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T R Walsh

Publications and source records attributed to T R Walsh.

At least 55 records · Page 3Linked to original sources
PubMed1998

The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution.

The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 A resolution by the multiwavelength anomalous dispersion (MAD) approach exploiting both the intrinsic binuclear zinc centre and incorporated selenomethionine residues. L1 is unique amongst all known beta-lactamases in that it exists as a tetramer. The protein exhibits the alphabeta/betaalpha fold found only in the metallo-beta-lactamases and displays several unique features not previously observed in these enzymes. These include a disulphide bridge and two substantially elongated loops connected to the active site of the enzyme. Two closely spaced zinc ions are bound at the active site with tetrahedral (Zn1) and trigonal bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydrolytic reaction. Ligation of the second zinc ion involves both residues and geometry which have not been previously observed in the metallo-beta-lactamases. Simulated binding of the substrates ampicillin, ceftazidime and imipenem suggests that the substrate is able to bind to the enzyme in a variety of different conformations whose common features are direct interactions of the beta-lactam carbonyl oxygen and nitrogen with the zinc ions and of the beta-lactam carboxylate with Ser187. We describe a catalytic mechanism whose principal features are a nucleophilic attack of the bridging water on the beta-lactam carbonyl carbon, electrostatic stabilisation of a negatively charged tetrahedral transition state and protonation of the beta-lactam nitrogen by a second water molecule co-ordinated by Zn2. Further, we propose that direct metal:substrate interactions provide a substantial contribution to substrate binding and that this may explain the lack of specificity which is a feature of this class of enzyme.

Bacillus↗
PubMed1998

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria.

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.

Aeromonas↗
PubMed1998

Overexpression, purification, and characterization of the cloned metallo-beta-lactamase L1 from Stenotrophomonas maltophilia.

The metallo-beta-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, with k(cat)/Km values ranging between 0.002 and 5.5 microM(-1) s(-1). These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-beta-lactamases isolated directly from S. maltophilia.

Chromatography, Gel↗
PubMed1997

Susceptibility testing of Stenotrophomonas maltophilia to carbapenems.

The susceptibility of 20 clinical isolates of Stenotrophomonas maltophilia to the carbapenems imipenem and meropenem was investigated by various methods. S. maltophilia appeared sensitive to meropenem but resistant to imipenem by disc testing in Iso-sensitest agar. Agar dilution MICs were performed using Iso-sensitest agar and with incubation under three sets of atmospheric conditions. MICs of meropenem were considerably lower than those of imipenem; this effect was maximal after incubation in 5% CO2 when the MIC of meropenem was approximately 64 times less than that of imipenem. Induction experiments showed that both carbapenems could induce production of the L1 carbapenemase. However, disc approximation tests showed that imipenem could induced resistance to meropenem. Partially stably derepressed mutants were readily selected in vitro. We conclude that, although S. maltophilia may give large zones of inhibition to meropenem on disc testing, resistant mutants are readily selected and therefore standard sensitivity tests may be poorly predictive of clinical outcome of treatment of S. maltophilia infections with meropenem.

Bacterial Proteins↗
PubMed1997

Distribution and expression of beta-lactamase genes among Aeromonas spp.

Clinical and environmental isolates of Aeromonas spp. (20 Aeromonas caviae, 33 Aeromonas veronii and 21 Aeromonas hydrophila) were examined for their ability to yield mutants derepressed for beta-lactamase production and for the distribution of the three chromosomally encoded beta-lactamase genes, ampS and cepS from A. veronii bv. sobria and cphA from A. hydrophila. Of these isolates, 100% and 96% of the isolates from A. hydrophila and A. caviae, respectively, yielded beta-lactamase derepressed mutants but only 38% of A. veronii isolates yielded the resistant phenotype at 37 degrees C. However, when tested at 30 degrees C, all isolates gave rise to the derepressed mutants, indicating a temperature effect on the control mechanism. All mutants had significantly higher beta-lactamase activity against ampicillin, oxacillin, cephaloridine and imipenem. Hybridization studies with cloned aeromonas beta-lactamase genes indicated that the cephalosporinase gene, cepS, is almost ubiquitous for the three species tested. The cphA gene cross-hybridized with all isolates of A. veronii and A. hydrophila but not to A. caviae isolates. In contrast, hybridization studies using ampS revealed that only 25% of A. caviae, 45% of A. veronii and 38% of A. hydrophila tested carried the ampS gene or one closely homologous to it. Nonetheless, strains that failed to hybridize with ampS showed two serine beta-lactamases when analysed by isoelectric focusing.

Aeromonas↗
PubMed1997

Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.

Amino Acid Sequence↗
PubMed1996

Enzyme kinetics and biochemical analysis of ImiS, the metallo-beta-lactamase from Aeromonas sobria 163a.

The metallo-beta-lactamase from Aeromonas sobria 163a, ImiS, was isolated in a two stage purification procedure using protein affinity columns. Enzyme kinetics show that ImiS hydrolyses the carbapenems but displays poor activity against other beta-lactams. ImiS possesses the narrowest spectrum of activity of the Group 3 enzymes that have been analysed. Sequencing of the 40 N-terminal amino acids show this region to be identical to that of the CphA metallo-beta-lactamase from Aeromonas hydrophila (Massidda, Rossolini & Satta, 1991). Light scattering analysis indicates that ImiS is functionally active as a monomer.

Aeromonas↗
PubMed1995

A clinical isolate of Aeromonas sobria with three chromosomally mediated inducible beta-lactamases: a cephalosporinase, a penicillinase and a third enzyme, displaying carbapenemase activity.

Hydrolytic profiles of Aeromonas sobria 163a, non-induced and induced, and of a derepressed mutant 163a-M, indicate that this strain has inducible beta-lactamases with activities against cephalosporins, penicillins and carbapenems. Three enzymes were identified and two of the beta-lactamase genes, ampS, encoding a penicillinase and cepS, encoding a cephalosporinase, were cloned into Escherichia coli, permitting analysis of the individual enzymes. Isoelectric focusing (IEF) analysis using inhibition profiles with EDTA and BRL 42715, confirmed AmpS (pI 7.9) and CepS (7.0) to be serine beta-lactamases. A third beta-lactamase displaying hydrolytic activity against the carbapenems was inhibited by EDTA. The carbapenemase had a pI of 9.3 and was detected on IEF gels by overlaying the gel with agarose containing imipenem and the chromogenic pH indicator, bromothymol blue. Co-inducibility and the recovery of a derepressed mutant in which all three enzymes were produced at high levels indicate that this isolate of A. sobria has three co-ordinately controlled beta-lactamase genes.

Aeromonas↗
PubMed1995

Sequence analysis of two chromosomally mediated inducible beta-lactamases from Aeromonas sobria, strain 163a, one a class D penicillinase, the other an AmpC cephalosporinase.

Two beta-lactamase genes from Aeromonas sobria, strain 163a, have been cloned and sequenced, one encoding a typical class C cephalosporinase, designated CepS, the other a class D penicillinase, designated AmpS. CepS is predicted to be a mature protein of 38 kDa with a pI value of 7.0. The amino acid sequence of CepS is most similar to that of AmpC from Pseudomonas aeruginosa (54.7%). AmpS is predicted to be a mature protein of 27 kDa with a pI value of 7.9 that mostly closely resembles BLAD from Klebsiella pneumoniae (42.2%), and OXA-1 from Escherichia coli (36.6%), beta-lactamases that are encoded by genes carried on multiresistant transposons. AmpS differs significantly from the other class D beta-lactamases in that it hydrolyses cloxacillin poorly and is inducible. Both genes exhibit a high overall GC content and possess a high NNC and NNG codon preference, similar to that of genes from Pseudomonas spp.

Aeromonas↗
PubMed1994

Sequence analysis of the L1 metallo-beta-lactamase from Xanthomonas maltophilia.

The amino acid sequence deduced from the L1 beta-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-beta-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 beta-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-beta-lactamases.

Amino Acid Sequence↗
PubMed1993

Response of normal and reperfused livers to glucagon stimulation: NMR detection of blood flow and high-energy phosphates.

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (P(i)) to ATP ratio in normal livers as measured by 31P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the P(i)/ATP ratio of 18%, 40%, and 40%, respectively. 19F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to decreases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.

Adenosine Triphosphate↗
PubMed1992

Changing etiology of iliopsoas abscess.

Over a 5-year period, iliopsoas abscesses were found in 11 patients. Although the most common underlying condition was Crohn's disease (3 of 11 patients), 5 abscesses resulted from hematogenous spread from a distant site. Each of these five patients was elderly, severely malnourished, or had an underlying chronic disease. Fever was a presenting sign in 8 of 11 patients, whereas all 4 patients who presented with back pain had nontuberculous lumbar osteomyelitis or disk space infections. No patient presented with the classic triad of fever, back pain, and anterior thigh or groin pain. Computed tomographic (CT) scans accurately established the clinical diagnosis in 10 of 11 patients. Two of the patients died. One patient was an intravenous drug abuser, whereas the other patient was being treated with steroids for systemic lupus erythematosus. Elderly patients, diabetics, and patients with chronic disease are susceptible to this kind of occult infection and may present with minimal clinical findings. Aggressive diagnosis using CT scanning and treatment with resection of involved bowel, complete drainage of the abscess, and prolonged antibiotics are required to salvage these patients.

Abscess↗
PubMed1992

An improved technique for isolated perfusion of rat livers and an evaluation of perfusates.

We have modified the apparatus for isolated rat liver perfusion (IPRL) in order to be able to perform two perfusions simultaneously. In addition, we studied the quality and stability of livers by comparison of five different perfusates: Blood (Group A), Original Krebs Henseleit buffer (Group B), Krebs buffer with glucose (Group C) or bovine serum albumin (BSA) added, (Group D). In a last group (E) albumin, glucose, and taurocholic acid were added to Krebs. After 180 min of perfusion, livers perfused with solutions including 2% albumin (Group D, E) had a significantly higher release of hepatocellular and endothelial cell (purine nucleoside phosphorylase) enzymes and lower bile production as compared to Groups A, B, and C (P less than 0.0001). Increasing levels of purine nucleoside phosphorylase (PNP), a reflection of damage to the microvascular endothelium preceded the increases in hepatocellular enzymes. Histologically, damages of sinusoidal endothelial cells and hepatocytes are appreciated moderate to severe in Groups D and E, slight to mild in Groups A and B, and not significant in Group C. These results suggest that BSA may have toxic effects to the perfused rat liver. These data also confirm that the IPRL modified for simultaneous perfusion of two livers is efficient, and that with this technique the rat liver can be optimally perfused for up to 3 hr with oxygenated Krebs Henseleit buffer without additives (Group B) and without blood. These two improvements should allow those performing studies with perfused rat livers to obtain data in a more efficient, accurate, and inexpensive fashion.

Animals↗
PubMed1990

Purine nucleoside phosphorylase: a new marker for free oxygen radical injury to the endothelial cell.

The effect of ischemia and reperfusion on purine nucleoside phosphorylase was studied in an isolated perfused rat liver model. This enzyme is localized primarily in the cytoplasm of the endothelial and Kupffer cells; some activity is associated with the parenchymal cells. Levels of this enzyme accurately predicted the extent of ischemia and reperfusion damage to the microvascular endothelial cell of the liver. Livers from Lewis rats were subjected to 30, 45 and 60 min of warm (37 degrees C) no flow ischemia that was followed by a standard reperfusion period lasting 45 min. Purine nucleoside phosphorylase was measured at the end of the no flow ischemia and reperfusion periods as was superoxide generation (O2-). Bile production was monitored throughout the no flow ischemia and reperfusion periods. Control perfusions were carried out for 120 min. A significant rise in purine nucleoside phosphorylase levels as compared with controls was observed at the end of ischemia in all the three groups. The highest level, 203.5 +/- 29.2 mU/ml, was observed after 60 min of ischemia. After the reperfusion period, levels of purine nucleoside phosphorylase decreased in the 30- and 45-min groups 58.17 +/- 9.66 mU/ml and 67.5 +/- 17.1 mU/ml, respectively. These levels were equal to control perfusions. In contrast, after 60 min of ischemia, levels of purine nucleoside phosphorylase decreased early in the reperfusion period and then rose to 127.8 +/- 14.8 mU/ml by the end of reperfusion (p less than 0.0001). Superoxide generation at the beginning of reperfusion was higher than in controls with similar values observed at the end of 30, 45 and 60 min of ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Lipid peroxidation is a nonparenchymal cell event with reperfusion after prolonged liver ischemia.

A proposed mechanism for irreversible ischemic liver damage has been peroxidation of membrane phospholipids by free radicals. However, the hepatocyte is laden with enzymes which are antioxidants and, therefore, ought to be relatively resistant to oxidative injury. To test the hypothesis that free radical damage from ischemia and reperfusion of the liver is a nonparenchymal cell process, we studied an in vivo model of ischemia. A point of transition from reversible to irreversible ischemia was defined at greater than or equal to 60 min of total ischemia by serial measurements of ATP at control, end of ischemia, and end of reperfusion periods (n = 6 each). Nonparenchymal cells were separated out of 10 livers in each ischemic group using a Percoll gradient. Second derivative spectroscopy did not detect conjugated dienes in any hepatocellular fraction, total cellular, mitochondrial, or microsomal, but did in the nonparenchymal cell fractions of livers from the 60- and 90-min ischemia groups. This in vivo study shows that irreversible ischemia in the rat liver is associated with free radical lipid peroxidation, but that the nonparenchymal cells rather than hepatocytes are the focus of this injury.

Adenosine Triphosphate↗
PubMed1990

Inhibition of free radical generation and improved survival by protection of the hepatic microvascular endothelium by targeted erythrocytes in orthotopic rat liver transplantation.

The capacity of specifically targeted erythrocytes to inhibit free radical-mediated injury to the endothelial cell after cold preservation, and improve liver function was studied in two experimental models: An isolated perfused rat liver (IPRL) system and syngeneic orthotopic rat liver transplantation. In the IPRL model, livers were preserved in University of Wisconsin solution for 24 h at 4 degrees C. At the end of the preservation period, livers were flushed with lactated Ringer's (control), immunoerythrocytes (IES), or blank intact erythrocytes prior to warm reperfusion for 2 h using an assanguinous Krebs-Henseleit buffer. Production of superoxide (O2-) anion during warm reperfusion in the IES-treated liver was reduced by 65% as compared with controls (P less than 0.001) and by 74% (P less than 0.001) when compared with blank erythrocyte-treated livers. Endothelial cell preservation, as assessed by levels of purine nucleoside phosphorylase (PNP), was much better in the IES-treated group (P less than 0.001) when compared with untreated livers. Hepatocellular preservation was markedly improved in the IES-treated livers. In the syngeneic liver transplantation model, livers were preserved in UW solution for 24 h at 4 degrees C. Prior to implantation, livers were flushed with 5 ml of cold lactated Ringer's or immunoerythrocytes. Survival after three weeks was 60% in the IES-treated group and 30% in the untreated group. Survival in the IES-treated group was not significantly different from a control (no preservation) group. IES-treated livers in both models demonstrated better endothelial cell integrity and ultimate liver function. IES treatment therefore appears to protect the hepatic microvascular endothelial cell from reperfusion injury and could prove to be an easy reproducible method of donor organ preparation after cold preservation.

Animals↗
PubMed1990

A modified apparatus for dual, sterilized, isolated perfusion of the rat liver.

The isolated perfused rat liver (IPRL) has proven to be a useful model for the study of physiology and pathology of the liver. For research in nonparenchymal cell (NPC) function that includes measurement of cytokine production (eg, TNF), it is necessary to have a sterilized perfusion system. We have modified the IPRL apparatus so as to be able to perform sterile perfusions of two livers simultaneously. The perfusion apparatus is a recirculating closed system in which the oxygenator is a plastic container separated into two chambers by a fenestrated plastic wall. A disposable macropore filter functions as both a bubble trap and perfusate filter. The sterilization process is done by immersing the various components in Benz-All solution. The tubing is disinfected by irrigation with 10% Clorox followed by 0.9% sodium chloride solution. The perfusate used is filter-sterilized Krebs buffer solution containing 0.5 g Mandol/250 mL perfusate. Not only can two organs be conveniently perfused simultaneously, but the entire system can be reliably sterilized for up to 20 consecutive perfusions. Bile production is higher and more stable with less leakage of intracellular enzymes. Many of the components are disposable and can be altered to suit the needs of a particular experiment.

Animals↗