Purification and characterization of arylacetonitrile-specific nitrilase of Alcaligenes sp. MTCC 10675.

Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 °C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec(-) , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase.

Optimization of arylacetonitrilase production from Alcaligenes sp. MTCC 10675 and its application in mandelic acid synthesis.

Alcaligenes sp. MTCC 10675 has been isolated from soil sample using enrichment method and has nitrilase catalytic system which is highly specific for the hydrolysis of arylaliphatic nitriles. Optimization of culture conditions using response surface methodology and inducer-mediated approach enhanced arylacetonitrilase production significantly (2.4-fold). Isobutyronitrile acted as an effective inducer for the induction of arylacetonitrilase, and it is highly specific for arylacetonitriles (phenyl acetonitrile and mandelonitrile). Arylacetonitrilase has no effect on its relative velocity (V r) up to 20 mM substrate (mandelonitrile) concentration and at 30 mM mandelonitrile, 23.4 % degree of inhibition (I d) was recorded. Half life of arylacetonitrilase of Alcaligenes sp. MTCC 10675 was 27.5 h at 25 °C. Hg(2+), Ag(+), Pb(3+), and Co(2+) were strong inhibitor of arylacetonitrilase activity which resulted into 100 %, 91 %, 84 %, and 83 % inhibition, respectively. Polar protic solvent (dichloromethane, dimethylsulphooxide, and n-butanol) reduce arylacetonitrilase activity up to 80-94 % at 10 % concentration. Alcaligenes sp. MTCC 10675 has higher biocatalytic activity, i.e., 3.9 gg(-1) dcw, which is highest in comparison to till reported organism. Arylacetonitrilase-mediated hydrolysis of racemic mandelonitrile resulted into R-(-) mandelic acid with 99.0 % enantiomeric excess (e.e.).

Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674.

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 µmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 µmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.

An isobutyronitrile-induced bienzymatic system of Alcaligenes sp. MTCC 10674 and its application in the synthesis of α-hydroxyisobutyric acid.

Alcaligenes sp. MTCC 10674 was isolated as acetone cyanohydrin hydrolyzing bacterium from soil of orchid gardens of Himachal Pradesh. Acetone cyanohydrin hydrolyzing activity of this organism comprised nitrile hydratase and amidase activities. It exhibited higher substrate specificity towards aliphatic hydroxynitrile (acetone cyanohydrin) in comparison to arylaliphatic hydroxynitrile. Isobutyronitrile (40 mM) acted as a carbon source as well as inducer for growth of Alcaligenes sp. MTCC 10674 and expression of acetone cyanohydrin hydrolyzing activity. Optimization of culture condition using response surface methodology increased acetone cyanohydrin hydrolyzing activity by 1.3-fold, while inducer mediation approach increased the activity by 1.2-fold. The half life of this enzyme was 25 h at 15 °C. V max and K m value for acetone cyanohydrin hydrolyzing enzyme was 0.71 µmol mg(-1) min(-1) and 14.3 mM, when acetone cyanohydrin was used as substrate. Acetone cyanohydrin hydrolyzing enzyme encountered product inhibition and IC50 and K i value were calculated to be 28 and 10.2 mM, respectively, when product α-hydroxyisobutyric acid was added in the reaction. Under optimized reaction conditions at 40 ml fed batch scale, 3 mg dcw ml (-) resting cells of Alcaligenes sp. MTCC 10674 fully converted 0.33 M acetone cyanohydrin into α-hydroxyisobutyric acid (1.02 g) in 6 h 40 min. The characterization of acetone cyanohydrins hydrolyzing activity revealed that it comprises bienzymatic nitrile hydrolyzing system, i.e. nitrile hydratase and amidase for the production of α-hydroxyisobutyric acid from acetone cyanohydrin and maximum 70 % yield is being reported for the first time.

The association between intraoperative correction of Dupuytren's disease and residual postoperative contracture.

The purpose of this study was to determine whether preoperative contracture and the amount of intraoperative correction can be used to predict the postoperative outcome of fasciectomy for Dupuytren's disease. A prospective study of 52 patients undergoing primary fasciectomy during an 18 month period was undertaken. The contracture of each joint was measured preoperatively, after fasciectomy during the operation and 6 months after surgery. Forty-two metacarpophalangeal (MCPJ) and 58 proximal interphalangeal (PIPJ) joints were treated surgically. Full intraoperative correction was achieved in 41 MCPJs. Thirty-seven had full correction at follow-up. Full intraoperative correction was obtained in 35 PIPJs and 13 had complete correction at follow-up. The extent of the preoperative deformity was a significant predictor of complete intraoperative correction. The extent of both preoperative deformity and intraoperative correction were significant predictors of loss of surgical correction after operation.

The prognosis of fasciectomy for abductor digiti minimi and pretendinous cords in Dupuytren's disease of the little finger.

Thirty-nine consecutive patients with little finger Dupuytren's contracture underwent open fasciectomy. Diseased abductor digiti minimi (ADM) pretendinous (PT) cords were identified. The mean pre-operative PIPJ contracture was 77 degrees in the PT group and 66 degrees in the ADM group. Mean residual deformity was 12 degrees in the PT group and 9 degrees in the ADM group. At six months, ten out of 27 patients had developed a recurrent deformity in the PT group (mean 24 degrees ) and seven out of 11 in the ADM group (mean 18 degrees ). There was no statistically significant difference between the two groups at any stage. Dupuytren's contracture of the little finger is as a result of an ADM cord in 29% of cases. In this series it led to an isolated contracture of the PIPJ in the majority of cases and rarely affected the MCPJ. Disease of the ADM cord was not associated with a difference in contracture or prognosis compared to a PT cord.

Comparison of intrapartum fetal monitor tracings of karyotypically abnormal and control infants.

To determine if there are any specific monitoring patterns associated with chromosomal abnormalities the intrapartum tracings of 17 postnatally diagnosed karyotypically abnormal infants were obtained. Controls were randomly selected from infants born on the same day at the same gestational age. The tracings from these two groups were independently evaluated. Comparison of the monitor interpretations showed: (1) the karyotypically abnormal group was more likely to have decreased or absent short- (p < 0.001) and long-term (p < 0.01) variability; (2) accelerations were more likely to be represented in the control group (p < 0.001); (3) no significant differences were found in the baseline heart rates or in the presence or frequency of mild, moderate, or severe variable or late decelerations. These findings may reflect underlying central nervous system abnormalities as opposed to uteroplacental insufficiency, but are not specific to karyotypically abnormal infants.

Use of intravenous amrinone in the short-term management of refractory heart failure in pregnancy.

The use of amrinone, a cardiac inotropic agent with vasodilatory properties, has not been described in human pregnancy. We report the use of amrinone in a woman at 18 weeks' gestation who had congestive heart failure secondary to bacterial endocarditis. Although her cardiac output and congestive heart failure improved, she developed hypoxemia, metabolic acidosis, and premature ventricular contractions. These effects resolved when the amrinone treatment was stopped.

Care of obstetric patients during the immediate postanesthesia period.

STUDY OBJECTIVE: To determine the level of care available to obstetric patients during the immediate postanesthesia period. DESIGN: Mail and telephone survey of members of anesthesia departments in Michigan. SETTING: All Michigan hospitals with licensed obstetric beds. PATIENTS: Patients recovering from general or major regional anesthesia following an operative delivery. INTERVENTIONS: The factors determining patient care were the physical suitability of the recovery site, skills and experience of personnel providing care in postanesthesia care units (PACUs), and adjustments in care patterns by anesthesia personnel. MEASUREMENTS AND MAIN RESULTS: Most obstetric PACUs are staffed by labor and delivery nurses whose assignment to the unit is only part of their overall patient care responsibilities within the labor and delivery area (88.2% of hospitals with more than 2,000 annual births and performing cesarean deliveries in the obstetric suite; 92.3% of hospitals with 500 to 1,999 annual births and performing cesarean deliveries in the obstetric suite). Obstetric PACUs in the remaining hospitals in either group are staffed by dedicated nurses who are permanently assigned to these units. Preparation of labor and delivery nurses for PACU duties varies greatly, but 60.0% of hospitals with more than 2,000 annual births and 30.8% of hospitals with 500 to 1,999 annual births provide no special training. Concern about the level of expertise available in obstetric PACUs staffed by labor and delivery nurses was expressed by almost every respondent and has led to a practice pattern followed by most anesthesia personnel of transferring patient care responsibility only after patients have regained consciousness, cardiovascular stability, and ventilatory adequacy. Several institutions also allow anesthesia personnel to summon nurses from the surgical PACU or to transfer patients to alternate recovery sites, such as the surgical PACU or the intensive care unit (ICU). CONCLUSIONS: In many obstetric PACUs, the level of expertise of personnel needs to be upgraded to ensure the safety of patients recovering from general or major regional anesthesia and to comply with existing care standards.

Is placenta previa a determinant of preeclampsia?

Low implantation of the placenta has been reported to be associated with a decreased risk for preeclampsia and this has been attributed to increased placental blood flow. However, placenta previa is known to be associated with separation and bleeding, intrauterine growth retardation, and elevated umbilical blood flow resistance by Doppler studies, suggesting decreased umbilical blood flow. To better evaluate the relationship of placenta previa and preeclampsia, 6576 consecutive patients who had ultrasound examination after 28 weeks gestation and delivery at our institution were studied. The placental location, parity, maternal weight, development of preeclampsia, and gestational age were evaluated by using frequency tables and stepwise discriminant analysis. Results showed that placenta previa is not a significant determinant of the development of preeclampsia, but parity, maternal weight, and gestational age contributed significantly to the development of preeclampsia. The finding of decreased incidence of preeclampsia with previa is explained not by increased placental blood flow but by associated maternal characteristics, and particularly by the strong association of previa with premature delivery.

Correlation of commonly used measures of intrauterine growth with estimated neonatal body fat.

Several morphometric measures have been used to identify infants at greatest risk from aberrant intrauterine growth. 119 near-term infants were studied to answer the more basic question of how well measures, such as birthweight percentile, ponderal index, the body mass index and the weight/length ratio reflect body fat in the neonate. Skinfold thicknesses were measured as an estimate of fat stores. Multiple regression analysis revealed that the weight/length ratio showed the strongest correlation with relative adiposity, explaining 52% of the variance. Further, sequential exponentiation of the crown-heel length in body mass index and ponderal index decreased the correlation with estimates of body fat. The simple weight/length ratio, exhibiting both a close correlation with body fat and independence of gestational age, race and sex, in near-term infants may be the best morphometric measure of the nutritional component of intrauterine growth in the neonate.

Bromocriptine mesylate for lactation suppression: a risk for postpartum hypertension?

The etiology of postpartum eclampsia and hypertension remains controversial. Recent reports have suggested a possible idiosyncratic hypertensive reaction associated with the use of bromocriptine mesylate. The purpose of this study was to determine whether bromocriptine, used for lactation suppression, is a risk factor for postpartum hypertension. In 1813 consecutively delivered staff patients, blood pressures at three separate home visits were obtained between 3-21 days postpartum. Postpartum hypertension, defined for the purposes of this study as systolic pressure of 140 mmHg or greater and/or diastolic pressure of 90 mmHg or greater on any of the three home visits, was the dependent variable; bromocriptine exposure was the independent variable. Covariates included age, race, chronic hypertension, pregnancy-induced hypertension, and antihypertensive medication. Discriminant analysis, including the first-order interactions, revealed that race, history of chronic hypertension, pregnancy-induced hypertension, and antihypertensive medication contributed significantly to postpartum hypertension (F (7, 1803) = 107.9; P less than .001, explained variance 30%). Of all the interaction terms, only the bromocriptine by pregnancy-induced hypertension interaction was significant. These findings support the contention that patients with antepartum pregnancy-induced hypertension who receive bromocriptine for lactation suppression are at increased risk for postpartum hypertension. Avoiding the elective use of this drug in patients with pregnancy-induced hypertension might constitute a reasonable clinical response to these findings.

Use of an HeO2 mixture in the management of upper airway obstruction during labor and delivery. A case report.

A woman presented in the late third trimester of pregnancy with upper airway obstruction. Pulmonary functions improved with the inhalation of a low-density mixture of He and O2. That gas mixture was employed to augment the air flow and to decrease the respiratory effort during labor and delivery.

First trimester diagnosis and early in utero treatment of obstructive uropathy.

The earliest diagnosis and treatment of obstructed fetal bladder is reported. Placement of vesico-amniotic shunt at 14.5 weeks of gestation enabled preservation of bilateral renal function and maintenance of normal amniotic fluid volume with normal pulmonary development. The shunt functioned adequately for more than 12 weeks. At birth, a mild 'prune' belly was the only deformity noted.

Effects of karyotype, gestational age and medium on the duration of amniotic fluid cell culturing.

Third-trimester genetic amniocentesis has often been underutilized because of the long period required to obtained results. While fetal cord sampling can be used to obtain rapid karyotypes, its availability is still very limited. With more defined media, the culture duration (CD) for amniotic fluid cells has decreased markedly. We investigated CD as a function of gestational age (GA) and karyotype and found that: (1) there is an inverse correlation of GA and CD; (2) aneuploid karyotypes, translocations and inversions all grow more slowly regardless of GA; (3) since late taps are performed most often on highly suspicious cases, the general impression of late-GA slow growth may be because of a higher likelihood of aneuploidy. Our data thus support increased utilization of third-trimester genetic amniocentesis in selected cases.

Pre-eclampsia, magnesium sulfate, and blood pressure evaluation during labor: a preliminary consideration.

The influences of uterine contractions and magnesium sulfate (MgSO4) infusion on blood pressure evaluation of the pre-eclamptic patient in labor have not been thoroughly investigated. Blood pressures were measured between and during contractions in 13 patients with pre-eclampsia, before and after MgSO4, and in 17 normotensive patients. Pre-eclampsia was associated with a mean 11 mm Hg increase in pulse pressure (P less than 0.001), which fell by 8 mm Hg following MgSO4 infusion (P less than 0.001). With pre-eclampsia, pressure increases up to 45/28 mm Hg were observed during contractions. Theoretically, reduced pulse pressure, resulting in decreased shearing force applied to vessel walls, could be a previously unrecognized therapeutic effect of MgSO4. Potentially dangerous pressure elevations could be missed by relying exclusively on diastolic values measured between contractions.

Mechanisms for reduced colloid osmotic pressure in preeclampsia.

The determinants of plasma colloid osmotic pressure were studied in 32 patients with preeclampsia and their matched control subjects. Although plasma colloid osmotic pressure was significantly related to preeclampsia, its severity, and proteinuria, it was most highly correlated with an elevated fibronectin level, suggesting that endothelial injury, rather than proteinuria, is the major mechanism of reduced colloid osmotic pressure in preeclampsia.