a specific marker of clotting, platelet counts (PLC) and mean platelet volumes (MPV), as well as beta-thromboglobulin (beta TG) and platelet factor 4 (Pf4), products of irreversible platelet activation in 24 preeclamptic patients and 24 controls matched for age, gestational age, labor status, and parity. In preeclampsia, FN and FPA were significantly elevated while PLC were significantly decreased (P less than 0.0001, less than 0.05 and less than 0.01, respectively). beta TG, Pf4, and MPV values did not show significant differences. These findings support the hypothesis that endothelial injury, clotting activation and platelet consumption are increased in preeclampsia. However, the much closer association of preeclampsia with FN levels as compared to FPA, beta TG, Pf4, suggests that endothelial injury is a more basic mechanism of preeclampsia than clotting or platelet activation.

Adolescent↗
PubMed1992

The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins.

The RTX group of exotoxins represents a branch of a family of exoproteins produced by Gram-negative bacteria which share the properties of being secreted by a leader-independent pathway and a tandemly-repeated nine-amino-acid sequence that is responsible for calcium binding. The Escherichia coli hemolysin (HlyA) is the prototype for the RTX exotoxin family which includes the leukotoxins of Pasteurella haemolytica and Actinobacillus actinomycetemcomitans and hemolysins from four Gram-negative genera. A review of the genetics, synthesis, export and target cell reactivity of the E. coli hemolysin is given. An evolutionary tree of the RTX toxin family based on amino acid sequence similarity is presented.

Animals↗
PubMed1992

A heterologous membrane protein domain fused to the C-terminal ATP-binding domain of HlyB can export Escherichia coli hemolysin.

The hydrophobic-rich NH2-terminal 34 amino acids of a tetracycline resistance determinant (TetC) were fused to the COOH-terminal 240 amino acids of the hemolysin transporter, HlyB, which contains a putative ATP-binding domain. This hybrid protein replaced the NH2-terminal 467-amino-acid portion of HlyB and could still export the Escherichia coli hemolysin (HlyA). Export by the hybrid protein was approximately 10% as efficient as transport by HlyB. Extracellular secretion of HlyA by the TetC-HlyB hybrid required HlyD and TolC. The extracellular and periplasmic levels of beta-galactosidase and beta-lactamase in strains that produced the hybrid were similar to the levels in controls. Thus, HlyA transport was specific and did not appear to be due to leakage of cytoplasmic contents alone. Antibodies raised against the COOH terminus of HlyB reacted with the hybrid protein, as well as HlyB. HlyB was associated with membrane fractions, while the hybrid protein was found mainly in soluble extracts. Cellular fractionation studies were performed to determine whether transport by the hybrid occurred simultaneously across both membranes like wild-type HlyA secretion. However, we found that HlyA was present in the periplasm of strains that expressed the TetC-HlyB hybrid. HlyA remained in the periplasm unless the hlyD and tolC gene products were present in addition to the hybrid.

Adenosine Triphosphate↗
PubMed1991

Pore-forming cytolysins of gram-negative bacteria.

A great deal is known about the structure, function and metabolic effects of enzymatic bacterial toxins such as the diphtheria, pertussis and cholera toxins. By comparison, our understanding of the pore-forming, cytolytic toxins, particularly those produced by Gram-negative bacterial pathogens, is far less complete. The genetics and biochemistry of a large, newly discovered family of calcium-dependent, pore-forming cytotoxins (RTX toxins) produced by different genera of the Enterobacteriaceae and Pasteurellaceae are discussed in this review. This toxin family is especially noteworthy because the individual toxins often exhibit different cell- and host-specificity. A brief review is also included of two ancestrally unrelated groups of calcium-independent, pore-forming toxins, the haemolysins produced by Proteus mirabilis and Serratia marcescens and the aerolysins secreted by species of Aeromonas. Their structure and function are contrasted with those of the RTX family members. Emerging questions about the role of cytolysins in pathogenesis are presented. Perhaps the most important issue raised is whether or not less attention should be paid to the lytic capacity of these cytotoxins, with more energy being devoted to the understanding of their non-lytic inhibitory activities against host cells.

Amino Acid Sequence↗
PubMed1991

Identification of RTX toxin target cell specificity domains by use of hybrid genes.

The Escherichia coli hemolysin (HlyA) and Pasteurella haemolytica leukotoxin (LktA) are cytolytic toxins encoded by genes belonging to the recently described RTX gene family. These cytotoxins are, respectively, 1,023 and 953 amino acids in length and are encoded by genes within identically organized operons. They share 45% amino acid sequence identities but differ in their target cell specificities. In vitro-derived recombinant hybrid genes between hlyA and lktA were constructed by using restriction endonuclease sites created by oligonucleotide site-directed mutagenesis. The cytolytic activity of hybrid proteins was investigated using as targets sheep erythrocytes and two cultured cell lines from different species (BL3, bovine leukemia-derived B lymphocytes; and Raji, human B-cell lymphoma cells). HlyA is cytolytic to all three cell types. LktA lyses only BL3 cells. Among the hybrid proteins displaying cytolytic activity, the striking finding is that the hemolytic activity of several LktA-HlyA hybrids was independent of any cytolytic activity against either cultured cell species. The hemolytic activity was associated with the HlyA region between amino acids 564 and 739. Structures that are critical for HlyA cytolytic activity against BL3 or Raji cells were destroyed when LktA-HlyA and HlyA-LktA hybrids were made, respectively, at amino acid positions 564 and 739 of HlyA. In contrast to HlyA, which lysed the two different cultured cell lines with equal efficiency, Lkt-HlyA hybrids possessing the amino-terminal 169 residues of LktA lysed BL3 cells more efficiently than Raji cells. This suggests that a significant but not exclusive element of the LktA ruminant cell specificity resides in the amino-terminal one-fifth of the protein. A molecular model of the functional domains of HlyA and LktA is presented.

Amino Acid Sequence↗