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Biomedical subjects

M J Lynch

Publications and source records attributed to M J Lynch.

At least 55 records · Page 3Linked to original sources
PubMed1991

Evidence for leucine zipper motif in lactose repressor protein.

Amino acid sequence homology between the carboxyl-terminal segment of the lac repressor and eukaryotic proteins containing the leucine zipper motif with associated basic DNA binding region (bZIP) has been identified. Based on the sequence comparisons, site-specific mutations have been generated at two sites predicted to participate in oligomer formation based on the three-leucine heptad repeat at positions 342, 349, and 356. Leu342----Ala, Leu349----Ala, and Leu349----Pro have been isolated and their oligomeric state and ligand binding properties evaluated. These mutant proteins do not form tetramers but exist as stable dimers with inducer binding comparable with the wild-type protein. Apparent operator affinities for lac repressor proteins with mutations in the proposed bZIP domain were significantly lower than the corresponding wild-type values. For these dimeric mutant proteins, the monomer-dimer equilibrium is linked to the apparent operator binding constant. The values for the monomer-monomer binding constant and for the intrinsic operator binding constant for the dimer cannot be resolved from measurements of the observed Kd for operator DNA. Further studies on these proteins are in progress.

Amino Acid Sequence↗
PubMed1991

Clinical pharmacokinetics of parenterally administered danofloxacin in cattle.

Danofloxacin is a new fluoroquinolone antibacterial, developed specifically for veterinary use. Its in vitro activity and pharmacokinetic properties have been investigated to assess its potential for use in the therapy of respiratory disease in cattle. The minimum inhibitory concentration of danofloxacin against 90% (MIC90) of contemporary European and North American field isolates of Pasteurella haemolytica, Pasteurella multocida and Haemophilus somnus, the most important bacterial respiratory pathogens of cattle, was 0.125 micrograms/ml. The plasma and lung kinetics of danofloxacin following parenteral administration of 1.25 mg/kg were evaluated in two studies. Danofloxacin was rapidly absorbed following intramuscular and subcutaneous injection and bioavailability was virtually complete (101% and 94% respectively). Plasma concentration profiles of danofloxacin were similar for intramuscular and subcutaneous routes with no significant differences in the area under the plasma concentration-time curves (AUC) following one, three or five consecutive daily doses, although slightly higher peak plasma concentrations were achieved by the intramuscular route. Following intramuscular administration, the mean peak lung concentration of danofloxacin was 4.1 times greater than that of plasma. Similarly, the AUC for lung tissue was 3.7 times greater than that for plasma. These data indicate that danofloxacin should be particularly appropriate for the therapy of bacterial respiratory disease in cattle.

Animals↗
PubMed1991

Use of an antibacterial powder spray to prevent post prostatectomy urinary infection.

To prevent ascending urinary infection in patients following transurethral prostatectomy, we have studied the use of a topical antibacterial agent applied to the groin and external genitalia. A prospective randomized controlled trial was conducted, with 50 patients being treated by daily application of a 2% polynoxylin powder, and 50 patients acting as untreated controls. Patient age and duration of catheterization were similar in the treated and untreated groups. No significant benefit was seen in the treated cohort (P greater than 0.05) in terms of reduction in postoperative urinary infection.

Administration, Topical↗
PubMed1991

Regulation by estrogen through the 5'-flanking region of the transforming growth factor alpha gene.

Expression of transforming growth factor alpha (TGF alpha) mRNA and protein can be stimulated by estrogens such as 17 beta-estradiol (E2) in estrogen-responsive rodent and human breast cancer cells. To ascertain if E2 can directly regulate TGF alpha expression through the 5'-flanking region of the human TGF alpha gene, E2-responsive MCF-7 or ZR-75-1 human breast cancer cells or E2-nonresponsive MDA-MB-231 breast cancer cells were transiently transfected with a plasmid containing an 1140-base pair (bp) Sac-I fragment of the TGF alpha 5'-flanking region ligated to the chloramphenicol acetyltransferase (CAT) gene. Cells that were transfected and subsequently treated with physiological concentrations of E2 (10(-11)-10(-8) M) for 24 h exhibited a 2- to 10-fold increase in CAT activity. The E2 stimulation of CAT activity was dose-dependent with an increase first found at 10(-10) M E2. The increase in CAT activity could be detected within 24-36 h after the addition of E2. There was no significant change in CAT activity in transiently transfected MDA-MB-231 cells as mediated through the TGF alpha 5'-flanking region after E2 treatment. MCF-7 cells were also transiently transfected with different fragments of the TGF alpha 5'-flanking region ligated to the luciferase gene. In the absence of E2 treatment, no detectable luciferase activity was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Recovery of the neonatal heart after normothermic ischemia. Effect of oxygen and catalase.

The objective of this study was to determine the effect of oxygen and the oxygen radical-scavenging enzyme catalase on the neonatal rabbit heart exposed to global ischemia. The experiments were performed with an isolated neonatal (7 to 10 days of age) working heart model in which normothermic (37 degrees C) ischemia was produced for 60 minutes. Left ventricular developed pressure, ratio of change of ventricular pressure to change in time, and aortic flow were measured before ischemia and 30 minutes after reperfusing the hearts with physiologic saline solution. In the control group (ischemia only), developed pressure and ratio of change of ventricular pressure to change in time recovered to 27% +/- 3% (mean +/- standard error of the mean) and 24% +/- 7% of baseline; the hearts were incapable of ejecting (aortic flow = 0). Treatment of hearts before and after ischemia with catalase (150 units/ml of perfusate) was studied in a second group (control plus catalase), but functional recovery (developed pressure = 32% +/- 1%; ratio of change of ventricular pressure to change in time = 24% +/- 2%, and aortic flow = 0) was not significantly different from the control group. The effect of washout midway through the ischemic period with a low oxygen (oxygen concentration less than 35 mm Hg) solution was measured in a third group (hypoxic physiologic saline solution). Functional recovery (developed pressure = 13% +/- 3%; ratio of change of ventricular su pressure to change in time = 13% + 2%; aortic flow = 0) was not significantly different from the control and control plus catalase groups. In marked contrast were the effects of washout with an oxygenated (oxygen concentration greater than 500 mm Hg) solution (oxygenated physiologic saline solution) in which functional recovery (developed pressure = 78% +/- 3%; ratio of change of ventricular pressure to change in time = 80% +/- 3%; aortic flow = 39% +/- 9%) was significantly better than in the control, control plus catalase, and hypoxic physiologic saline solution groups. Use of modified St. Thomas' Hospital cardioplegic solution (cardioplegic solution group) during the ischemic period also resulted in substantial functional recovery (developed pressure = 80% +/- 3%; ratio of change of ventricular pressure to change in time = 78% +/- 5%; aortic flow = 64% +/- 7%) that did not differ significantly from that in the oxygenated physiologic saline solution group.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Morganella morganii urease: purification, characterization, and isolation of gene sequences.

Morganella morganii, a very common cause of catheter-associated bacteriuria, was previously classified with the genus Proteus on the basis of urease production. M. morganii constitutively synthesizes a urease distinct from that of other uropathogens. The enzyme, purified 175-fold by passage through DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and Superose 6 chromatography resins, was found to have a native molecular size of 590 kilodaltons and was composed of three distinct subunits with apparent molecular sizes of 63, 15, and 6 kilodaltons, respectively. Amino-terminal analysis of the subunit polypeptides revealed a high degree of conservation of amino acid sequence between jack bean and Proteus mirabilis ureases. Km for urea equalled 0.8 mM. Antiserum prepared against purified enzyme inhibited activity by 43% at a 1:2 dilution after 1 h of incubation. All urease activity was immunoprecipitated from cytosol by a 1:16 dilution. Antiserum did not precipitate ureases of other species except for one Providencia rettgeri strain but did recognize the large subunits of ureases of Providencia and Proteus species on Western blots (immunoblots). Thirteen urease-positive cosmid clones of Morganella chromosomal DNA shared a 3.5-kilobase (kb) BamHI fragment. Urease gene sequences were localized to a 7.1-kb EcoRI-SalI fragment. Tn5 mutagenesis revealed that between 3.3 and 6.6 kb of DNA were necessary for enzyme activity. A Morganella urease DNA probe did not hybridize with gene sequences of other species tested. Morganella urease antiserum recognized identical subunit polypeptides on Western blots of cytosol from the wild-type strain and Escherichia coli bearing the recombinant clone which corresponded to those seen in denatured urease. Although the wild-type strain and recombinant clone produced equal amounts of urease protein, the clone produced less than 1% of the enzyme activity of the wild-type strain.

Amino Acid Sequence↗
PubMed1990

Neutrophils are not necessary for induction of ischemia-reperfusion lung injury.

Ischemia-reperfusion lung injury limits lung transplantation. Neutrophil activation and/or xanthine oxidase-mediated purine degradation may cause toxic oxygen metabolite production and lung injury. We investigated whether circulating blood elements are involved in the pathogenesis of ischemia-reperfusion lung injury. Isolated rat lungs were perfused with physiological salt solution (PSS) stabilized with Ficoll until circulating blood elements were not detected in the lung effluent. Lungs were then rendered ischemic by stopping ventilation and perfusion for 45 min at room temperature. Lung injury occurred and was quantitated by the accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid during reperfusion. Lung injury occurred, in the absence of circulating blood elements, when ischemic lungs were reperfused with PSS-Ficoll solution alone. Reperfusion with whole blood or PSS-Ficoll supplemented with human or rat neutrophils did not increase lung injury. Furthermore, during lung ischemia, the presence of neutrophils did not enhance injury. Experiments using PSS-albumin perfusate and quantitating lung injury by permeability-surface area product yielded similar results. Microvascular pressures were not different and could not account for the results. Toxic O2 metabolites were involved in the injury because addition of erythrocytes or catalase to the perfusate attenuated the injury. Thus reperfusion after lung ischemia causes injury that is dependent on a nonneutrophil source of toxic O2 metabolites.

Animals↗
PubMed1989

Immortalization of primary baby rat kidney cells by retroviral mediated gene transfer of adenovirus E1A genes.

We describe a retroviral mediated gene transfer system that facilitates the transfer of heterologous genes into primary epithelial cells. Using a series of retroviral vectors containing the genes coding for the 12S and 13S mRNAs from the E1A region of adenovirus type 5 and the hygromycin resistance gene as a selectable marker, we demonstrate that, when packaged as helper virus-free amphotropic particles, these recombinant retroviruses infect murine fibroblasts and rat and human epithelial cells. Moreover, one of these constructs, containing genes for either the 12S or 13S product, is able to immortalize primary baby rat kidney epithelial cells in vitro and, thus, when used in combination with optimized culture conditions, may facilitate the establishment in culture of primary epithelial cells from a wide variety of tissues and species.

Animals↗
PubMed1989

Extended standard curve stability on the CCD Magic Lite immunoassay system using a two-point adjustment.

Ciba Corning Diagnostics has developed a two-point adjustment algorithm for use with the Magic Lite System which allows for extended stability of a full 7-10 point calibration curve over the life of a kit. This adjustment is accomplished by using a master calibration curve established during manufacturing along with two calibrators for each assay. Most conventional non-automated immunoassays contain anywhere from 6 to 10 calibrators which are included with each run of an assay. Eliminating the need to run full standard curves and using the two-point adjustment algorithm results in significant savings in both labour and reagent usage.

Algorithms↗
PubMed1989

Distribution of cardioplegic solution infused antegradely and retrogradely in normal canine hearts.

The adequacy of retrograde delivery of cardioplegic solution to the right ventricle and interventricular septum is controversial. To address this issue quantitatively, we infused blood cardioplegic solution labeled with radioactive microspheres (15 microns diameter) into the coronary sinus (n = 8 dogs) at a pressure of 51 +/- 1 mm Hg (mean +/- standard error of the mean) to be compared with the same quantity of labeled cardioplegic solution (20 ml/kg) delivered through the aorta (n = 6 dogs) at 97 +/- 7 mm Hg. Both methods of delivery produced cardiac arrest, but retrograde infusion required a significantly longer time to complete the infusion (6.2 +/- 0.8 minutes versus 1.5 +/- 0.1 minutes, p less than 0.01). Greater than 99% of the microspheres passing through the vasculature of the left ventricle were trapped in the left ventricular myocardium with antegrade infusion, and the distribution of the cardioplegic solution was uniform. Antegrade delivery (cardioplegic flow x infusion time) averaged approximately 3.0 to 4.0 ml/gm, except at the apex, where delivery averaged approximately 2.0 ml/gm. With retrograde infusion, 93% of the microspheres perfusing the left ventricle were trapped and delivery of the cardioplegic solution was not uniform. In the anterolateral free wall, delivery of cardioplegic solution averaged between 1.5 and 2.9 ml/gm (p less than 0.001 compared with antegrade) and only 0.6 to 0.8 ml/gm in the posteroseptal region of the basal left ventricle (p less than 0.001 compared with the antegrade group and anterolateral samples of the retrograde group). In the middle portion of the right ventricle, antegrade trapping of microspheres was 99% and delivery of cardioplegic solution averaged approximately 2.0 ml/gm. With retrograde delivery, only 16.5% (range 11.8% to 26.0%) of the microspheres passing through the right ventricular vasculature were trapped in the right ventricular myocardium, which indicates that substantial shunting had occurred. Corrected for the high shunt fraction, retrograde delivery of cardioplegic solution to the middle portion of the right ventricle averaged only 0.5 ml/gm (p less than 0.01). Retrograde delivery to the atrial septum and right atrium was also low. Because retrograde delivery of cardioplegic solution was markedly nonuniform, we conclude that inadequate cardioplegic delivery to the middle portion of the right ventricle and posteroseptal portion of the left ventricle could result with cardioplegic infusion through the coronary sinus.

Animals↗
PubMed1989

Hypothermia or continuous ventilation decreases ischemia-reperfusion injury in an ex vivo rat lung model.

The susceptibility of lung tissue to ischemia-reperfusion injury has made distant procurement of heart-lung allografts difficult. The effects of hypothermia, ventilation without perfusion, and various reperfusion solutions (PSS/Ficoll or whole blood) on the development of ischemia-reperfusion lung injury were investigated. Use of an ex vivo rat lung model in which the above variables were individually varied permitted a direct approach for these studies. Normothermic ischemia for 1 hour caused significant damage, documented by increased iodine 125 bovine serum albumin (125I-BSA) in alveolar lavage fluid and lung parenchyma compared with nonischemic controls. Hypothermic (4 degrees C) ischemia for 4 hours in lungs reperfused with salt solution and for as many as 12 hours in lungs reperfused with whole blood caused no significant increase in 125I-BSA in alveolar lavage fluid and lung parenchyma compared with nonischemic controls. Lungs ventilated without perfusion showed no increase in 125I-BSA leakage compared with controls. The ex vivo rat lung model is excellent for studying ischemia-reperfusion injury. It is reproducible, allows for variance of reperfusion solutions, and permits change in temperature and ventilation easily.

Animals↗
PubMed1988

Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury.

Ischemic-reperfusion lung injury is a factor potentially limiting the usefulness of distant organ procurement for heart-lung transplantation. Toxic oxygen metabolites are considered a major etiologic factor in reperfusion injury. Although oxygen-free radicals may be generated by many mechanisms, we investigated the role of xanthine oxidase in this injury process by using lodoxamide, a xanthine oxidase inhibitor, to inhibit ischemic-reperfusion injury in an isolated rat lung model. Isolated rat lungs were perfused with physiologic salt solution (PSS) osmotically stabilized with Ficoll until circulating blood elements were nondetectable in the pulmonary venous effluent. Lungs were rendered ischemic by interrupting ventilation and perfusion for 2 hr at 37 degrees C. After the ischemic interval, the lungs were reperfused with whole blood and lung injury was determined by measuring the accumulation of 125I-bovine serum albumin in lung parenchyma and alveolar lavage fluid as well as by gravimetric measurements. Lung effluent was collected immediately pre- and postischemia for analysis of uric acid by high-pressure liquid chromatography. Lodoxamide (1 mM) caused significant attenuation of postischemic lung injury. Uric acid levels in the lung effluent confirmed inhibition of xanthine oxidase. Protection from injury was not complete, however, implying that additional mechanisms may contribute to ischemic-reperfusion injury in the lung.

Animals↗
PubMed1988

Protection of the neonatal heart following normothermic ischemia: a comparison of oxygenated saline and oxygenated versus nonoxygenated cardioplegia.

Optimal methods of myocardial preservation in the neonate remain unknown. Hypothermia and cardioplegia have been shown to protect neonatal hearts, but few studies have examined the effects of cardioplegia when administered at normothermia. Accordingly, the role of 37 degrees C St. Thomas' cardioplegic solution in protecting the neonatal heart during 1 hour of ischemia in an isolated working rabbit heart model was examined. Both oxygenated and nonoxygenated cardioplegic solutions (CSs) were evaluated and compared with an oxygenated physiological saline solution (PSS). Following ischemia, control hearts were characterized by severely impaired left ventricular function, whereas all three treatment groups recovered well, indicating that the treatments provided substantial protection. Aortic flow recovered to 62, 63, and 57% of preischemic values for the oxygenated CS, nonoxygenated CS, and oxygenated PSS groups, respectively. Similarly, rate of change of pressure recovered to 76, 80, and 76% of preischemic values for oxygenated CS, nonoxygenated CS, and oxygenated PSS groups. All values were significantly greater than those for the control group. Recovery of developed pressure was significantly improved in all three groups. End-diastolic pressure rose markedly following ischemia in control hearts, was not increased after ischemia in hearts receiving oxygenated and nonoxygenated CS, but was increased in the oxygenated PSS group. These data indicate that crystalloid cardioplegia and oxygenated PSS provide substantial protection in neonatal rabbit hearts, even when delivered at 37 degrees C. No additional benefit was seen when the cardioplegic solution was oxygenated. Therefore, either method of balancing the oxygen supply/demand ratio appears to be beneficial: supplying oxygen intermittently during ischemia (oxygenated PSS group) or decreasing oxygen demand during the ischemic period (cardioplegia groups).

Animals↗
PubMed1988

Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli.

Recombinant urease from Providencia stuartii has been expressed in and purified from Escherichia coli, and the genetic organization of the structural genes has been determined. Urease expression was induced by urea and repressed by nitrogen-rich components in the medium. The urease protein was purified 331-fold by DEAE-Sepharose, phenyl-Sepharose, Mono-Q, and phenyl-Superose chromatographies with a 7.3% yield. The enzyme possessed a Km for urea of 9.3 mM and hydrolyzed urea at a Vmax of 7,100 mumol/min per mg. P. stuartii urease is composed of three polypeptides (Mrs, 73,000, 10,0000, and 9,000) denoted by alpha, beta, and gamma. The native enzyme is best described as (alpha 1 beta 2 gamma 2)2, based on a native Mr of 230,000, obtained by gel filtration chromatography, and on the Coomassie blue staining intensities of the individual subunits. Atomic absorption analysis of the pure protein revealed 1.9 +/- 0.1 nickel ions per alpha 1 beta 2 gamma 2 unit. In vitro transcription-translation analysis of transposon insertion mutants of the recombinant urease demonstrated that the urease peptides are encoded on adjacent DNA sequences and transcribed as a polycistronic mRNA in the order gamma, beta, and then alpha. Three urease-defective insertion mutants were identified that did not affect synthesis of urease subunit polypeptides, indicating that some nickel processing, enzyme activation, or other function may also be necessary for producing an active urease.

Autoradiography↗
PubMed1988

Squamous cell cancer of the liver arising from a solitary benign nonparasitic hepatic cyst.

A case of squamous cell carcinoma arising from a solitary benign nonparasitic hepatic cyst (SBNHC) causing bile duct obstruction is presented. A review of the literature regarding SBNHC suggests that, although these lesions may appear benign, they may also undergo metaplastic and subsequent malignant transformation. This appears to be particularly true when the SBNHC is lined with squamous epithelium. Once squamous cell carcinoma arises from one of these lesions, the prognosis is extremely grave, despite all forms of surgical and medical management.

Carcinoma, Squamous Cell↗
PubMed1988

The effect of hypothermic ischemia on recovery of left ventricular function and preload reserve in the neonatal heart.

Neonatal and adult myocardium respond differently to ischemia. In addition, the neonatal heart possesses a limited preload reserve. The effect of uninterrupted hypothermic ischemia on recovery of left ventricular function and preload reserve was studied in two groups of isolated rabbit hearts: group 1 (neonates, n = 8), 7 to 10 days old; group 2 (adults, n = 15), 6 to 12 months old. Peak left ventricular systolic pressure, the first derivative of left ventricular systolic pressure, and heart rate were measured at left ventricular pressures of 0, 5, 10, and 15 mm Hg before and after 120 minutes of global ischemia at 27 degrees C. Before ischemia, left ventricular systolic pressure increased significantly at each increment of left ventricular end-diastolic pressure for both groups of hearts. After hypothermic ischemia, recovery of left ventricular systolic pressure was significantly reduced at each level of left ventricular end-diastolic pressure among neonatal hearts (range 75% to 79% of control values). The postischemic recovery of left ventricular systolic pressure in the adult hearts was markedly reduced from baseline values (range 43% to 53% of control values) and was significantly worse than that of neonatal hearts at each level of left ventricular end-diastolic pressure (p less than 0.001). Both groups were able to respond to increasing preload after ischemia. The slope of the curve describing the relationship between left ventricular end-diastolic pressure and percent recovery of left ventricular systolic pressure was not different from zero for neonatal hearts but was significantly greater than zero among the adults (0.22 +/- 0.21 versus 0.73 +/- 0.07, p = 0.0056). After ischemia, the first derivative of left ventricular systolic pressure fell significantly from control values among neonatal hearts (71% of control values). The reduction was considerably greater, however, among the adult hearts (54% of control values). These data indicate that the neonatal heart recovers systolic function better than the adult heart after global ischemia with moderate hypothermia.

Aging↗
PubMed1987

Emergence of resistance to imipenem in Pseudomonas aeruginosa.

The emergence of resistance to imipenem by Pseudomonas aeruginosa was investigated with four pairs of isolates. Each pair represented pretherapy (susceptible) and posttherapy (resistant) specimens. In all cases, the imipenem-resistant isolates did not demonstrate changed susceptibilities to other beta-lactams. Agarose gel electrophoresis revealed no change in plasmid profiles between any pair of isolates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the Sarkosyl-insoluble membrane protein revealed the loss of an outer membrane protein of apparent molecular mass 48 to 49 kilodaltons in posttherapy strains when grown with imipenem selection (5 micrograms/ml). There was no significant difference in the binding of [14C]imipenem to the penicillin-binding proteins of the pre- and posttherapy strains. Trichloroacetic acid precipitation of membranes isolated after growth in the presence of [14C]imipenem revealed that significantly less drug was bound to Sarkosyl-soluble membrane protein in three of the four posttherapy strains than the membrane proteins of the respective pretherapy strains. beta-Lactamase activity against imipenem at 100 or 3 microM was not detected in any isolate either with or without induction. These data suggest that resistance to imipenem is associated with the loss of a 48- to 49-kilodalton outer membrane protein accompanied by, in three of four cases, decreased penetration of the antibiotic across the outer membrane.

Bacterial Outer Membrane Proteins↗