Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library--construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine).

Plaster cast burns: the reality.

An in vivo experimental study was performed of the temperatures produced by a setting plaster cast using hot and cold water. The results confirmed a statistical difference in the temperatures reached using hot and cold water. Forearm and below knee plasters did not represent a burn risk. Lower limb cylinder casts reached sufficient temperatures to cause burns with hot water but did not remain at these temperatures long enough to pose a clinical risk.

Prospective comparison of sevoflurane and desflurane in formerly premature infants undergoing inguinal herniotomy.

BACKGROUND: Formerly premature infants having inguinal herniotomy have been at a high risk of postoperative apnoea, newer less soluble anaesthetic agents may reduce this risk. METHODS: Thirty infants, under 37 weeks gestation and under 47 weeks post-conceptional age, undergoing inguinal herniotomy had an inhalational induction with sevoflurane and were randomly allocated to sevoflurane (group S) or desflurane (group D) for maintenance. All infants received i.v. atracurium 0.5 mg kg(-1), rectal acetaminophen 20 mg kg(-1) and caudal bupivacaine 0.25% 1 ml kg(-1). Infants were monitored for apnoeas (using nasal thermistry and impedance), haemoglobin oxygen desaturations and bradycardias for 12 h before and after operation with an Alice 4 polysomnograph. Emergence timings were recorded. RESULTS: There was no difference between pre- and postoperative incidence of apnoeas in either group, and no group difference between desflurane and sevoflurane in terms of pre- and postoperative ventilatory events or in the number of apnoeas in the postoperative period (nine patients in group D and five patients in group S had apnoeas). Median times to first movement, tracheal extubation, eye opening and first cry were all faster with group D (group D: 3.0, 10.0, 9.0 and 11.0 min and group S: 7.0, 15.1, 13.5 and 16.1 min, respectively). No infant had problems with airway irritation on emergence and no infant required airway intervention for apnoea. CONCLUSIONS: Infants wake faster from general anaesthesia when maintained with desflurane as compared with sevoflurane, but no difference in postoperative respiratory events was demonstrated between the groups.

Emergence and recovery in children after desflurane and isoflurane anaesthesia: effect of anaesthetic duration.

BACKGROUND: We hypothesized that increasing duration of inhalation anaesthesia is associated with slower emergence and recovery in children, and that this effect would be less marked with desflurane in comparison with isoflurane. METHODS: Fifty-four infants and children assigned in groups according to age and expected length of operation were prospectively randomized to receive either isoflurane (I) or desflurane (D) for anaesthesia. After standard induction, the anaesthesia was maintained using an age-related 1.0 minimum alveolar concentration (MAC) equivalent for either agent in air and oxygen. Local analgesia was used as appropriate. End-tidal volatile agent concentration was recorded until extubation. Clinical evaluation of recovery was made by observers, blinded to group allocation. RESULTS: For patients <4 yr of age, the median (95% CI) times in minutes to first movement [5.27 (D), 9.22 (I)], eye opening [9.42(D), 13.3(I)] and extubation [7.18 (D), 12.5 (I)] were significantly shorter (P<0.05) for desflurane. In the group >4 yr of age, the median (95% CI) times in minutes to first movement [4.42 (D), 11.6 (I)], eye opening [8.55(D), 18.0(I)] and extubation [7.08 (D), 16.7 (I)] were significantly shorter (P<0.001) for desflurane. Times to leave recovery were not significantly different for the group <4 yr of age, but were significantly shorter for desflurane in the group >4 yr of age (P<0.01). The isoflurane, but not desflurane, had a time-dependent effect on arousal. There were no significant differences in incidence of airway irritation or emergence delirium between the two agents. CONCLUSIONS: The rate of recovery in children after exposure to desflurane was faster than those patients receiving isoflurane; recovery from desflurane, but not isoflurane, was relatively unaffected by the duration of anaesthesia.

Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase.

Lactate dehydrogenase (LDH) interconverts pyruvate and lactate with concomitant interconversion of NADH and NAD(+). Although crystal structures of a variety of LDH have previously been described, a notable absence has been any of the three known human forms of this glycolytic enzyme. We have now determined the crystal structures of two isoforms of human LDH-the M form, predominantly found in muscle; and the H form, found mainly in cardiac muscle. Both structures have been crystallized as ternary complexes in the presence of the NADH cofactor and oxamate, a substrate-like inhibitor. Although each of these isoforms has different kinetic properties, the domain structure, subunit association, and active-site regions are indistinguishable between the two structures. The pK(a) that governs the K(M) for pyruvate for the two isozymes is found to differ by about 0.94 pH units, consistent with variation in pK(a) of the active-site histidine. The close similarity of these crystal structures suggests the distinctive activity of these enzyme isoforms is likely to result directly from variation of charged surface residues peripheral to the active site, a hypothesis supported by electrostatic calculations based on each structure. Proteins 2001;43:175-185.

Effector sites in the three-dimensional structure of mammalian sperm beta-acrosin.

BACKGROUND: Proacrosin is a serine protease found specifically within the acrosomal vesicle of all mammalian spermatozoa. During fertilization proacrosin autoactivates to form beta-acrosin, in which there is a "light" chain cross-linked to a "heavy" chain by two disulphide bonds. beta-acrosin is thought to be multifunctional with roles in acrosomal exocytosis, as a receptor for zona pellucida proteins, and as a protease to facilitate penetration of spermatozoa into the egg. RESULTS: The crystal structures of both ram and boar beta-acrosins have been solved in complex with p-aminobenzamidine to 2.1 A and 2.9 A resolution, respectively. Both enzymes comprise a heavy chain with structural homology to trypsin, and a light chain covalently associated in a similar manner to blood coagulation enzymes. In crystals of boar beta-acrosin, the carboxyl terminus of the heavy chain is inserted into the active site of the neighboring molecule. In both enzyme structures, there are distinctive positively charged surface "patches" close to the active site, which associate with carbohydrate from adjacent molecules and also bind sulfate ions. CONCLUSIONS: From the three-dimensional structure of beta-acrosin, two separate effector sites are evident. First, proteolytic activity, believed to be important at various stages during fertilization, arises from the trypsin-like active site. Activity of this site may be autoregulated through intermolecular associations. Second, positively charged regions on the surface adjacent to the active site may act as receptors for binding zona pellucida glycoproteins. The spatial proximity of these two effector sites suggests there may be synergy between them.

Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-beta binding alcohol dehydrogenase (ABAD).

Human type II hydroxyacyl-CoA dehydrogenase/amyloid-beta binding alcohol dehydrogenase (HADH II/ABAD) is an oxidoreductase whose salient features include broad substrate specificity, encompassing 3-hydroxyacyl-CoA derivatives, hydroxysteroids, alcohols and beta-hydroxybutyrate, and the capacity to bind amyloid-beta peptide, leading to propagation of amyloid-induced cell stress. In this study, we examine the structure and enzymatic activity of the homologous rat HADH II/ABAD enzyme. We report the crystal structure of rat HADH II/ABAD as a binary complex with its NADH cofactor to 2.0 A resolution, as a ternary complex with NAD(+) and 3-ketobutyrate (acetoacetate) to 1.4 A resolution, and as a ternary complex with NADH and 17 beta-estradiol to 1.7 A resolution. This first crystal structure of an HADH II confirms these enzymes are closely related to the short-chain hydroxysteroid dehydrogenases and differ substantially from the classic, type I 3-hydroxyacyl-CoA dehydrogenases. Binding of the ketobutyrate substrate is accompanied by closure of the active site specificity loop, whereas the steroid substrate does not appear to require closure for binding. Despite the different chemical nature of the two bound substrates, the presentation of chemical groups within the active site of each complex is remarkably similar, allowing a general mechanism for catalytic activity to be proposed. There is a characteristic extension to the active site that is likely to accommodate the CoA moiety of 3-hydroxyacyl-CoA substrates. Rat HADH II/ABAD also binds amyloid-beta (1-40) peptide with a K(D) of 21 nM, which is similar to the interaction exhibited between this peptide and human HADH II/ABAD. These studies provide the first structural insights into HADH II/ABAD interaction with its substrates, and indicate the relevance of the rodent enzyme and associated rodent models for analysis of HADH II/ABAD's physiologic and pathophysiologic properties.

Purification, co-crystallization and preliminary X--ray analysis of the natural aspartic proteinase inhibitor IA3 complexed with saccharopepsin from Saccharomyces cerevisiae.

The vacuolar aspartic proteinase from baker's yeast, saccharopepsin, has been co-crystallized with its natural inhibitor I(A)3, found in the cytosol. The I(A)3-saccharopepsin complex crystals belong to the space group P6(2)22, with unit-cell parameters a = b = 192.1, c = 59. 80 A and one molecule per asymmetric unit. The initial X-ray analysis of the complex indicates that the crystals diffract to 5.0 A, similar to native saccharopepsin crystals. This is probably a consequence in part of glycosylation of the native saccharopepsin. Full structural analysis of the complex crystal is in progress.

Chloroquine binds in the cofactor binding site of Plasmodium falciparum lactate dehydrogenase.

Although the molecular mechanism by which chloroquine exerts its effects on the malarial parasite Plasmodium falciparum remains unclear, the drug has previously been found to interact specifically with the glycolytic enzyme lactate dehydrogenase from the parasite. In this study we have determined the crystal structure of the complex between chloroquine and P. falciparum lactate dehydrogenase. The bound chloroquine is clearly seen within the NADH binding pocket of the enzyme, occupying a position similar to that of the adenyl ring of the cofactor. Chloroquine hence competes with NADH for binding to the enzyme, acting as a competitive inhibitor for this critical glycolytic enzyme. Specific interactions between the drug and amino acids unique to the malarial form of the enzyme suggest this binding is selective. Inhibition studies confirm that chloroquine acts as a weak inhibitor of lactate dehydrogenase, with mild selectivity for the parasite enzyme. As chloroquine has been shown to accumulate to millimolar concentrations within the food vacuole in the gut of the parasite, even low levels of inhibition may contribute to the biological efficacy of the drug. The structure of this enzyme-inhibitor complex provides a template from which the quinoline moiety might be modified to develop more efficient inhibitors of the enzyme.

Prenatal maternal blood pressure response to stress predicts birth weight and gestational age: a preliminary study.

OBJECTIVE: The objective was to test the prospective association between prenatal maternal circulatory responses to a standardized psychologic challenge and birth outcome. STUDY DESIGN: We examined the relationship between blood pressure responses to a cognitive arithmetic stressor and birth outcome in 40 healthy primigravid women. Pregnant women between 18 and 37 years old were recruited from the University of Kentucky Prenatal Service Clinic for participation. All women performed an interactive arithmetic task while maternal heart rate and blood pressures were determined. Subsequent birth outcome parameters of birth weight and gestational age were obtained for prospective analyses. RESULTS: Results indicated that maternal systolic and diastolic blood pressures and heart rates were significantly increased during the arithmetic task (p < or = 0.01). Regression analyses suggested that women with larger diastolic blood pressure responses during stress had infants with lower birth weights (p < 0.01) and decreased gestational age (p < 0.05). CONCLUSIONS: This effect was specific to psychologic stress reactivity and was not related to maternal age, maternal race, baseline blood pressures, the trimester of stress testing, nor expired carbon monoxide. The relationship between maternal blood pressure response and birth outcome may reflect the transplacental impact of individual differences in systemic stress responsivity.

Risk factors for preterm delivery in a healthy cohort.

To examine whether risk factors differed among subgroups of preterm (< 37 weeks of gestation) deliveries, we studied a cohort of 1,825 enlisted servicewomen who delivered from 1987 through 1990 at four U.S. Army medical centers. Preterm deliveries were classified by length of gestation (< 29 weeks, 29-32 weeks, 33-36 weeks) and clinical course [medical indication, idiopathic preterm labor, or preterm rupture of membranes (PROM)]. We abstracted medical records for information on age, race, army rank, marital status, gravidity, parity, the baby's sex, maternal prepregnancy height and weight, gestation at entry to prenatal care, alcohol drinking and smoking, time since and outcome of preceding pregnancy, surgery performed during pregnancy, anemia, and diagnoses of uterine abnormalities, sexually transmitted diseases, and urinary tract infections. We used proportional hazards analysis to evaluate associations for each subgroup of preterm delivery. The relative odds associated with a history of preterm delivery in the preceding pregnancy ranged from 3.1 for deliveries due to preterm labor or PROM to 6.2 for deliveries that occurred during 29-32 weeks; none of the other factors was consistently associated across the subgroups of preterm delivery. The paucity of associations is consistent with the conclusion of other investigators that most of the causes of preterm delivery are unknown.

Prevalence of low birth weight and preterm delivery in relation to the interval between pregnancies among white and black women.

BACKGROUND: The higher mortality rate among black infants than among white infants in the United States results largely from the greater frequency of low birth weight and prematurity among black infants. Higher rates of low birth weight and preterm delivery have been associated with shorter intervals between pregnancies. METHODS: We studied a racially mixed population of women in military families, who had access to free, high-quality health care. A total of 1992 white and black women had two consecutive, singleton pregnancies during the study period. We determined the outcome of the second of each pair of pregnancies and the length of time between the pregnancies. RESULTS: Short interpregnancy intervals (calculated from delivery to the next conception) were more frequent among black than among white women. A total of 7.7 percent of the 298 black women and 3.2 percent of the 1628 white women delivered premature, low-birth-weight infants (P < 0.001). Among the black women, an interpregnancy interval of less than nine months was associated with a significantly greater prevalence of preterm delivery and low birth weight in the neonates (11.6 percent, vs. 4.4 percent for longer interpregnancy intervals; P = 0.020). Among the white women, only intervals of less than three months between pregnancies were associated with a greater prevalence of prematurity and low birth weight in the infants (11.8 percent vs. 2.8 percent; P < 0.001). Of the black women, 46.3 percent had interpregnancy intervals of less than nine months; 4.2 percent of the white women had interpregnancy intervals of less than three months. CONCLUSIONS: A short interval between pregnancies is a risk factor for low birth weight and preterm delivery, and such intervals are more common among black than among white women. The relative frequency of intervals of less than nine months between pregnancies may be an important factor in the wide disparity in pregnancy outcomes between white and black women in the United States.

Antenatal hospitalization among enlisted servicewomen, 1987-1990.

OBJECTIVE: To describe the prevalence of, and indications for, antenatal hospitalization among women who delivered live and stillborn infants. METHODS: We reviewed the records of a cohort of 1825 black and white enlisted women who delivered from 1987-1990 at the four largest Army medical centers in the United States. Women with multiple gestations and those whose pregnancies ended before 20 weeks' gestation were excluded. Records of all women with preterm deliveries and a one-third sample of women with term deliveries were abstracted. RESULTS: Overall, 26.8 +/- 1.6% (mean +/- standard error) of the women were hospitalized antenatally. Of the estimated 702 antenatal hospitalizations, 44.0 +/- 3.4% were related to preterm labor, 10.3 +/- 1.9% to preeclampsia, 5.5 +/- 1.5% to hyperemesis, and 4.7 +/- 1.5% to urinary tract or kidney infection. The prevalence of hospitalization was lowest before 20 weeks (5.0 +/- 0.8%) and highest at 33-36 weeks (12.2 +/- 1.2%). Small and probably clinically insignificant differences between black and white women were noted in the overall prevalence of antenatal hospitalization and in the indications for hospitalization. CONCLUSION: As measured by hospitalization, severe antenatal morbidity is common in this population of healthy enlisted women.

Preterm delivery among black and white enlisted women in the United States Army.

OBJECTIVE: To examine black-white differences in preterm delivery in a healthy population who had unrestricted access to prenatal care. METHODS: We conducted a retrospective cohort study of 842 black and 1026 white enlisted servicewomen who delivered a singleton infant of 20 or more weeks' gestation from July 1, 1987 through September 30, 1990 at four Army Medical Centers in the United States. Data were collected by reviewing maternal and newborn records. We used logistic and proportional hazards regression models to analyze outcomes defined by length of gestation, cause of preterm delivery, and jointly by length and cause. RESULTS: Black enlisted women had a cumulative probability of preterm delivery (13.5%) that was higher than that for white enlisted women (10.5%) (hazard ratio 1.31, 95% confidence interval [CI] 1.002-1.70). However, the ratio of black-to-white hazards was not uniform. Black-white differences were small and nonsignificant from 33-36 weeks' gestation, when most preterm deliveries occur. The differences were also small and nonsignificant for deliveries related to spontaneous rupture of membranes or idiopathic preterm labor, the most common causes of preterm delivery. The black-to-white hazard ratios were greatest for all deliveries before 33 weeks' gestation and for medically indicated preterm deliveries. CONCLUSIONS: Efforts to reduce black-white differences in preterm delivery must go beyond providing prenatal care and eliminating recreational drug use. Future studies should consider black-white differences in environments during the mother's own development and in psychosocial and physical stresses during pregnancy.

Risk of chromosomal abnormalities in patients with idiopathic polyhydramnios.

This prospective investigation was designed to assess the incidence of chromosomal abnormalities in patients with idiopathic polyhydramnios. Polyhydramnios was defined as 25 cm or greater in total vertical height in all four quadrants (amniotic fluid index) in any nonreferral patient (ie, primary care population) undergoing sonographic examination with a singleton pregnancy, normal fetal anatomical survey, normal glucose screening, and negative antibody screen. During the 2-year period from May 1, 1988 through April 30, 1990, 5038 gravidas delivered at Madigan Army Hospital Center. Unexplained polyhydramnios was detected sonographically in 125 patients, an incidence of 2.5%. After obtaining informed written consent, amniocentesis was performed in all patients. Within this group (N = 125), four chromosomal abnormalities (incidence of 3.2%) were detected. There were two trisomy 18 and two trisomy 21 fetuses. None of the four patients had maternal serum alpha-fetoprotein screening performed. The incidence of aneuploidy in patients with idiopathic polyhydramnios (3.2%) is much higher than the reported incidence of major karyotype abnormalities in live births (0.59%). We conclude that fetal chromosomal analysis should be considered in all obstetric patients with sonographic evidence of idiopathic polyhydramnios.

The association of antiphospholipid antibodies with pregnancies complicated by fetal growth restriction.

The association of antiphospholipid antibodies with fetal growth restriction is often cited, but the published evidence for this is based on few patients and comes primarily from patient histories, not study groups. In this prospective study, we evaluated a subgroup of our population with fetuses whose estimated weights at ultrasound were at or below the tenth percentile for gestational age. Plasma and serum testing was performed to determine the presence of antiphospholipid antibodies, specifically lupus anticoagulant and anticardiolipin antibodies, respectively. From March 1990 through March 1991, 55 women were followed for suspected fetal growth restriction. Intensive monitoring of the fetal condition and modification of the mother's activity were recommended, resulting in 100% compliance. Despite this, 37 newborns were confirmed by birth weight to be at or below the tenth percentile, and all were below the 45th percentile. Fifteen of 55 women (27%) were positive for anticardiolipin antibodies, as were nine of 37 (24%) with correctly diagnosed fetal growth restriction. Five of 15 women whose newborns had ponderal indexes below the tenth percentile tested positive for anticardiolipin antibodies. None of the women had a positive lupus anticoagulant test. The prevalence of anticardiolipin antibodies in this study group was significantly higher than in our general population. We conclude that there is a statistically significant association between the presence of circulating maternal anticardiolipin antibodies and fetal growth restriction.

A simple method of separation of Pneumocystis carinii from rat lung.

Existing methods for purification of Pneumocystis carinii are unsatisfactory. Dialysis is a simple, non-stressful treatment which aided purification of organisms from rat cells. Yields were quantitated using a Petroff-Hausser chamber and nuclear staining. Following dialysis overnight, three times the yields of similar, undialysed preparations were obtained, with less than 0.2% contamination by lung cells. Overnight incubation at 4 degrees C also improved separation, but yields were only half those following dialysis. Dialysis is now used routinely in the preparation of pure Pneumocystis carinii suspensions.

Screening for illicit drug use in a military obstetric population.

The purpose of our investigation was to determine the prevalence of illicit drug use within our socioeconomically heterogeneous obstetric population, in order to assess the need for institution of universal screening. Five hundred consecutive new obstetric registrants had urine collected for routine culture. Following removal of a small aliquot of urine for culture, the samples were sent to the Armed Forces Institute of Pathology, Division of Forensic Toxicology. Each specimen was screened for the presence of alcohol, cocaine metabolites, cannabinoids, opiates, and amphetamines using fluorescent polarization immunoassay techniques. All positive screening tests were confirmed by gas chromatography mass spectrometry. Thirty samples were either lost in processing or of insufficient quantity to test. Five of the 470 samples (1.06%) tested were positive. One subject was taking prescription narcotics, so the correlated prevalence was 0.85%. Three tested positive for tetrahydrocannabinol and two for opiates. Analysis of our data demonstrates that our obstetric population has a significantly lower prevalence of illicit drug use than other populations reported previously (P less than .01). We recommend that each institution providing obstetric services determine its specific prevalence of illicit drug use.