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PubMed1996

Reversed shunting across the ductus arteriosus or atrial septum in utero heralds severe congenital heart disease.

OBJECTIVES: This study was performed to define the significance of Doppler color flow mapping in demonstrating reversal of the direction of the normal physiologic flow across the atrial septum and ductus arteriosus in the human fetus. BACKGROUND: Reversal of the physiologic shunting across the ductus arteriosus or atrial septum in utero (i.e., left to right) can be readily identified by Doppler color flow mapping, complemented by pulsed and continuous wave Doppler information. METHODS: We reviewed echocardiograms recorded at our three institutions from 1988 to 1993, which displayed reversal of flow by Doppler color flow in 53 fetuses of gestational age 18 weeks to term. The diagnoses were confirmed by postnatal echocardiography, operation or autopsy. Reversal of shunting was consistently associated with severe heart disease. RESULTS: Reversed atrial shunting was found with severe left heart obstructive lesions, including 19 with hypoplastic left heart syndrome, 3 with critical aortic stenosis, 2 with double-outlet right ventricle and 1 each with an interrupted aortic arch, atrioventricular septal defect and severe left ventricular dysfunction due to dilated cardiomyopathy. Reversed ductus arteriosus shunting was found with severe right heart obstructive lesions, including nine fetuses with pulmonary atresia, six with severe obstructive tricuspid valve abnormalities, five with severe tetralogy of Fallot, four with Ebstein's anomaly and two with single ventricle and pulmonary stenosis. Associated cardiac lesions were common in both groups. Only 3 of the 15 infants who were delivered alive from the reverse ductus arteriosus shunt group and 4 of 12 from the reverse atrial shunt group still survive. CONCLUSIONS: The finding of reversed flow by Doppler color flow mapping during fetal life provides a key to subsequent accurate diagnosis and denotes a spectrum of diseases with a very poor prognosis.

Ductus Arteriosus↗
PubMed2006

Enzymatic hydrolysis of microcrystalline cellulose in reverse micelles.

The activities of cellulases from Trichoderma reesei entrapped in three types of reverse micelles have been investigated using microcrystalline cellulose as the substrate. The reverse micellar systems are formed by nonionic surfactant Triton X-100, anionic surfactant Aerosol OT (AOT), and cationic surfactant cetyltrimethyl ammonium bromide (CTAB) in organic solvent media, respectively. The influences of the molar ratio of water to surfactant omega0, one of characteristic parameters of reverse micelles, and other environmental conditions including pH and temperature, on the enzymatic activity have been studied in these reverse micellar systems. The results obtained indicate that these three reverse micelles are more effective than aqueous systems for microcrystalline cellulose hydrolysis, and cellulases show "superactivity" in these reverse micelles compared with that in aqueous systems under the same pH and temperature conditions. The enzymatic activity decreases with the increase of omega0 in both AOT and Triton X-100 reverse micellar systems, but reaches a maximum at omega0 of 16.7 for CTAB reverse micelles. Temperature and pH also influence the cellulose hydrolysis process. The structural changes of cellulases in AOT reverse micelles have been measured by intrinsic fluorescence method and a possible explanation for the activity changes of cellulases has been proposed.

Biochemistry↗
PubMed2002

Impact of daily life myocardial ischemia in patients with chronic reversible and irreversible myocardial dysfunction.

Repetitive myocardial ischemia during daily life has been suggested as the underlying mechanism of reversible myocardial dysfunction, which may progress into a hibernating state. Thirty-seven patients with ischemic cardiomyopathy (ejection fraction 35 +/- 7%) underwent positron emission tomography (N-13 ammonia and 18-F-fluoro-2-deoxy-glucose [FDG]) and exercise testing before coronary artery bypass grafting (CABG) and 48- hour ambulatory electrocardiographic monitoring to detect ischemia before CABG and 6 months postoperatively. Reversibility of regional myocardial dysfunction was detected by echocardiographic follow-up at 5 days, 2 months, and 6 months after the operation. Preoperatively, ischemic episodes during daily activities were more common (2 [25th to 75th percentiles 0 to 4] vs 0 episodes, p <0.01) and duration of ischemia longer (9 [25th to 75th percentiles 0 to 37] vs 0 [25th to 75th percentiles 0 to 1] minutes, p <0.02) in patients with reversible dysfunction (n = 15) than in patients with irreversible dysfunction (n = 22). The number of ischemic episodes per patient correlated with the numbers of reversibly dysfunctional segments (p = 0.003), viable segments as seen by positron emission tomography (p <0.05), and flow-metabolic mismatch segments (p <0.05). CABG eliminated ambulatory ischemic episodes in patients with reversible dysfunction (0 episodes, p <0.05 vs before CABG). Preoperatively, all patients with reversible dysfunction had a positive exercise test (14 of 15 patients), whereas daily life ischemia was present in 60% of patients. Reversibly dysfunctional segments in patients with ambulatory ischemia had faster recovery of function (15 of 28 patients vs 2 of 12 patients recovered at 5 days, p <0.05), higher FDG uptake (0.86 +/- 0.19% vs 0.71 +/- 0.24%, p <0.05) than in patients without ambulatory ischemia, whereas perfusion was similar (0.63 +/- 0.20 and 0.62 +/- 0.19 ml/g/min). Thus, exercise-induced myocardial ischemia is associated with reversibility of myocardial dysfunction, but not all patients with reversible ischemic cardiomyopathy have ischemic attacks during daily life.

Activities of Daily Living↗
PubMed2001

The decompensated detrusor V: molecular correlates of bladder function after reversal of experimental outlet obstruction.

PURPOSE: Calcium ion homeostasis has a significant role in smooth muscle function. Its regulation requires complex storage and release mechanisms via ion pumps and channels located within intracellular storage sites (sarcoplasmic reticulum) and at the plasma membrane. We have previously reported a dramatic loss of the 2 major sarcoplasmic reticulum proteins sarcoplasmic endoplasmic reticulum calcium magnesium adenosine triphosphatase (SERCA2) and the ryanodine sensitive ion channel, also called the ryanodine receptor, after outlet obstruction. In our current study we investigated the correlation of the expression of these 2 major sarcoplasmic reticulum components with bladder function recovery after the reversal of outlet obstruction. METHODS AND METHODS: Standard partial bladder outlet obstruction was created in adult New Zealand White rabbits. Voiding patterns were monitored 2 and 4 weeks postoperatively, and rabbits were selected for outlet obstruction reversal based on a voiding pattern consistent with a decompensated state, as indicated by a frequency of greater than 30 voids daily and an average voided volume of less than 4 cc. Bladder biopsy was done when outlet obstruction was reversed. Voiding performance was monitored postoperatively and the animals were sacrificed 2 weeks later. Voiding patterns and muscle strip studies enabled us to define 2 functional outcome categories after reversal, namely normal versus minimally improved. Microsomal membrane protein fractions were prepared from the same bladder tissues before and after reversal, and probed by Western blot analysis for SERCA2 and ryanodine receptor expression. RESULTS: Western blot analysis revealed a major loss of SERCA2 and ryanodine receptor expression at the time of reversal and biopsy. In 65% of bladders obstruction reversal resulted in a normalized voiding pattern with a recovery of ryanodine receptor expression that was 15% to 65% of control values. In contrast, in the 35% of bladders with persistent voiding symptoms there was minimal recovery of ryanodine receptor expression. SERCA2 expression increased slightly in each group after reversal but did not differ in bladders with normalized versus improved function. CONCLUSIONS: Bladder decompensation is highly associated with a loss of sarcoplasmic reticulum function. Furthermore, the decompensated detrusor recovers function after obstruction reversal, which is associated with the recovery of these sarcoplasmic reticulum components.

Animals↗
PubMed1998

Neutralase reverses the anti-coagulant but not the anti-thrombotic activity of heparin in a rabbit model of venous thrombosis.

Neutralase (heparinase I; E.C. 4.2.2.7) is a heparin-degrading enzyme undergoing clinical evaluation as an alternative to protamine for reversing the anticoagulant effects of heparin in coronary bypass surgery. The objective of this study was to assess the relative effects of Neutralase and protamine on reversal of heparin-dependent elevations in coagulation parameters and inhibition of clot formation in a rabbit vena caval stasis model. Rabbits were treated with saline or heparin (300 U/kg) for 10 minutes, followed by saline, protamine (2.6 mg/kg), or Neutralase (10 or 30 microg/kg, representing 1.23 IU/kg and 3.69 IU/kg, respectively). Twenty minutes later, venous stasis was induced, and vena caval clots were excised, weighed, and characterized. Coagulation parameters [activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT)] and antiFactor IIa and Xa levels were measured throughout the protocol. Both protamine and Neutralase reversed heparin-mediated increases in aPTT (>300 seconds to 26-35 seconds) and TCT (>300 seconds to 29-56 seconds) to values that were not different from saline-treated, nonheparinized animals. Thrombus weight in the nonheparinized saline group was 62+/-7 mg; heparin-treated animals had no detectable clots. Protamine reversal of heparin was associated with clot formation (89+/-20 mg) while Neutralase reversal was not (no clots). Heparin-induced increases in antiFactor IIa activity were reversed similarly by protamine and Neutralase (from 4.3-8.8 U/ml to 0.2-0.3 U/ml) while antiFactor Xa activity was differentially reversed (from 3.9-5.9 U/ml to 0.7-1.3 U/ml Neutralase; 5.5 U/ml to 0.02 U/ml protamine). These results are consistent with a hypothesis that Neutralase cleaves heparin into fragments, which are devoid of antiFactor IIa activity that retain modest antiFactor Xa activity, resulting in reversal of anticoagulant, but not antithrombotic, heparin activity. This property of Neutralase may be beneficial in reducing post-surgical thrombotic events after reversal of heparin.

Animals↗
PubMed1998

Reversal of MRP-mediated vincristine resistance in KB cells by buthionine sulfoximine in combination with PAK-104P.

The mechanism of multidrug resistance protein (MRP)-mediated multidrug resistance (MDR) is still unclear. MRP reportedly transports some GSH conjugates. Recently, we demonstrated that a pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4 -(3-nitrophenyl)-3-pyridinecarboxylate P-oxide (PAK-104P), that reversed P-glycoprotein (P-gp)-mediated MDR directly interacted with MRP and completely reversed the vincristine (VCR) resistance in MRP-mediated MDR C-A120 cells. We investigated the reversing effect of PAK-104P in C-A120 cells in combination with buthionine sulfoximine (BSO), another MDR-reversing agent with a different reversing mechanism. In immunoblots, MRP was overexpressed in C-A120 cells. The level of ATP-dependent [3H]VCR uptake was high in membrane vesicles from KB-C2 cells, but low in those from C-A120 and parental KB-3-1 cells. The sensitivity to VCR of C-A120 cells, but not of KB-C2 cells, was considerably increased by 100 microM BSO. VCR accumulation in C-A120 cells, but not in KB-C2 cells, was also enhanced by BSO. BSO did not inhibit ATP-dependent [3H]LTC4 uptake in C-A120 vesicles. The combination of BSO with PAK-104P at their low concentrations resulted in complete reversal of VCR resistance in C-A120 cells. These findings suggested that BSO might not directly interact with MRP and reversed resistance in MRP-mediated MDR cells by reducing the intracellular glutathione (GSH) level that was needed for the transport of drugs by MRP and suggested a role for the combination of drug resistance-modulating agents with different reversing mechanisms in the reversal of MRP-mediated MDR.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

Monoaminergic changes associated with socially induced sex reversal in the saddleback wrasse.

The process of sex reversal in fishes is socially mediated and requires a total reorganization of the hypothalamo-pituitary-gonadal axis. When the ratio of males to females in a population of saddleback wrasse (Thalassoma dupperrey) is too low, the largest female becomes male over the course of 6 to 8 weeks. This event requires the conversion of external social cues into internal chemical cues. In an attempt to investigate the role monoamines might play in this process, two females were housed together in floating enclosures in order to induce sex reversal in the larger. Brains were sampled at various time points throughout the process of sex reversal. Monoamines were measured in the amygdala, preoptic area, ventral hypothalamus, locus coeruleus and raphe nucleus. Changes were demonstrated in monoamine metabolism for all brain regions examined. The most important changes in monoamine-system activation were seen during the first week of sex reversal. It is during this time that transitional animals undergo behavioral sex reversal. There is an increase in serotonergic activity in the amygdala which is likely related to territorial acquisition. The absence of male aggression results in a less stressful environment for the female and a reduction in serotonergic activity in the preoptic area allowing for an increase in noradrenergic activity potentially triggering the reorganization of the reproductive axis. In the ventral hypothalamus, there is a decrease in noradrenergic and increase in dopaminergic activity associated with this change from female to male. The locus coeruleus shows an increase in noradrenergic activity later in the process of sex reversal which is probably a response to more circulating androgens. In the raphe nucleus, there is a decrease in serotonergic activity at the time of behavioral sex reversal. This decrease in serotonergic activity is linked to the behavioral component of sex reversal. This study suggests that monoamines play a very important role in both behavioral and gonadal sex reversal in the saddleback wrasse, the former under the control of serotonin in the raphe and the latter mediated via serotonergic effects on norepinephrine in the preoptic area.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1997

A novel protamine variant reversal of heparin anticoagulation in human blood in vitro.

PURPOSE: Protamine reversal of heparin anticoagulation during cardiovascular surgery may cause severe hypotension and pulmonary hypertension. A novel protamine variant, [+18RGD], has been developed that effectively reverses heparin anticoagulation without toxicity in canine experiments. Heretofore, human studies have not been undertaken. This investigation hypothesized that [+18RGD] would effectively reverse heparin anticoagulation of human blood in vitro. METHODS: Fifty patients who underwent anticoagulation therapy during vascular surgery had blood sampled at baseline and 30 minutes after receiving heparin (150 IU/kg). Activated clotting times were used to define specific quantities of [+18RGD] or protamine necessary to completely reverse heparin anticoagulation in the blood sample of each patient. These defined amounts of [+18RGD] or protamine were then administered to the heparinized blood samples, and percent reversals of activated partial thromboplastin time, thrombin clotting time, and antifactor Xa/IIa levels were determined. In addition, platelet aggregation assays, as well as platelet and white blood cell counts were performed. RESULTS: [+18RGD] and protamine were equivalent in reversing heparin as assessed by thrombin clotting time, antifactor Xa, antifactor IIa levels, and white blood cell changes. [+18RGD], when compared with protamine, was superior in this regard, as assessed by activated partial thromboplastin time (94.5 +/- 1.0 vs 86.5 +/- 1.3% delta, respectively; p < 0.001) and platelet declines (-3.9 +/- 2.9 vs -12.8 +/- 3.4 per mm3, respectively; p = 0.048). Platelet aggregation was also decreased for [+18RGD] compared with protamine (23.6 +/- 1.5 vs 28.5 +/- 1.9%, respectively; p = 0.048). CONCLUSIONS: [+18RGD] was as effective as protamine for in vitro reversal of heparin anticoagulation by most coagulation assays, was statistically more effective at reversal than protamine by aPTT assay, and was associated with lesser platelet reductions than protamine. [+18RGD], if less toxic than protamine in human beings, would allow for effective clinical reversal of heparin anticoagulation.

Aged↗
PubMedDate not supplied

Observations on Doppler mid-diastolic mitral flow reversal.

Mitral regurgitation is detected occasionally in diastole during severe aortic regurgitation, hypertrophic cardiomyopathy and atrioventricular block. We have noticed mitral mid-diastolic flow reversal in both patients and many normal subjects. To evaluate this flow phenomenon, pulsed Doppler mitral flow velocity and M-mode echocardiographic recordings were obtained in 38 normal subjects (age range, 16 to 61 years). Fifteen of 38 subjects (40%) had mid-diastolic flow reversal, defined as reversed flow occurring in mid-diastole with a duration greater than 50 msec. Mid-diastolic flow reversal was more common in subjects with longer RR intervals (1031 versus 893 msec), more rapid M-mode echocardiographic EF (early diastolic deceleration) slopes of mitral valve anterior leaflet motion (141 versus 93 mm/sec), and more rapid deceleration of early diastolic mitral flow velocities (612 versus 426 cm/sec2). Mid-diastolic flow reversal by Doppler color flow mapping was recorded in the left atrium in all subjects, even in subjects without mid-diastolic flow reversal shown by pulsed Doppler echocardiography. However, subjects with mid-diastolic flow reversal detected by pulsed Doppler echocardiography demonstrated greater extension of flow into left atrium (10.4 versus 4.1 mm) and longer duration (112 versus 69 msec) of color flow reversal. These data suggest that mid-diastolic flow reversal represents a physiologic intravalvular flow that is possibly the result of reflected flow from the vigorous early diastolic mitral semiclosure.

Adolescent↗
PubMed2004

Reversal of negative charges on the surface of Escherichia coli thioredoxin: pockets versus protrusions.

Recent studies of proteins with reversed charged residues have demonstrated that electrostatic interactions on the surface can contribute significantly to protein stability. We have used the approach of reversing negatively charged residues using Arg to evaluate the effect of the electrostatics context on the transition temperature (T(m)), the unfolding Gibbs free energy change (DeltaG), and the unfolding enthalpy change (DeltaH). We have reversed negatively charged residues at a pocket (Asp9) and protrusions (Asp10, Asp20, Glu85), all located in interconnecting segments between elements of secondary structure on the surface of Arg73Ala Escherichia coli thioredoxin. DSC measurements indicate that reversal of Asp in a pocket (Asp9Arg/Arg73Ala, DeltaT(m) = -7.3 degrees C) produces a larger effect in thermal stability than reversal at protrusions: Asp10Arg/Arg73Ala, DeltaT(m) = -3.1 degrees C, Asp20Arg/Arg73Ala, DeltaT(m) = 2.0 degrees C, Glu85Arg/Arg73Ala, DeltaT(m) = 3.9 degrees ). The 3D structure of thioredoxin indicates that Asp20 and Glu85 have no nearby charges within 8 A, while Asp9 does not only have Asp10 as sequential neighbor, but it also forms a 5-A long-range ion pair with the solvent-exposed Lys69. Further DSC measurements indicate that neutralization of the individual charges of the ion pair Asp9-Lys69 with nonpolar residues produces a significant decrease in stability in both cases: Asp9Ala/Arg73Ala, DeltaT(m) = -3.7 degrees C, Asp9Met/Arg73Ala, DeltaT(m) = -5.5 degrees C, Lys69Leu/Arg73Ala, DeltaT(m) = -5.1 degrees C. However, thermodynamic analysis shows that reversal or neutralization of Asp9 produces a 9-15% decrease in DeltaH, while both reversal of Asp at protrusions and neutralization of Lys69 produce negligible changes. These results correlate well with the NMR analysis, which demonstrates that only the substitution of Asp9 produces extensive conformational changes and these changes occur in the surroundings of Lys69. Our results led us to suggest that reversal of a negative charge at a pocket has a larger effect on stability than a similar reversal at a protrusion and that this difference arises largely from short-range interactions with polar groups within the pocket, rather than long-range interactions with solvent-exposed charged groups.

Alanine↗
PubMedDate not supplied

Renaturation and interaction of ribonuclease A with AOT surfactant in reverse micelles.

This study extended works on effects of solute on the percolation of reverse micelles to the effects of interactions between protein and surfactants on protein refolding by reverse micelles. The changes in percolation behavior were identified and attributed to the position of solutes in the core aqueous phase and the interaction between the solute and the surfactants. The percolation behavior of reverse micelles with solutes was related to protein renaturation and the reverse micelle. This study aims to highlight the involvement of the interface and the interaction of the protein with the surfactant during protein refolding. Ribonuclease A and AOT reverse micelles together constitute a model system considered here. The systemic parameters of the reverse micelle, water content (W(o)) and pH value, were applied to modify the interaction between the denatured protein molecules and the surfactant interface. The interactions and the locations of the protein molecules were determined from changes in percolation temperature measured by conductivity. The percolation and protein activity show that a stronger interaction of the protein molecules with surfactant corresponds to superior recovery of protein activity. The investigation concludes that the refolding of protein by reverse micelles is not only facilitated by the isolation of reverse micelles but also by the interaction due to the interface of the reverse micelle.

Dioctyl Sulfosuccinic Acid↗
PubMedDate not supplied

Extraction behavior of hemoglobin using reversed micelles by dioleyl phosphoric acid.

A new surfactant, dioleyl phosphoric acid (DOLPA), has been applied to the extraction of hemoglobin using reversed micelles. The reversed micelles formed by DOLPA can easily extract hemoglobin from aqueous to reversed micellar solutions. DOLPA is the first surfactant to extract hemoglobin completely without using any cosurfactants. On the basis of the difference between DOLPA and AOT reversed micelles in the forward extraction behavior of hemoglobin, the nature of the interfacial complex that would be formed between surfactants and hemoglobin at the oil--water interface was found to be the dominant factor in determining the extraction efficiency of hemoglobin be reversed micelles. In addition, back-transfer studies of hemoglobin from the DOLPA reversed micelles were also carried out by the phase transfer method. It was found that hemoglobin, once dissolved into the DOLPA reversed micelles, is not transferred to a fresh aqueous solution even when the conditions are adjusted to not allow the forward transfer of hemoglobin. However, the addition of several kinds of alcohol drastically improved the yield in the back-transfer of hemoglobin. The efficiency in the back-transfer of hemoglobin strongly depends on the aqueous conditions that are in contact with the reversed micelles, such as pH, ionic strength, and alcohol concentration. A pH higher than the pI of hemoglobin, a salt concentration lower than that of the water pool, and the proper concentration of alcohol are required for the recovery aqueous phase to ensure the back-transfer of hemoglobin from the DOLPA reversed micelles.

Alcohols↗
PubMedDate not supplied

Extraction and activity of chymotrypsin using AOT-DOLPA mixed reversed micellar systems.

Novel reversed micellar solutions formulated with a mixture of AOT (dioctyl sulfosuccinate) and DOLPA (dioleyl phosphoric acid) show good potential for use in reversed micellar protein extraction operations. Chymotrypsin is easily extracted from an aqueous phase into organic isooctane containing 10 mM AOT and DOLPA in a 4:1 ratio. The extraction ability of the mixed reversed micelles of 10 mM was higher than that of 200 mM AOT alone. The results of extraction indicated that the AOT-DOLPA mixed reversed micelles are very useful for separating and enriching chymotrypsin. Back-extraction of chymotrypsin from the organic phase to a fresh aqueous phase is also accomplished by adding an alcohol to the organic phase. Although the back-transfer of chymotrypsin from the reversed micelles formed by AOT alone is very slow and difficult, in the AOT-DOLPA mixed reversed micelles, the back-extraction can be achieved completely by addition of 10% (v/v) isobutyl alcohol to the reversed micellar phase. The time to attain to the equilibrium of back-extraction was reduced from more than 24 to 2 h by adding the alcohol. On the basis of the activity data, the best composition of AOT and DOLPA was a 4:1 ratio and the total surfactant concentration was 10 mM. The activity of chymotrypsin recovered from the mixed reversed micelles was higher than that of the initial protein before the forward-transfer. This result means that the novel mixed reversed micellar solutions are useful not only in separation but also in purification of proteins.

Animals↗
PubMed1998

Solvent kinetic isotope effects of human placental alkaline phosphatase in reverse micelles.

Human placental alkaline phosphatase was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2-ethylhexyl) sulphosuccinate (Aerosol-OT) in 2,2, 4-trimethylpentane. This microemulsion system provides a convenient instrumental tool to study the possible kinetic properties of the membranous enzyme in an immobilized form. The pL (pH/p2H) dependence of hydrolysis of 4-nitrophenyl phosphate has been examined over a pL range of 8.5-12.5 in both aqueous and reverse micellar systems. Profiles of log V versus pL were Ha-bell shaped in the acidic region but reached a plateau in the basic region in which two pKa values of 9.01-9.71 and 9.86-10.48, respectively, were observed in reverse micelles. However, only one pKa value of 9.78-10.27 in aqueous solution was detected. Profiles of log V/K versus pL were bell-shaped in the acidic region. However, they were wave-shaped in the basic region in which a residue of pKa 9.10-9.44 in aqueous solution and 8.07-8.78 in reverse micelles must be dehydronated for the reaction to reach an optimum. The V/K value shifted to a lower value upon dehydronation of a pKa value of 9.80-10.62 in aqueous solution and 11.23-12.17 in reverse micelles. Solvent kinetic isotope effects were measured at three pL values. At pL 9.5, the observed isotope effect was a product of equilibrium isotope effect and a kinetic isotope effect; at pL 10.4, the log V/K value was identical in water and deuterium. The deuterium kinetic isotope effect on V/K was 1.14 in an aqueous solution and 1.16 in reverse micelles. At pL 11.0 at which the log V values reached a plateau in either solvent system, the deuterium kinetic isotope effect on V was 2.08 in an aqueous solution and 0.62 in reverse micelles. Results from a proton inventory experiment suggested that a hydron transfer step is involved in the transition state of the catalytic reaction. The isotopic fractionation factor (pi) for deuterium for the transition state (piT) increased when the pH of the solution was raised. At pL 11.0, the piT was 1.07 in reverse micelles, which corresponds to the inverse-isotope effect of the reaction in this solvent system. Normal viscosity effects on kcat and kcat/Km were observed in aqueous solution, corresponding to a diffusional controlled physical step as the rate-limiting step. We propose that the rate-limiting step of the hydrolytic reaction changes from phosphate releasing in aqueous solution to a covalent phosphorylation or dephosphorylation step in reverse micelles.

Alkaline Phosphatase↗
PubMed2003

Reversibility to a beta2-agonist in COPD: relationship to atopy and neutrophil activation.

Chronic obstructive pulmonary disease (COPD) is characterised by limited bronchial reversibility and chronic neutrophilic inflammation. However, in some cases of COPD, eosinophilic inflammation is present. We investigated the relationship between reversibility to beta2-agonist with atopy and neutrophil activation in patients with stable COPD. For this purpose, 38 outpatients with COPD (mean age: 64 years) 12 with asthma (mean age: 51 years) and 13 healthy controls (mean age: 49 years) were tested using increasing doses of inhaled salbutamol (up to 3100 microg). According to their reversibility, COPD patients were divided into two groups: reversible COPD (deltaFEV1 > or = 12% pred, n = 16) and non-reversible COPD (deltaFEV1 < 12% pred, n = 22). Atopy, assessed by skin prick, was found at similar frequencies in both COPD groups. Total serum IgE was higher in COPD patients vs. controls, but did not differ significantly between the COPD groups. The blood eosinophil count was significantly higher in the reversible COPD group than in the non-reversible COPD, and correlated with deltaFEV % pred (Rs = 0.54, P < 0.05), as well as in asthmatics. The non-reversible COPD group had a higher level of spontaneous neutrophil activation (by reduction of nitroblue tetrazolium) versus controls. We conclude that airway reversibility in COPD patients is associated with the degree of blood eosinophilia, but not with the degree of blood neutrophil activation.

Adrenergic beta-Agonists↗
PubMed1991

The p15 carboxyl-terminal proteolysis product of the human immunodeficiency virus type 1 reverse transcriptase p66 has DNA polymerase activity.

The reverse transcriptase of human immunodeficiency virus type 1 is a heterodimeric protein consisting of two polypeptides with masses of 66 and 51 kDa and has, as a second enzymatic activity, RNase H activity. The 66-kDa polypeptide can be cleaved by the virus-encoded protease to yield polypeptides of 51 and 15 kDa. The latter has been characterized as possessing RNase H activity [Hansen, J., Schultze, T., Mellert, W. & Moelling, K. (1988) EMBO J. 7, 239-243]. We have purified simultaneously the heterodimeric reverse transcriptase/RNase H containing the 66/51-kDa polypeptides and the 15-kDa RNase H from Escherichia coli containing the expression vector pJS 3.7 by a procedure including chromatography on DEAE-cellulose, phosphocellulose, and heparin-Sepharose. Two RNase H and reverse transcriptase peaks were separated on phosphocellulose, one coinciding with the heterodimeric protein and the other with the 15-kDa protein. On the basis of the following findings it appears that the 15-kDa polypeptide has both RNase H and reverse transcriptase activities: (i) it copurified with both activities; (ii) it functioned as a reverse transcriptase in an in situ assay after SDS/polyacrylamide gel electrophoresis; (iii) polyclonal antibodies raised against the 66-kDa polypeptide reacted in immunoblots exclusively with a 15-kDa polypeptide, reacted in immunoblots exclusively with a 15-kDa polypeptide, while no immunoreactive bands in the range of 51-66 kDa were seen in the 15-kDa polypeptide preparation; (iv) the p15 and the p66/51 reverse transcriptase could be quantitatively pelleted in an enzymatically active form only when antibodies specific for the p66 carboxyl terminus were used; and (v) the p15 protein had bona fide properties of a reverse transcriptase and could enzymatically synthesize a high molecular weight, alkali-resistant product. The two reverse transcriptases appear to have different behaviors on various template/primer systems tested. Conceivably different forms of human immunodeficiency virus type 1 reverse transcriptases might be used in individual steps of (+)- and (-)-strand replication.

Blotting, Western↗
PubMed2006

Reverse gyrase functions as a DNA renaturase: annealing of complementary single-stranded circles and positive supercoiling of a bubble substrate.

Reverse gyrase is a hyperthermophile-specific enzyme that can positively supercoil DNA concomitant with ATP hydrolysis. However, the DNA supercoiling activity is inefficient and requires an excess amount of enzyme relative to DNA. We report here several activities that reverse gyrase can efficiently mediate with a substoichiometric amount of enzyme. In the presence of a nucleotide cofactor, reverse gyrase can readily relax negative supercoils, but not the positive ones, from a plasmid DNA substrate. Reverse gyrase can completely relax positively supercoiled DNA, provided that the DNA substrate contains a single-stranded bubble. Reverse gyrase efficiently anneals complementary single-stranded circles. A substoichiometric amount of reverse gyrase can insert positive supercoils into DNA with a single-stranded bubble, in contrast to plasmid DNA substrate. We have designed a novel method based on phage-mid DNA vectors to prepare a circular DNA substrate containing a single-stranded bubble with defined length and sequence. With these bubble DNA substrates, we demonstrated that efficient positive supercoiling by reverse gyrase requires a bubble size larger than 20 nucleotides. The activities of annealing single-stranded DNA circles and positive supercoiling of bubble substrate demonstrate that reverse gyrase can function as a DNA renaturase. These biochemical activities also suggest that reverse gyrase can have an important biological function in sensing and eliminating unpaired regions in the genome of a hyperthermophilic organism.

Animals↗
PubMed2000

Long-term outcome after medical reversal of transtentorial herniation in patients with supratentorial mass lesions.

OBJECTIVE: To determine the short- and long-term outcomes after successful reversal of transtentorial herniation by medical treatment. Although it has been recognized that aggressive medical management can reverse transtentorial herniation, it is believed that overall outcome in such patients is poor. DESIGN: Prospective cohort study. SETTING: Neurocritical care unit of a university hospital. PATIENTS: A total of 28 consecutive patients who underwent an episode of transtentorial herniation (defined as decrease in level of consciousness accompanied by pupillary dilation) secondary to a supratentorial mass lesion followed by successful reversal. INTERVENTION: Herniation was reversed by using a combination of hyperventilation, mannitol and hypertonic saline. MEASUREMENTS AND MAIN RESULTS: The following outcomes were analyzed: risk of second herniation, radiologic evidence of structural damage or vascular compromise related to herniation on post-herniation computed tomographic scan, in-hospital mortality, and long-term functional outcome using Rankin score and Barthel index. A total of 32 episodes of transtentorial herniations were reversed in 28 patients during a 14-month period. The most common precipitating cause were edema (n = 23) or new/expanding intracerebral hematoma (n = 5). After first reversal of transtentorial herniation in 28 patients, a second herniation episode was observed in 16 patients after a mean interval of 88.2 hrs (range, 23-432 hrs); four were successfully reversed. On follow-up computed tomographic scan, hypodense lesion in midbrain (n = 6), temporal lobe contusion (n = 2), posterior cerebral artery (n = 3), and middle cerebral artery (n = 1) infarction were visualized in a minority of patients. The in-hospital mortality was 60% (n = 15) with brain death being the cause of death in 13 patients; care was withdrawn in eight patients. Second episode of herniation (p = .002) and midbrain involvement during herniation (p = .02) were associated with in-hospital mortality. During a mean follow-up period of 11.4+/-4.2 months, two patients died of cerebral neoplasm and human immunodeficiency virus-related sepsis, respectively. Of the 11 survivors, 7 were functionally independent (Rankin score <3 and Barthel index >60). CONCLUSIONS: Although mortality after transtentorial herniation is high, we found a prominent potential for meaningful recovery with aggressive medical reversal of transtentorial herniation. Our study implies that timely medical intervention for reversing transtentorial herniation can result in preservation of neurologic function.

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