Structure of beta-lactam synthetase reveals how to synthesize antibiotics instead of asparagine.

The enzyme beta-lactam synthetase (beta-LS) catalyzes the formation of the beta-lactam ring in clavulanic acid, a clinically important beta-lactamase inhibitor. Whereas the penicillin beta-lactam ring is generated by isopenicillin N synthase (IPNS) in the presence of ferrous ion and dioxygen, beta-LS uses ATP and Mg2+ as cofactors. According to sequence alignments, beta-LS is homologous to class B asparagine synthetases (AS-Bs), ATP/Mg2+-dependent enzymes that convert aspartic acid to asparagine. Here we report the first crystal structure of a beta-LS. The 1.95 A resolution structure of Streptomyces clavuligerus beta-LS provides a fully resolved view of the active site in which substrate, closely related ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP) and a single Mg2+ ion are present. A high degree of substrate preorganization is observed. Comparison to Escherichia coli AS-B reveals the evolutionary changes that have taken place in beta-LS that impede interdomain reaction, which is essential in AS-B, and that accommodate beta-lactam formation. The structural data provide the opportunity to alter the synthetic potential of beta-LS, perhaps leading to the creation of new beta-lactamase inhibitors and beta-lactam antibiotics.

Autistic spectrum disorders in Möbius sequence: a comprehensive study of 25 individuals.

The prevalence of autistic disorder was analysed in 25 individuals with Möbius sequence, a disorder with brain-stem dysfunction. The sample consisted of 18 males and seven females (20 participants were aged 2 to 22 years, and five were aged 1, 19 and 23 months, and 55 years old). Participants were recruited after a nationwide call and were part of a multidisciplinary study of individuals with Möbius sequence. They were given a meticulous neuropsychiatric examination including standardized autism diagnostic interviews. Ten individuals had an autistic spectrum disorder. Six of these met all diagnostic criteria for autism. In 23 individuals cognitive development could be assessed. Eight of those 23 patients had clear learning disability and six individuals were functioning in the normal but subaverage range. Autistic spectrum disorder and learning disability occurred in more than a third of the examined patients. Considering the hospital-based nature of the sample, these findings may be overestimates. Nevertheless, awareness of this coexistence is important in the diagnosis and habilitation care of children with Möbius sequence. Moreover, the results provide further support for the notion of a subgroup of autistic spectrum disorders being caused by first trimester brain-stem damage.

Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide.

A reduction in nitric oxide (NO) synthesis has been suggested to play a role in pregnancy-induced hypertension. We have recently reported that normal pregnancy in the rat is associated with significant increases in whole-body NO production and renal protein expression of neuronal and inducible NO synthase. The purpose of this study was to determine whether whole-body and renal NO production is reduced in a rat model of pregnancy-induced hypertension produced by chronically reducing uterine perfusion pressure starting at day 14 of gestation. Chronic reductions in uterine perfusion pressure resulted in increases in arterial pressure of 20 to 25 mm Hg, decreases in renal plasma flow (<23%) and glomerular filtration rate (<40%), but no difference in urinary nitrite/nitrate excretion relative to control pregnant rats. In contrast, reductions in uterine perfusion pressure in virgin rats resulted in no significant effects on arterial pressure. Renal endothelial (<4%) and inducible (<11%) NO synthase protein expression did not decrease significantly in the chronically reduced uterine perfusion pressure rats relative to normal pregnant rats; however, significant reductions in neuronal NO synthase were observed (<30%). The results of this study indicate that the reduction in renal hemodynamics and the increase in arterial pressure observed in response to chronic decreases in uterine perfusion pressure in pregnant rats are associated with no change in whole-body NO production and a decrease in renal protein expression of neuronal NO synthase.

Assessment of multiple-choice questions in selected test banks accompanying text books used in nursing education.

The purpose of this study was to assess multiple-choice questions used in test-banks accompanying selected nursing textbooks. A random sample of 2,913 questions was selected from a convenience sample of 17 test banks. Questions were evaluated on (a) adherence to generally accepted guidelines for writing multiple-choice questions; (b) cognitive level as defined by Bloom's (1961) taxonomy; and (c) distribution of correct answers as A, B, C, or D. The results were 2,233 violations of item-writing guidelines, most of which were minor but some were serious. A large number of questions (47.3%) were written at the knowledge level and only 6.5% were written at the analysis level. The correct answers were evenly distributed: c2s ranged from 0.00 to 4.84; chi square value needed to reach .05 probability was 26.30. Faculty are encouraged to evaluate multiple-choice questions from test banks carefully before using them for exams.

Induction of anchorage independent growth by heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product. To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells. HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells. Furthermore, NRK cells treated with either E. coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG. We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence.

The möbius sequence: a relook.

BACKGROUND: The constellations of findings often referred to as Mobius syndrome might be better described as Mobius sequence, because the term sequence defines a cascade of secondary events after an embryonic insult from heterogeneous causes. Classic clinical findings include evidence of sixth and seventh cranial nerve involvement, often with associated malformations of limbs, craniofacial structures, and other cranial nerves. METHODS: A prospective study was undertaken in Sweden of 25 patients who showed characteristic findings of Mobius sequence. RESULTS: Of the patients who did not have strabismus surgery, 10 patients had straight eyes in the primary position, 7 had esotropia, 2 had exotropia, and 1 had hypertropia. All had significant limitation of abduction, except 1 patient with exotropia who showed minimal underaction on abduction but a large limitation of adduction. In the description in these early cases, some patients manifested a clinical pattern resembling a horizontal gaze paresis. Narrowing of the palpebral fissure on adduction similar to that seen in Duane syndrome was observed in a few cases. Two patients had ptosis. Nineteen patients had diminished facial expression bilaterally, often asymmetric, and 6 cases appeared to be unilateral. Seven patients had abnormal tearing. Associated systemic findings included Poland anomaly (2), club feet or other limb anomalies (8), micrognathia (8), tongue malformations (17), cleft palate (5), and speech problems (18). An unusual finding was autism syndrome (6) or autism-like syndrome (1). CONCLUSIONS: The associated findings in Mobius sequence may give further clues to the location and timing of the developmental disturbance. The wide range of ocular motility patterns suggests that the previous concept of a lesion solely in the sixth nerve nucleus is an inadequate explanation for these findings.

Chest wall arteriovenous fistula: an unusual complication after chest tube placement.

Posttraumatic arteriovenous fistulas can form between vessels of the thorax that have sustained loss of integrity to the vessel wall. Although most are caused by injuries as a consequence of missile penetration or stab wounds, iatrogenic damage is a potential cause. Herein we present a case of a systemic arteriovenous fistula involving an intercostal artery and subcutaneous vein after chest tube placement.

Differential expression of renal nitric oxide synthase isoforms during pregnancy in rats.

Alterations in nitric oxide (NO) production have been suggested to play a role in mediating changes in renal function during normal pregnancy and in pregnancy-induced hypertension. Although NO production is enhanced during normal pregnancy, the mechanisms for the increase are unknown. The purpose of this study was to determine whether the elevation in NO production during pregnancy is associated with increases in renal expression of endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) nitric oxide synthases. To achieve this goal we examined systemic and renal hemodynamics, urinary excretion of nitrate/nitrite, and renal protein expression of the three NOS isoforms in prepregnant rats, pregnant rats at days 6, 13, and 19 of gestation and at day 4 postpartum. Mean arterial pressure decreased by 14% in late pregnancy whereas the glomerular filtration rate and renal plasma flow increased by 21% and 24%, respectively, in mid pregnancy. Excretion of nitrate/nitrite increased throughout pregnancy with a 3.4-fold increase present at day 19 (12.2+/-0.7 to 41.1+/-1.3 micromol/24 h). Renal eNOS protein expression decreased by 39% during pregnancy with the lowest level resulting at day 19 and returning to virgin levels by day 4 post partum. In contrast, renal iNOS and nNOS protein expression increased 31% and 25%, respectively, with highest expression occurring for both at day 13 of pregnancy. These data suggest that the increased NO production and renal hemodynamics associated with pregnancy in rats may be caused by the upregulation of iNOS and nNOS in the kidney.

Role of nitric oxide in modulating renal function and arterial pressure during chronic aldosterone excess.

Chronic aldosterone (Aldo) excess is associated with transient sodium retention, extracellular fluid volume expansion, renal vasodilation, and hypertension. The purpose of this study was to determine the role of nitric oxide (NO) in mediating the renal vasodilation and the escape from the sodium-retaining actions of Aldo. To achieve this goal, we examined the long-term effects of Aldo (15 microgram. kg-1. min-1 for 7 days) in conscious, chronically instrumented control dogs (n = 9) and in dogs (n = 12) pretreated with the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 microgram. kg-1. min-1). In control dogs, Aldo caused a transient sodium retention (126 +/- 6 to 56 +/- 2 meq/day) followed by a return of sodium excretion to normal levels. Aldo also increased renal plasma flow by 15% (205 +/- 13 to 233 +/- 16 ml/min), glomerular filtration rate by 20% (72 +/- 3 to 87 +/- 5 ml/min), and arterial pressure from 90 +/- 3 to 102 +/- 3 mmHg. Aldo increased urinary nitrate/nitrite excretion by 60% in the control dogs. Although the sodium-retaining (144 +/- 7 to 56 +/- 7 meq/day) and arterial pressure (122 +/- 6 to 136 +/- 5 mmHg) responses to Aldo were the same in dogs pretreated with L-NAME compared with control, the renal hemodynamic response was markedly attenuated. The results of this study suggest that NO plays an important role in mediating the renal vasodilation during chronic Aldo excess.

Infection in surgical patients: effects on mortality, hospitalization, and postdischarge care.

In-hospital mortality, length of stay (LOS), and level of postdischarge care in infected and noninfected surgical patients were studied. An analysis was conducted of a database that included diagnostic, procedure, and drug data collected when surgical patients were discharged from the hospital. Hospitals consisted of 90 nongovernment, nonspecialty, teaching, and nonteaching acute care hospitals of more than 100 beds. Patients in the database included 288,906 patients of all ages hospitalized between July and September 1994. Patients selected of those who had undergone procedures likely to pose a moderate to high risk of infection. Of the 288,906 patients, 12,384 had undergone a moderate- to high-risk procedure; of these, 1,479 (11.9%) had had an infection during their hospitalization. Infection rates ranged from 1.9% to 25.4%, depending on the procedure. The in-hospital mortality rate in infected patients was 14.5%, versus 1.8% for noninfected patients. Similarly, LOS in infected patients (median, 14 days) was substantially greater than in noninfected patients (4 days). About 24% of infected patients required additional professional care after discharge, compared with 7% of noninfected patients. Infection occurs in a substantial portion of surgical patients and is associated with a higher death rate, longer hospitalization, and more intense postdischarge care.

Angiotensin II stimulates synthesis of endothelial nitric oxide synthase.

Previous studies have suggested that NO may play an important role in protecting the renal vessels from angiotensin II (ANGII)-mediated vasoconstriction. One possible mechanism for this interaction is that ANGII could stimulate NO production in the kidney by increasing endothelial NO synthase (NOS III). The present studies were performed in rats to determine whether acute or chronic elevations in ANGII are associated with enhanced renal NOS III mRNA or protein synthesis. In both acute and chronic studies captopril (20 microg/kg/min) was given I.V. to inhibit endogenous ANGII production. Acute suprarenal infusion of ANGII (8 ng/kg/min) for 110 minutes had no effect on arterial pressure but decreased GFR and renal plasma flow by 20% and 30%, respectively, and increased renal vascular resistance by 70%. Acute ANGII increased renal NOS III mRNA by 70% (as determined by ribonuclease protection assay), but had no effect on renal NOS III protein concentration (as detected by Western blot analyses). In contrast, chronic infusion of ANGII (5 ng/kg/min) for 10 days, increased arterial pressure by 30% and tended to reduce GFR and renal plasma flow. Chronic ANGII had no effect on renal NOS III mRNA levels, but increased NOS III protein by 90%. These data suggest that ANGII can stimulate NOS III synthesis and suggest that this may be one of the mechanisms whereby AngII may enhance NO production.

Systemic hemodynamics and regional blood flow during chronic nitric oxide synthesis inhibition in pregnant rats.

Pregnancy-induced hypertension in women is associated with severe vasoconstriction and reductions in organ blood flow and cardiac output. Recent studies have indicated that nitric oxide (NO) synthesis inhibition during mid to late gestation in pregnant rats results in severe hypertension and proteinuria. The purpose of this study was to determine the systemic hemodynamic and regional blood flow alterations associated with chronic NO synthesis inhibition in the pregnant rat. The study was conducted in four groups of rats: virgin rats (n=6), pregnant rats (n=10), virgin rats treated with L-NAME (n=6), and pregnant rats treated with L-NAME (n=11). Rats were treated with L-NAME in drinking water at a dose of 1 mg/d for a week starting from day 13 of gestation in pregnant rats or an equivalent time for virgins. Mean arterial pressure (MAP), cardiac output, total peripheral resistance (TPR), and regional flows were measured by tracing radiolabeled microspheres in conscious rats. Pregnant rats that were given L-NAME showed significantly higher MAP (137+/-6 versus 96+/-2 mm Hg), higher TPR (5.08+/-0.58 versus 2.90+/-0.44 mm Hg/mL/min/100 g), and lower cardiac output (87.4+/-8.4 versus 113.3+/-11.1 mL/min) than pregnant controls. Chronic NO synthesis inhibition decreased the renal blood flow in pregnant rats at a significantly greater magnitude than in virgin rats. Significant reductions in regional blood flow to the heart, lungs, liver, diaphragm, and skeletal muscles were also observed in pregnant rats treated with L-NAME. The results of this study indicate that NO may play a role in mediating the alterations in systemic hemodynamics and regional blood flow in late pregnant rats.

Endothelin-A receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats.

The aim of this study was to examine the role of endothelin-A (ET(A)) receptors in mediating the hypertension and renal injury associated with high salt intake in Dahl salt-sensitive (DS) rats. To achieve this goal, we examined the effects of chronic selective ET(A) antagonist (A-127722) treatment at a dose of 10 mg/kg/d on arterial pressure, renal function, and morphology in DS and Dahl salt-resistant (DR) rats placed on a high sodium (8% NaCl) diet (HSD) for 3 weeks. Placement of DS rats (n=13) on HSD for 3 weeks caused a progressive increase in systolic pressure (from 118+/-3 to 186+/-15 mm Hg). The increase in systolic pressure was significantly attenuated (from 125+/-4 to 167+/-12 mm Hg) in DS rats treated with A-127722 (n=13). Mean arterial pressure (MAP) measured directly at the end of the study was also significantly lower by 18 mm Hg (P<.02) in the DS rats treated with A-127722. The slope of the chronic pressure-natriuresis curve was shifted to the right in DS rats and to the left by chronic ET(A) receptor blockade in DS rats. The hypertension in DS rats was associated with marked proteinuria (from 4.1+/-1.1 to 74.3+/-5.3 mg/24 h/100 g body weight) that was significantly attenuated (from 5.7+/-1.2 to 55.2+/-6.5 mg/24 h/100 g body weight) in DS rats treated with A-127722. The percentage of glomeruli displaying fibrosis, hypercellularity, and hyalinization was also significantly reduced after treatment with A-127722 in DS rats. Arterial pressure, protein excretion, renal hemodynamics, and morphologic structure were not significantly changed in response to ET(A) receptor blockade in DR rats placed on HSD. These data indicate that endothelin-A receptor activation may play a role in the exacerbation of hypertension and development of renal injury in DS rats.

The puzzle of autism: an ophthalmologic contribution.

PURPOSE: A previous study of 86 thalidomide-affected subjects with ophthalmic manifestations revealed the unexpected finding of autism in 4 of the 5 severely retarded individuals. The subjects had anomalies associated with an early gestational effect of thalidomide, including facial nerve palsy and incomitant strabismus. Because autism has been observed in a few cases of Möbius sequence (Möbius syndrome), a condition characterized by involvement of the sixth and seventh cranial nerves, the similarity to early thalidomide embryopathy suggested a relation between cranial nerve involvement and autism. The present study was undertaken to further evaluate the association of autism with patients manifesting findings of Möbius syndrome. METHODS: A prospective study of 25 Swedish patients with Möbius sequence was conducted. The patients had a complete multidisciplinary evaluation, including ophthalmologic and psychiatric examinations and standard testing for autism. Findings associated with autism were compared with the ocular and systemic anomalies of the 4 thalidomide-affected subjects. RESULTS: In the Möbius group 6 patients had autism, achieving the criteria for autism according to all the diagnostic manuals that were used. One patient showed autistic-like conditions meeting fewer numbers of the criteria. A few were too young to be meeting evaluated. Incomitant strabismus ranging from primary abduction defects alone to a horizontal gaze paresis pattern was noted in these patients, in addition to characteristic findings of seventh nerve paresis. Aberrant lacrimation was observed in many cases, especially often associated with autism. CONCLUSION: The common group of anomalies noted in both cases of thalidomide embryopathy and Möbius sequence suggests that brain-stem damage probably early in embryogenesis can sometimes be associated with autism.