PubMed Health⌕ Search

Biomedical subjects

A J Pope

Publications and source records attributed to A J Pope.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. I: Kinetic mechanism of the substrate activation reaction studied by transient and steady-state techniques.

The kinetic mechanism for the amino acid activation reaction of Staphylococcus aureus isoleucyl-tRNA synthetase (IleRS; E) has been determined from stopped-flow measurements of the tryptophan fluorescence associated with the formation of the enzyme-bound aminoacyl adenylate (E.Ile-AMP; Scheme 1). Isoleucine (Ile) binds to the E.ATP complex (K4 = 1.7 +/- 0.9 microM) approximately 35-fold more tightly than to E (K1 = 50-100 microM), primarily due to a reduction in the Ile dissociation rate constant (k-1 approximately 100-150 s-1, cf. k-4 = 3 +/- 1.5 s-1). Similarly, ATP binds more tightly to E.Ile (K3 = approximately 70 microM) than to E (K2 = approximately 2.5 mM). The formation of the E.isoleucyl adenylate intermediate, E.Ile-AMP, resulted in a further increase in fluorescence allowing the catalytic step to be monitored (k+5 = approximately 60 s-1) and the reverse rate constant (k-5 = approximately 150-200 s-1) to be determined from pyrophosphorolysis of a pre-formed E.Ile-AMP complex (K6 = approximately 0.25 mM). Scheme 1 was able to globally predict all of the observed transient kinetic and steady-state PPi/ATP exchange properties of IleRS by simulation. A modification of Scheme 1 could also provide an adequate description of the kinetics of tRNA aminoacylation (kcat,tr = approximately 0.35 s-1) thus providing a framework for understanding the kinetic mechanism of aminoacylation in the presence of tRNA and of inhibitor binding to IleRS.

Adenosine Triphosphate↗
PubMed1998

Characterization of isoleucyl-tRNA synthetase from Staphylococcus aureus. II. Mechanism of inhibition by reaction intermediate and pseudomonic acid analogues studied using transient and steady-state kinetics.

The interactions of isoleucyl-tRNA synthetase (IleRS, E) from Staphylococcus aureus with both intermediate analogues and pseudomonic acid (PS-A) have been investigated using transient and steady-state techniques. Non-hydrolyzable analogues of isoleucyl-AMP (I) were simple competitive inhibitors (Ile-ol-AMP, Ki = 50 nM and Ile-NHSO2-AMP, Ki = 1 nM;). PS-A (J) inhibits IleRS via a slow-tight binding competitive mechanism where E.J (Kj = approximately 2 nM), undergoes an isomerization to form a stabilized E*.J complex (K*j = 50 pM). To overcome tight-binding artifacts when K*j << [E], K*j values were estimated from PPi/ATP exchange where [S] >> Km, thus raising K*j,app well above [E]. Using [3H]PS-A, it was confirmed that binding occurs with 1:1 stoichiometry and is reversible. Formation of inhibitor complexes was monitored directly through changes in enzyme tryptophan fluorescence. For Ile-ol-AMP and Ile-NHSO2-AMP, the fluorescence intensity of E.I was identical to that when E.Ile-AMP forms catalytically. Binding of PS-A induced only a small change in IleRS fluorescence that was characterized using transient kinetic competition. SB-205952, a PS-A analogue, produced a 37% quenching of IleRS fluorescence upon binding as a result of radiationless energy transfer. Inhibitor reversal rates were obtained by measuring relaxation between spectroscopically different complexes. Together, these data represent a comprehensive solution to the kinetics of inhibition by these compounds.

Anti-Bacterial Agents↗
PubMed1998

Effects of substrate and inhibitor binding on proteolysis of isoleucyl-tRNA synthetase from Staphylococcus aureus.

Binding of ligands to isoleucyl-tRNA synthetase (IleRS; E) from Staphylococcus aureus was investigated through effects on proteolytic digestion. Approximately 50-fold higher concentrations of protease (trypsin or chymotrypsin) were required to inactivate IleRS after incubation with substrates and formation of the E. Ile-AMP intermediate compared with free E. Binding of pseudomonic acid A (PS-A) or isoleucynol adenylate (Ile-ol-AMP) also induced resistance to proteolysis and altered the patterns of IleRS cleavage fragments in an inhibitor-class specific manner. The determinants for PS-A binding were investigated via proteolysis of E.[3H]PS-A. Limited proteolysis of E.[3H]PS-A (excising residues 186-407) could be achieved without significant loss of bound inhibitor, eliminating this region as contributing to inhibitor binding. Assays were developed which allowed IleRS proteolysis to be readily followed using fluorescence polarization. Inhibitor-protected IleRS was labeled with fluorescein isothiocyanate with only a small effect upon catalytic activity (Fl-IleRS). The (pseudo) kinetics of proteolytic cleavage of Fl-IleRS could be measured at low nanomolar Fl-IleRS concentrations in 96/384-well microtiter plates, allowing real-time monitoring of dose-dependent protection from proteolysis. Thus, inhibitor (and substrate) binding could be reproducibly assessed in the absence of measurements of catalytic acitvity. This could potentially form the basis of novel screening assays for ligands to other proteins.

Adenosine Triphosphate↗
PubMed1998

Effect of potent urease inhibitor, fluorofamide, on Helicobacter sp. in vivo and in vitro.

The therapeutic potential of urease inhibition of Helicobacter pylori has been studied by examining the effect of the potent urease inhibitor, fluorofamide (N-(diaminophosphinyl)-4-fluorobenzenamide), on urease activity and bacterial survival in vivo and in vitro. In culture, acid protection in H. pylori was shown to be due to changes in the pH of the medium brought about by the release of ammonia. Both the acid protection and the ammonia release were completely blocked by fluorofamide at low doses (ED50 = approximately 100 nM). However, fluorofamide was unstable under acidic conditions (T1/2 = 5.7 min at pH 2). Despite this, fluorofamide was the best available compound to test in vivo. In ferrets naturally infected with H. mustelae, a single dose (50 mg/kg, per os) of fluorofamide completely inhibited bacterial urease. In repeat dosing studies, fluorofamide (50 mg/kg per os, three times a day) was compared with the Helicobacter triple therapy regime (amoxycillin, metronidazole, and bismuth subcitrate). Fluorofamide failed to eradicate the H. mustelae infection, compared to 80% eradication with triple therapy. However, histological samples showed a profound reduction in bacterial numbers following fluorofamide treatment. A combination of fluorofamide and amoxycillin was dosed to ferrets (seven days of treatment with 50 mg/kg fluorofamide plus 10 mg/kg amoxycillin per os twice a day); however, this failed to eradicate the infection, despite there being a reduction in bacterial numbers in 3/5 ferrets after 21 days after dosing stopped. It was concluded that urease inhibitors (either alone or in combination with antibiotics) are unlikely to have therapeutic potential for Helicobacter pylori infections. This is probably because, in vivo, some bacteria (perhaps dormant forms) are not entirely dependent upon urease for survival. However, given the acid instability of fluorofamide, the possibility that more stable urease inhibitors might have therapeutic potential, cannot be excluded.

Amoxicillin↗
PubMed1996

Extraperitoneal laparoscopy: anatomical dissections with experimental balloon dilators.

OBJECTIVES: To develop balloon dilators in vitro and in cadavers and to evaluate the anatomy of the extraperitoneal spaces and their surroundings created by such balloons. MATERIALS AND METHODS: Balloons of three different materials (condoms, fingers of surgical gloves, whole surgical gloves and 'party' balloons) were tested in an in vitro study and compared with a prototype balloon dilator used in cadavers. The balloon dilators were used to create extraperitoneal spaces in the pelvic, the loin and the iliac regions through stab incisions. The incisions were extended after dilatation to examine the spaces created and the consequent anatomical changes. RESULTS: In vitro, all balloons were found to have capacities of more than 10 L, except that made from the finger of a surgical glove. Early rupture after insertion and during dilatation was a problem in condom and party balloons. In cadavers, there was good exposure of anatomical structures within the extraperitoneal spaces created. Reflections of peritoneum in the loin and the iliac regions were unmoved by balloon dilatation. CONCLUSIONS: The prototype balloon dilator possessed all the characteristics desired of an ideal instrument for creating extraperitoneal spaces. Good exposure of anatomical structures by the balloon dilators allows a minimal dissection in extraperitoneal laparoscopic procedures in the aforementioned regions. That the peritoneal reflection was unmoved despite adequate expansion of the balloons also means that the exact placement of secondary laparoscopic ports is crucial in preventing peritoneal and colonic trauma.

Catheterization↗
PubMed1995

Properties of the reversible K(+)-competitive inhibitor of the gastric (H+/K+)-ATPase, SK&F 97574. I. In vitro activity.

SK&F 97574 (3-butyryl-4-(2-methylamino)-8-(2-hydroxyethoxy)quinoline), is a potent inhibitor of the (H+/K+)-ATPase in membrane vesicles isolated from porcine gastric mucosa. It inhibits (H+/K+)-ATPase activity in lyophilised vesicles in a kinetically competitive manner with respect to the activating cation, K+, with an inhibition constant (Ki) of 0.46 +/- 0.003 microM. Inhibition of (H+/K+)-ATPase activity is freely reversible. Binding of SK&F 97574 was shown to be mutually exclusive and the previously reported reversible (H+/K+)-ATPase inhibitors, SCH 28080 and MDPQ. Therefore, despite its structural dissimilarity, SK&F 97574 appears to bind to the same lumenal region of the (H+/K+)-ATPase identified as the binding site for these compounds. SK&F 97574 is a weak base (pKa = 6.86), and would therefore be expected to accumulate in the acidic compartment at the lumenal face of the parietal cell. In intact gastric vesicles (which have the lumenal face of the ATPase on the interior), SK&F 97574 inhibited ATP-dependent H(+)-transport with a similar potency to ATPase activity. SK&F 97574 is therefore relatively membrane permeable, and would be predicted to gain access readily to its site of action in vivo. The effect of pH on inhibition of H+/K(+)-ATPase activity by SK&F 97574 is consistent with its being active only in its protonated form. The selectivity of SK&F 97574 for the gastric (H+/K+)-ATPase was tested by examining its ability to inhibit a closely related p-class pump, the (Na+/K+)-ATPase from dog kidney. SK&F 97574 was found to have a 60-fold greater sensitivity for the former enzyme. The (Na+/K+)-ATPase was not inhibited in a K(+)-competitive manner by SK&F 97574, indicating an entirely different, probably nonspecific, mechanism.

Adenosine Triphosphate↗
PubMed1995

Properties of the reversible, K(+)-competitive inhibitor of the gastric (H+/K+)-ATPase, SK&F 97574. II. Pharmacological properties.

SK&F 97574 [3-Butryl-4-(2-methylamino)-8-(2-hydroxyethoxy) quinoline] is a potent, reversible inhibitor of the gastric (H+/K+)-ATPase. In an anaesthetised lumen-perfused rat preparation, it inhibited pentagastrin-stimulated gastric acid secretion with intravenous and intraduodenal inhibitory ED50 values of 2.40 mumol/kg and 4.43 mumol/kg, respectively. In the conscious fistula rat model, doses of 10 mumol/kg IV and 25 mumol/kg PO produced mean peak inhibitions of basal acid output of 91% and 97%, respectively. In these experiments, the duration of action of SK&F 97574 was much shorter than that of the covalent (H+/K+)-ATPase inhibitor, omeprazole. In the conscious Heidenhain pouch dog, SK&F 97574 inhibited histamine-stimulated gastric acid secretion after both intravenous and oral administration with ED50 values of 0.49 mumol/kg and 0.89 mumol/kg, respectively. In this model, duration of action studies showed that significant residual inhibition of acid secretion remained 8 hours after intravenous dosing with SK&F 97574 (producing peak inhibition of 92%). However, 24 hours after oral dosing of SK&F 97574 (10 mumol/kg), no significant inhibition remained. These data indicate that the duration of action of SK&F 97574 is longer than that of the histamine H2 receptor antagonists such as cimetidine, but shorter than that of covalent (H+/K+)-inhibitors such as omeprazole. Overall, the pharmacological properties of SK&F 97574 suggest that it could be a potentially useful clinical treatment for acid-related diseases.

Aminoquinolines↗
PubMed1995

Transurethral Needle Ablation of the prostate (TUNA): clinical results and ultrasound, endoscopic, and histologic findings in pilot study of patients in urinary retention.

Transurethral Needle Ablation of the prostate (TUNA) is a new thermal ablation treatment for benign prostatic hyperplasia (BPH) utilizing radiofrequency electric current delivered by needles into the depth of the prostate to produce an area of coagulative necrosis. A pilot study of 10 patients in urinary retention was undertaken to assess the procedure. After treatment, nine patients voided at a median time of 3 days, although a further two required transurethral resection because of chronic infection in one and chronic urinary retention in the other. At 3 months' follow-up, the mean Qmax was 13.0 mL/sec, the mean AUA Symptom Score was 9.1, and the mean quality of life score was 1.6. The mean Pdet fell from 73.3 to 39.0 cm H2O. On transrectal ultrasound at 3 months, cystic lesions were seen in two patients, with a third having large cavities. A 10.2% reduction in mean prostatic volume, from 48.8 cc to 43.8 cc, was noted but considered to be not significant. On endoscopy at 3 months, mucosal retraction was seen in seven patients, with cavities in two patients. Histologic study in patients undergoing transurethral resection 4 to 6 months after TUNA showed necrosis and fibrosis. It is considered that an area of coagulative necrosis is produced by TUNA that resolves either by scar formation with retraction or by liquefaction with cyst formation. If the lesion communicates with the urethra, a cavity may form, which is undesirable. Our early experience is encouraging. The TUNA treatment is effective, can be given without anesthesia, and should be either a day case or an office procedure. It should prove to have a significant place in the treatment of BPH.

Aged↗
PubMed1994

Laparoscopic ureterolithotomy: the results of an initial series, and an evaluation of its role in the management of ureteric calculi.

OBJECTIVE: To evaluate the place of laparoscopic ureterolithotomy in the management of ureteric calculi for which extracorporeal shockwave lithotripsy (ESWL) and endourological techniques are unsuitable. PATIENTS AND METHODS: Laparoscopic ureterolithotomy was attempted in nine patients (eight men, one woman) with an age range of 26-81 years (mean 55.5) who had large, long standing and impacted calculi in the upper and mid ureter. The stone size ranged from 5 to 28 mm (mean 13.2) and stone duration ranged from one to 24 months (mean 8.2). The transperitoneal route was used in six patients and the extraperitoneal route in three but was converted to a transperitoneal approach in two. RESULTS: The stone was successfully removed in all nine patients. The operative time ranged from 80 to 260 minutes (mean 158). No intra-operative complication was encountered and no patient required a blood transfusion. Post-operative complications included urinary leak and fever. The post-operative stay ranged from 2 to 13 days (mean 5.2). CONCLUSIONS: Laparoscopic ureterolithotomy has definite advantages over open ureterolithotomy. Furthermore, the more difficult a stone is for treatment by ESWL and endourological techniques, the more suitable it is for laparoscopic removal. For large, hard, long-standing and impacted ureteric calculi, one laparoscopic ureterolithotomy as initial therapy may be preferable to multiple endourological and ESWL procedures.

Adult↗
PubMed1993

Alternatives to urinary diversion with a bag: results in 100 patients.

One hundred patients underwent surgery to free them from a cutaneous urinary diversion (n = 59) or to avoid having one (n = 41). The reconstruction was tailored to the needs of the patient: 7 had a simple undiversion, 35 a complex undiversion and 58 a continent suprapubic diversion. The outcome was satisfactory in 97%. Two patients were rediverted to an ileal conduit. The simpler the reconstruction the lower the re-operation rate: simple undiversion 0%, complex undiversion 20%, Mitrofanoff 42%, Kock pouch 69%. In spite of the high complication rate, there is a strong demand for such reconstructions and a high satisfaction rate amongst patients.

Adolescent↗
PubMed1992

Kinetics of the Vacuolar H-Pyrophosphatase : The Roles of Magnesium, Pyrophosphate, and their Complexes as Substrates, Activators, and Inhibitors.

The responses of the vacuolar membrane (tonoplast) proton-pumping inorganic pyrophosphatase (H(+)-PPase) from oat (Avena sativa L.) roots to changes in Mg(2+) and pyrophosphate (PPi) concentrations have been characterized. The kinetics were complex, and reaction kinetic models were used to determine which of the various PPi complexes were responsible for the observed responses. The results indicate that the substrate for the oat root vacuolar H(+)-PPase is Mg(2)PPi and that this complex is also a non-competitive inhibitor. In addition, the enzyme is activated by free Mg(2+) and competitively inhibited by free PPi. This conclusion differs from that reached in previous studies, in which it was proposed that MgPPi is the substrate for plant vacuolar H(+)-PPases. However, models incorporating MgPPi as a substrate were unable to describe the kinetics of the oat H(+)-PPase. It is demonstrated that models incorporating Mg(2)PPi as the substrate can describe some of the published kinetics of the Kalanchoë daigremontiana vacuolar H(+)-PPase. Calculations of the likely concentrations of Mg(2)PPi in plant cytoplasm suggest that the substrate binding site of the oat vacuolar H(+)-PPase would be about 70% saturated in vivo.

Journal Article↗
PubMed1991

Characterisation of the effects of anthranilic and (indanyloxy) acetic acid derivatives on chloride transport in membrane vesicles.

The effects of the Cl- channel blockers, NPPB, IAA94/95 and a number of related compounds on 36Cl- transport in membrane vesicles from bovine kidney cortex and rabbit ileum mucosa brush borders have been studied. These vesicles have been previously shown to be enriched in Cl- channel and Cl-/anion cotransport activity, respectively. Chloride transport was assayed in both types of vesicles by measuring the uptake of 36Cl- in response to an outwardly-directed Cl- concentration gradient. In kidney microsomes, a large proportion of the observed 36Cl- uptake was mediated by an electrogenic uniport and could be substantially reduced by clamping the membrane potential at zero mV using K+ and valinomycin. Chloride uptake was inhibited by both NPPB and IAA94/95 with apparent IC50 values of around 10 microM under optimal conditions (i.e., 4 min uptake at 4 degrees C). Under other conditions (e.g., 10 min uptake at 25 degrees C), where uptake had reached a steady-state level, much higher concentrations of inhibitor were required to cause inhibition. Therefore, previous differences in the reported potency of these compounds may, in part, have been due to the conditions under which Cl- uptake was measured. In addition, both NPPB and, to a lesser extent, IAA94/95 were found to have other effects on the vesicles, in that, when added at a concentration of 100 microM, they induced a leakage of pre-accumulated 36Cl-. This was probably caused by either dissipation of membrane potential or damage to the vesicle membranes. The sulphonic acid derivatives of NPPB and IAA94/95 (NPPB-S and ISA94/95, respectively) blocked 36Cl- uptake with around the same potency as NPPB and IAA94/95, but did not cause any non-specific Cl- leakage, when added at concentrations up to 100 microM. Inhibition of 36Cl- uptake by all four compounds was almost completely reversible. However, when vesicles were incubated with the inhibitors in the presence of an outward Cl- concentration gradient, or if vesicles were freeze/thawed in the presence of the compounds, inhibition could be only partially reversed. In rabbit brush border membrane vesicles, 36Cl- uptake was not reduced when the vesicles were voltage clamped using valinomycin and K+, and was therefore probably mediated by Cl-/Cl- exchange. However, despite the lack of effect of valinomycin, 36Cl- uptake was inhibited by both NPPB (approx. 80% inhibition at 100 microM) and, to a lesser extent, by IAA94/95 (approx. 30% inhibition at 100 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Acetates↗
PubMed1991

The morphological and functional changes in rat bladder following photodynamic therapy with phthalocyanine photosensitization.

We have studied photodynamic therapy (PDT) in the rat bladder with a new photosensitizer, aluminium sulfonated phthalocyanine (AlSPc) given intravenously and intravesically. The microscopic distribution of photosensitizer fluorescence in the bladder wall was studied by laser fluorescence microscopy. Prior to PDT the bladder capacity and compliance were assessed by filling cystometry. Intravesical red light (675 nm.) from a copper vapour pumped dye laser was used to activate the photosensitizer using light doses of 20 to 200 J/cm2. Urodynamic and histologic changes were studied at intervals for up to three months. The fluorescence studies showed that AlSPc was eliminated from the deeper muscle layers more quickly than from the superficial layers of the bladder wall so that by 24 hours there was four times as much fluorescence from the mucosa and lamina propria compared to the deeper muscle. Control bladders illuminated with laser light alone showed no effects at these light doses. Animals treated 24 hours after sensitization showed a reduction in bladder capacity of up to 78% (20 J/cm2. light and 1.5 mg./kg.AlSPc). An initial reduction in compliance recovered in two weeks after low doses (0.5 mg./kg.) of AlSPc but was still abnormal at three months after higher doses (1.5 mg./kg.); though there was no long term histologic abnormality seen. Aluminium sulfonated phthalocyanine is a promising photosensitizer for bladder photodynamic therapy and using low doses of the drug it is possible to produce a superficial necrosis without muscle damage across a range of light doses. This heals by epithelial regeneration with no long term functional impairment. Direct absorption of this photosensitizer following intravesical administration seems unreliable.

Animals↗
PubMed1991

The detection of phthalocyanine fluorescence in normal rat bladder wall using sensitive digital imaging microscopy.

The ability to detect photosensitisers in tissue at a microscopical level is important when studying photodynamic therapy (PDT) in both normal and malignant tissue. We have studied the fluorescence distribution of aluminium sulphonated phthalocyanine (A1SPc) in the normal rat bladder using a cooled CCD (charge coupled device) imaging system with computerised image processing. This system makes it possible to carry out a quantitative assessment of photosensitiser fluorescence in the various layers of the bladder wall. The highest fluorescence intensities were obtained within 1 h of intravenous administration but there was little selectivity of uptake between layers. A1SPc was eliminated from the deeper muscle layers more quickly than from the superficial layers of the bladder wall so that by 24 h a 4:1 ratio of fluorescence intensity was apparent which persisted at least until 72 h, although the absolute amount of photosensitiser declined. Following irradiation by red light (675 nm), photobleaching of the sensitiser in the deeper layers further increased this ratio. Direct absorption of A1SPc by the bladder wall following intravesical administration proved unreliable.

Administration, Intravesical↗
PubMed1991

Photodynamic therapy.

Explore the source record for details and available documents.

Dose-Response Relationship, Radiation↗
PubMed1991

A user's guide to flexible cystoscopes.

This survey, carried out on behalf of the BAUS Instruments Committee, aims to assist the urologist in choosing a flexible cystoscope. The 5 most commonly available flexible cystoscopes (from ACMI, Olympus, Pentax, Storz and Wolf) were assessed in a clinical setting for handling, image quality and user satisfaction. Several technical characteristics, including deflection range, irrigant flow rate and optics, were tested in the laboratory. Whilst all instruments were satisfactory, the favourite of our testing panel was the Olympus CYF-2, which also came out best in the technical assessment and has the most comprehensive service support. The cheaper Storz instrument also performed well.

Cystoscopes↗