Neonatal hyperbilirubinemia associated with glucose-6-phosphate dehydrogenase deficiency in Sephardic-Jewish neonates: incidence, severity, and the effect of phototherapy. Glucose-6-phosphate dehydrogenase (G-6-PD) deficiency is frequently associated with neonatal hyperbilirubinemia, and sometimes kernicterus, often in the absence of any identifiable trigger or hematological evidence of hemolysis. The aim of this study was to compare the incidence and severity of, and the effect of phototherapy on, jaundice in G 6-PD-deficient vs G-6-PD-normal neonates in the Sephardic-Jewish community. Healthy term newborns, born to mothers of families stemming from geographic areas known to be "at risk" for G-6-PD deficiency, were screened for the condition and surveyed for hyperbilirubinemia. Seventy-five G-6-PD-deficient neonates formed the study group, while 266 neonates with normal levels of the enzyme formed the control group. Neonates with any other identifiable cause for jaundice were excluded. Phototherapy was commenced when the serum bilirubin levels reached 16 mg/dL (274 mumol/L) or more, and it was discontinued at 12 mg/dL (205 mumol/L) or less. Hyperbilirubinemia developed in 27 (36%) of the deficient neonates (serum total bilirubin greater than 13.9 mg/dL [238 mumol/L]), compared with 50 (18.8%) of control neonates (P = .002), while 20 (26.7%) of the study group required phototherapy, compared with 31 (11.7%) of control neonates (P = .002). Two neonates in the study group required exchange transfusion (serum bilirubin greater than 20 mg/dL [342 mumol/L]), vs 0 in the control group (not significant).(ABSTRACT TRUNCATED AT 250 WORDS)


Community-based evaluation of the effect of breast-feeding on the risk of microbiologically confirmed or clinically presumptive shigellosis in Bangladeshi children. To assess the association between breast-feeding and the risk of microbiologically confirmed or clinically presumptive shigellosis, the authors performed a case-control analysis of Bangladeshi children younger than 3 years of age who were followed up for 1 month after exposure to Shigella in their residential neighborhoods. Two hundred sixty-nine cases with culture-confirmed shigellosis (n = 119) or clinically presumptive shigellosis (culture-negative dysentery, n = 150) were compared with 819 controls without Shigella diarrhea or other invasive diarrheal illnesses. The odds ratio relating breast-feeding to confirmed or presumptive shigellosis, adjusted for potentially confounding variables, was 0.48 (95% confidence interval = 0.32 to 0.72; P less than .001), suggesting a substantial protective effect. The protective association decreased with age but was still significant during the third year of life; appeared to be directly related to the degree of stunting; and was equivalent for confirmed and presumptive shigellosis. Notably, the protective association remained substantial against episodes due to Shigella which were resistant to at least one of the antibiotics customarily used for treatment of Shigella diarrhea (age-adjusted odds ratio = 0.40; 95% confidence interval = 0.22 to 0.74; P less than .01). These data suggest that breast-feeding confers a high level of protection against shigellosis throughout the first 3 years of life, especially among nutritionally compromised children, and thereby underscore the importance of promotion of breast-feeding as a central component of Shigella control programs in less developed settings.


Microwave heating of infant formula: a dilemma resolved. Microwave heating of infant formula is a common practice despite concerns of infant scalding. Beyond the issue of physical safety, little is known about the effects on nutrient content of microwave heating of infant formula. Casein-predominant infant formula in 120- and 240-mL glass and plastic nursing bottles of varying colors were heated for 40 seconds and 60 seconds, respectively. Temperature profiling was monitored during the heating cycle. Analysis of riboflavin and vitamin C was made prior to and after heating. Topmost portions reached a mean temperature of 44.7 +/- 1.7 degrees C and 43.0 +/- 2.4 degrees C for all types of 240-mL and 120-mL bottles, respectively. Topmost temperatures were significantly hotter than temperatures reached at other sites. Routine mixing resulted in formula temperatures which could safely be fed to infants (35.4 +/- 0.3 degrees C and 33.9 +/- 0.2 degrees C for 240-mL and 120-mL bottles, respectively). There was no significant loss of either riboflavin or vitamin C. Protocols for microwave heating are given.


Brush-culture method for diagnosing tinea capitis. Using traditional methods to verify the existence of a fungal infection in children with suspected tinea capitis is a cumbersome process. Scraping scale and pulling hairs for culture or microscopic examination can be time-consuming and uncomfortable for the child. This study is the first comparison of this method with an alternate brush-culture method for diagnosing tinea capitis. In 70 children with symptoms suggestive of tinea capitis, there was strong agreement between methods in detecting disease or lack thereof (McNemar's test, P less than .2). In the 51 paired positive cultures, those obtained by brush culture turned positive significantly faster (t test, P less than .01) than samples obtained by the traditional method. The brush method is a reliable, painless, and more expedient way to obtain cultures from children with suspected tinea capitis.


Growth and development of children conceived by in vitro fertilization. To assess the physical and mental development of infants born after in vitro fertilization (IVF), we performed a general physical and developmental examination (Bayley and Stanford-Binet scales) on a cohort of 116 IVF children, conceived and born at our institution between February 1985 and March 1989, and on 116 non-IVF matched controls. Study and control groups were each composed of 66 singletons, 19 pairs of twins and 4 sets of triplets, whose age at examination ranged from 12 to 45 months. The developmental indices of IVF infants were within the normal range and did not differ from those of their matched controls. The indices were positively correlated to gestational age, birth weight, head circumference at birth and at examination, and mother's education. Mean birth weight, gestational age, and birth weight percentile of IVF infants were lower than the mean of the healthy population. Mean percentiles of weight and length at examination (mean age 22.4 months) were equally low but did not differ from those of the matched controls. However, mean percentiles of head circumference at birth and at examination compare well with the normal mean, both in IVF and control groups. Twins and triplets (IVF and controls) had significantly lower physical and mental indices as compared to singletons.


Effect of maternal hypertension on neonatal neutropenia and risk of nosocomial infection. Neonatal neutropenia occurs in approximately 50% of newborns delivered by women with pregnancy-induced hypertension. It is thought to be transient, independent of birth weight and gestational age, and unassociated with significant risks, including infection. It recently was suggested that neonatal neutropenia occurs primarily in smaller, younger neonates, is related to the severity of pregnancy-induced hypertension, and importantly, may be associated with an increased risk for nosocomial infection. We examined these points in a large inborn population in consecutive years, performing retrospective (n = 110, 1989) and prospective (n = 151, 1990) studies in low birth weight (less than or equal to 2200 g) neonates delivered by women with pregnancy-induced hypertension. Overall, 40% to 50% of neonates studied developed neonatal neutropenia, and they were younger and smaller (P less than .01) than non-neutropenic neonates. In the prospective study, neutropenic neonates were more likely to have mothers with severe pregnancy-induced hypertension (P less than .001), and the incidence of neonatal neutropenia was primarily among neonates less than 30 weeks of gestation and less than 1500 g birth weight, approximately 80% vs 35% to 45% in older, larger neonates or infants (P less than .001). Although nosocomial infection occurred more frequently among the group of neutropenic neonates in the prospective study (P less than .02), the incidence was similar to that in matched non-neutropenic controls delivered of normotensive women. Thrombocytopenia (less than 100,000/mm3) was not more frequent in neutropenic neonates.(ABSTRACT TRUNCATED AT 250 WORDS)


Diagnostic imaging in the evaluation of the first urinary tract infection in infants and young children. The evaluation of infants and children after the first urinary tract infection has undergone change in recent years. Standard diagnostic imaging studies are being utilized on a more frequent basis, because these procedures can provide information which often has a direct impact on patient care. Selection of the proper tests requires an understanding of how they are performed and the basis for their choice. The rationale for the use of different imaging studies and their application to patient care are discussed.


Urine sodium excretion in response to an oral glucose tolerance test in obese and nonobese adolescents. To determine if physiologic changes of insulin are capable of inducing sodium retention in insulin-resistant patients, we evaluated the ability of an oral glucose tolerance test to alter urine sodium excretion in 32 obese subjects (13.3 +/- 1 years, weight 82 +/- 5 kg, mean arterial pressure 89.3 +/- 1.5 mm Hg) and 13 nonobese subjects (13.8 +/- 2 years, weight 46 +/- 4 kg, mean arterial pressure 74.5 +/- 2.6 mm Hg). After an overnight fast, subjects were placed in water diuresis and fasting insulin and glucose levels were drawn. Three 30-minute urine collections were obtained for baseline urine sodium excretion. The oral glucose tolerance test was administered with glucose and insulin levels drawn at 15, 30, 45, 60, 90, and 120 minutes. During the oral glucose tolerance test, four 30-minute urine collections were obtained for urine sodium excretion. Serum glucose levels at baseline and throughout the glucose tolerance test did not differ between obese and nonobese subjects. Baseline insulin levels were elevated significantly in the obese (20 +/- 3 microU/mL) compared with the nonobese (5 +/- 0.7 microU/mL) subjects. Furthermore, insulin levels remained significantly elevated in the obese subjects compared with the nonobese subjects throughout the glucose tolerance test (118 +/- 19 vs 49 +/- 6 microU/mL, obese vs nonobese subjects at 1-hour post-glucose tolerance test).(ABSTRACT TRUNCATED AT 250 WORDS)


A prospective study of cognitive functioning following low-dose cranial radiation for bone marrow transplantation. This study prospectively examined the effects of low doses of cranial irradiation on psychological development in children. The subjects were 22 children receiving bone marrow transplantation. Fifteen children receiving cranial doses ranging from 350 to 1200 cGy as part of their conditioning regimen were contrasted to seven children who were not irradiated. Measures of intellectual and psychosocial development were administered to all subjects prior to transplant and again at one-year posttransplant. There were no decrements in psychological functioning at the one-year follow-up, regardless of the dose of cranial radiation received or the age at which radiation was administered. In addition, there were no significant differences in test scores relative to baseline in a smaller cohort of children followed up for 3 years. Although no significant between-group findings were found, examination of individual cases revealed a high degree of variability, with decrements in IQ of 10 points or more found in 7 children at the one-year follow-up. Findings suggest that doses less than 1500 cGy may prove to be relatively well tolerated with respect to long-term cognitive and psychosocial development, even in young children, although caution is urged in light of the limited length of follow-up.


Screening for fungal endophthalmitis in children at risk. To evaluate the efficacy of screening ophthalmologic examinations in high-risk children, we reviewed the medical records for all patients hospitalized from 1985 through 1989 at The Hospital for Sick Children, Toronto, Ontario, who underwent ophthalmological consultation to rule out endogenous fungal endophthalmitis (n = 176). The patients were divided into groups: Group 1 (n = 47), those with deep-tissue fungal infection, and Group 2 (n = 129), those at risk for invasive fungal disease. Group 2 was subdivided further into two subgroups: Group 2a (n = 48), those with evidence of superficial fungal colonization (positive fungal culture) but no deep-tissue involvement, and Group 2b (n = 81), those with no evidence of fungal colonization (negative fungal culture). Of these 176 patients, 7 were diagnosed with endogenous fungal endophthalmitis: 6 from Group 1, 1 from Group 2a, and 0 from Group 2b. We found a significant association between the development of endogenous fungal endophthalmitis and the status of the fungal culture result (P less than .005). The odds ratio indicated the risk of endogenous fungal endophthalmitis in Group 1 patients with deep-tissue infection was at least 19 times that of Group 2 at-risk patients. The risk of endogenous fungal endophthalmitis in Group 1 patients was at least 7 times that of Group 2a colonized patients and 12 times that of Group 2b patients with no positive fungal culture. Our study confirms the necessity of careful dilated ophthalmoscopic examination in patients with invasive fungal disease and suggests screening for those at-risk patients with superficial fungal colonization.(ABSTRACT TRUNCATED AT 250 WORDS)


Basics and recommendations for brain SPECT. Task Group "Neurology" of the European Association of Nuclear Medicine. In this article guidelines for single-photon emission computerized tomography (SPECT) of the brain are presented by the Task Group "Neurology" of the European Association of Nuclear Medicine (EANM). Under separate headings are discussed: the pathophysiologic principles of various brain diseases, pharmacokinetics and pharmacodynamics of various radiopharmaceuticals, physical principles of SPECT imaging, computerized data processing, clinical protocols and the evaluation of SPECT results.


[Is a TSH screening for the diagnosis or exclusion of functional thyroid autonomy meaningful?]. A normal baseline TSH serum concentration is accepted as excluding thyroid dysfunction. The present study examined the usefulness of that concentration as a screening test for functional autonomy. 310 patients were retrospectively examined. They were all clinically euthyroid and had normal peripheral thyroid hormone concentrations. The positive predictive value for the confirmation of autonomy was 55.5%, the negative predictive value for the exclusion of autonomy 87.7%, using 0.5 mU/l as a lower limit. The probability for the presence of thyroid autonomy in patients with a TSH serum concentration below 0.3 mU/l was 72.5%.


Late cervical lymph node recurrence in differentiated thyroid carcinoma. An inherent problem of 131I beta dose distribution in small tumor deposits? In differentiated thyroid carcinoma, postoperative radioiodine therapy is an undisputed element of the integral therapeutic concept. Sometimes, however, and years after initial diagnosis and therapy, cervical lymph node metastases occur without any evidence of local relapse or distant metastases, indicating that radioiodine therapy has not achieved definite tumor cell sterilization. A Monte Carlo computer simulation of the spatial energy dose distribution of 131I in small functioning tumor manifestations was performed to explain this phenomenon. It appeared that only a small fraction of the total beta-energy dose can be deposited inside the tumor if the latter becomes smaller than 1 mm in diameter: for tumors with homogeneous 131I uptake and diameters of 1.0, 0.5, 0.2, 0.1, 0.05 and 0.02 mm, this value does not exceed 86, 73, 39, 16, 8 and 4%, respectively. These data suggest a potential therapeutic gap for small tumor manifestations not apparent at the time of initial diagnosis and could explain the occasional occurrence of late cervical lymph node metastases.


Variability in teniposide plasma protein binding is correlated with serum albumin concentrations. Teniposide is a widely used anticancer drug that is extensively bound to plasma proteins (greater than 95%). We evaluated the drug's plasma protein binding in nine patients with acute lymphocytic leukemia who were in their first complete remission, and in a second group of nine patients at the time of relapse and subsequently after achieving another complete remission. Plasma protein binding was assessed by equilibrium dialysis, with direct high-performance liquid chromatographic measurement of total and free teniposide. The mean unbound fraction was 0.44% (0.21-0.88%) in the plasma of patients in first remission. It was significantly higher in patients at the time of relapse (mean = 0.86%; range 0.68-1.08%) and after achieving another complete remission (mean = 1.25%; range 0.51-2.11%). Serum albumin values were significantly lower at the time of relapse (mean = 4.6 vs 4.0 mg/dl; p less than 0.014), and decreased further during intensive postremission therapy containing L-asparaginase (mean = 3.2; p less than 0.05). For all 18 patients, a significant negative correlation (r2 = 0.667; p less than 0.001) was found between serum albumin and unbound teniposide, with low albumin being associated with higher unbound fraction. Such patients have higher systemic exposure to unbound (presumably active) teniposide at any given total plasma concentration of the agent.


Prospective evaluation of risk factors for antibiotic-associated bleeding in critically ill patients. A prospective surveillance program was initiated to determine the relative role of antibiotics containing N-methylthiotetrazole (NMTT) versus patient risk factors in producing antibiotic-associated bleeding. Five hundred forty-six critically ill patients with serum albumin 30 g/L or below were evaluated for evidence of a bleeding event as documented by clinical observation, hemoglobin changes, and transfusions. Bleeding events occurred in 16% of patients receiving an aminoglycoside combination, 10% receiving antibiotics with the NMTT side chain, and 14.5% receiving antibiotics not containing NMTT (p greater than 0.05). The bleeding rate was highest in febrile patients with cancer (14.5%) and lowest in those with a suspected or documented abdominal infection (10%) (p = 0.04), but within each patient group there was no difference among the antibiotics. We conclude that the use of NMTT-containing antibiotics is not an independent risk factor for bleeding, but the role of severity of illness may be underappreciated.


Combination use of sulfonylureas and insulin in the treatment of noninsulin-dependent diabetes mellitus. Combination therapy with insulin and sulfonylureas for treatment of noninsulin-dependent diabetes mellitus has been evaluated frequently. However, many of the trials either lacked proper control or were of inadequate duration, and none compared combination therapy to intensive insulin therapy using several daily injections. Such a combination decreases, but does not return to normal, glycosylated hemoglobin and fasting glucose concentrations in comparison to therapy with one or two daily injections of insulin in some subjects. Alternatively, adding a sulfonylurea to insulin therapy may allow a reduction in the insulin dosage without compromising glycemic control. When considering the use of the two agents, the possible advantage of a simplified insulin regimen must be weighed against the disadvantages of an increased potential for adverse effects and increased cost.


Cerebral blood flow changes with enalapril. Patients with carotid artery occlusive disease (CAOD) may be at increased risk of iatrogenic cerebral hypoperfusion. Using the 133xenon-inhalation technique, we evaluated the effects of enalapril on regional cerebral blood flow (CBF) in 14 patients with chronic hypertension, 7 with CAOD and 7 without CAOD (no CAOD). Regional CBF and blood pressure were measured before and 60 minutes after a single dose of enalapril. Changes in mean arterial pressure after enalapril were not significantly different between the two groups: CAOD -4.67 +/- 8.7 mm Hg, no CAOD -6.18 +/- 8.2 mm Hg. Changes in mean CBF after enalapril were also not statistically different: CAOD -1.0 +/- 3.9, no CAOD 1.0 +/- 2.8. In the CAOD group only, however, changes in CBF were significantly related to increasing age (r = -0.9253, p less than 0.01), such that in patients 65 years or older CBF tended to decrease, whereas in younger patients it increased. Elderly patients with CAOD may be at increased risk of iatrogenic cerebral hypoperfusion, and it may be appropriate to evaluate prospectively the effects of antihypertensive medications on CBF.


Theophylline--an alternative therapy for bradyarrhythmia in the elderly. Treating elderly debilitated patients with symptomatic bradyarrhythmia may be difficult. Traditional therapy includes use of limited resources (intensive care unit monitoring) and expensive interventions (permanent pacemaker insertion). When a pacemaker is not ideal or appropriate, yet intervention is desired to improve quality of life, theophylline may be a safe and effective alternative therapy. We expanded this use of theophylline in a series of 11 patients admitted 14 consecutive times with hemodynamically compromising bradyarrhythmias, including atropine-resistant complete heart block with idioventricular rhythm. Three patients experienced deterioration of status and recurrence of bradycardia with the withdrawal or reduction of theophylline, and subsequent recovery of heart rate and conduction with its resumption.


Chronic cough responsive to ibuprofen.

Chronic cough responsive to ibuprofen. A 57-year-old woman believed that ibuprofen, prescribed for back pain, improved her idiopathic chronic cough that had been resistant to inhaled and oral corticosteroids. To confirm this observation, we performed an n-of-1 clinical trial with four treatment periods, each separated by a 4-day washout. Ibuprofen 1800 mg/day for 6 days or placebo was randomly allocated in a double-blind fashion with a block size of 4. The number of coughs during the first 30 minutes after awakening were counted daily throughout the study using a tape recorder. Sixty-two coughs/hour occurred while taking ibuprofen, compared with 164 with placebo. We conclude that ibuprofen may be effective in idiopathic chronic cough, and suggest that prostaglandins may be pathogenic factors in some patients.


Increase in serum digoxin concentrations after indomethacin therapy in a full-term neonate. Digoxin was administered to an 18-day-old infant who showed evidence of cardiac failure. When a Doppler echogram revealed a patent ductus, indomethacin was administered for medical management. Therapeutic digoxin doses then resulted in toxic serum concentrations of 8.2 ng/ml. Serum creatinine rose accordingly. Although this patient did not manifest signs of digoxin toxicity, practitioners should be alerted to the potential complications of these commonly used agents.


Quality of life measurements in oncology. Quality of life (QOL) has transcended from an implicit concern to an explicit measurement in oncology. In contemporary clinical research, QOL can now often be identified and objectively measured in health as well as in illness. The major domains which comprise QOL in patients with cancer include symptoms and side effects, social function, physical function, and psychological status. One of the preferred methods for measuring QOL consists of self-administered questionnaires. A number of instruments have been developed which vary in features such as type of domains included, number of items, response options, specificity for disease type, and psychometric assessments. The role of QOL assessment is becoming increasingly important in clinical oncology.


Parenteral nutrition support in patients with cancer. In the patient with cancer, malnutrition may result from the disease itself or from its treatment. Total parenteral nutrition (TPN) has been used for many years to treat or prevent malnutrition in the patient with cancer. There have been few studies, however, that demonstrate significant benefit from TPN therapy in these patients. Patients receiving high-dose chemotherapy (as in bone marrow transplantation), and patients with solid tumors who have documented malnutrition (cachexia, weight loss) prior to cancer surgery, may benefit from TPN. No other groups of patients with cancer appear to derive significant benefit from TPN, and some groups may actually be harmed by its use. Practical considerations in the use of TPN include periodic calorie-protein assessment, electrolyte management, and monitoring for drug-TPN interactions.


Natural and modified history of isolated ventricular septal defect: a 17-year study. We studied 882 cases of isolated ventricular septal defect (VSD) diagnosed from 1971 to 1988 with a mean follow-up period of 9.5 years. They represent 22.5% of all congenital heart defects diagnosed in this period. Six hundred eighty-three children (77.4%) did not develop symptoms, had small defects, and the rate of spontaneous closure was high (40.2%). The remaining 199 children presented symptoms; at the initial catheterization 25, 65, 107, and 2 cases were grouped in hemodynamic groups 2, 3, 4, and 5-6, respectively, on the basis of pulmonary flow and resistance. Only seven patients (0.7%) developed aortic regurgitation, and only five patients (0.5%) developed infective endocarditis. Complete surgical correction was performed in 137 children (15.5% of the total cases), with surgical mortality decreasing from 21.4% before 1983 to 3.5% afterward. Overall mortality was 3% for the entire cohort, 0% for hemodynamic groups 1 and 2, 3% for group 3, and 25% for hemodynamic groups 4-6. About two thirds of the deaths took place between 1 month and 1 year of life, and one thirds of the deaths occurred before surgical treatment. Surgical mortality rates for hemodynamic groups 2, 3, and 4 were 0, 4.7, and 15.3%, respectively. Actuarial survival curves show an important improvement in the prognosis after 1983. Our results stress the importance of early surgical complete correction on patients with large defects and severe hemodynamic changes.


Bacterial endocarditis in children: trends in its diagnosis, course, and prognosis. In a population-based study of 41 children with bacterial endocarditis (BE), diagnosed in the period 1970 through 1989 in eastern Denmark, we analyzed trends in the diagnosis of BE and in mortality, and searched for possible prognostic factors. During this period the delay in diagnosis from first symptom to treatment did not change, but the delay from admission to treatment was significantly prolonged from 0 to 3 days, despite the introduction of echocardiography (ECHO). There was a significant improvement in the prognosis, the mortality rate having decreased from 40 to 0% [95% confidence limits: 12-74 vs. 0-26 (0.01 less than p less than 0.02)]. The improved prognosis was not explained by changes in the etiology or pattern of antibiotic resistance and may reflect a milder course of BE in children. Children with "mild anomalies"--such as bicuspid aortic valve (n = 5), coarctation of the aorta (n = 2), and prolapse of the mitral valve (n = 2)--had a significantly poorer prognosis than children with other forms of congenital heart disease (CHD) (p = 0.004), a reminder of the importance of suspecting BE in all children with unexplained long-lasting or intermittent fever, because some may have unrecognized "mild" CHD.


HLA and rheumatic fever in Turkish Children. We report the results of research on human leukocyte allo-antigen (HLA) and rheumatic fever (RF), the first published study to be carried out among Turkish children with RF. Ninety-three Turkish children, aged between 6 and 16 years (mean: 8 +/- 2.6), with RF participated in the study. Of the total, 26 patients had their first attack and 39 had acute rheumatic activity at the time of registration. The results demonstrate (1) negative but not significant association between HLA-A2 and RF; (2) a positive association between HLA-DR4 and RF (p less than 0.001); (3) a significant association between HLA-DR4 and carditis, but not with isolated arthritis. These results corroborate the concept of race-specific genetically determined familial susceptibility to the development of rheumatic heart disease.


Acute renal failure complicating pediatric cardiac surgery: a comparison of survivors and nonsurvivors following acute peritoneal dialysis. The outlook for patients with cardiac surgery complicated by acute renal failure (ARF) is poor, with a reported mortality of 50-67%. In addition to assessing the impact of recent advances in pediatric cardiac surgery on the mortality rate and renal outcome of surgery complicated by ARF requiring peritoneal dialysis (PD), this study compares preoperative, operative, and postoperative variables in patients who survived surgery and those who did not survive. From 1982 through 1988, 44 postoperative cardiac patients developed ARF, and 40 (age: 2 days to 15 years) required PD. Seventeen of 40 patients survived (mortality 57.5%) and 16 of these patients recovered normal renal function. Preoperative variables, including operative age and weight, did not appear to directly influence survival. Operative profiles, including length of cardiopulmonary bypass, aortic cross-clamp time, and hypotension immediately off bypass, did not distinguish surviving patients from those that did not survive. Postoperative variables, such as postoperative hypotension treatment, arrhythmias, hematologic status, cardiac arrest with resuscitation, did not differentiate survivors from nonsurvivors. The mean duration of PD was less than 2 weeks, and complications were infrequent. Renal status following PD in survivors was usually normal. We conclude that recent advances in pediatric cardiac surgery have not further increased the high mortality of surgery complicated by ARF. Survival is associated with renal recovery and thus aggressive treatment using PD is warranted.


Interrupted aortic arch in infancy: a 10-year experience. Fifty infants with interrupted aortic arch (IAA), admitted between 1979 and 1988, were reviewed. They usually presented early in severe cardiac failure or shock. In the initial 5-year period, 17 of the 21 infants underwent diagnostic or confirmatory cardiac catheterization, in contrast with the latter 5 years when only eight of the subsequent 29 patients underwent catheterization. Since 1987, all patients underwent surgery after cross-sectional echocardiography. Fifteen infants had a type A IAA and 35 had type B. All had associated cardiac anomalies. Four infants were not operated on. In the initial 5-year period, of 17 infants who were surgically treated, four had a one-stage total repair, the remaining had a two-stage repair with initial reconstruction of the arch and pulmonary artery banding. There was an overall surgical mortality of 65%, reflecting the precarious state of many of these infants before surgery with a significant contribution from unrelieved subaortic stenosis. In the latter 5-year period, 29 underwent surgery, 22 had a one-stage total repair. There were three deaths, all in infants whose active treatment was withdrawn. The outcome of the survivors has generally been good, subsequent surgery being mainly related to the associated anomalies (e.g., recurrent subaortic stenosis, conduit replacement). Over this 10 year period the greater accuracy of noninvasive diagnoses, and perioperative intensive care, have led to an improvement in the preoperative state of these infants. Single-stage total repair is our procedure of choice.


Aneurysm of the ductus arteriosus in the neonate: three case reports with a review of the literature. Ductus arteriosus aneurysm was demonstrated in three neonates by echocardiography. One case was already diagnosed in utero. One patient needed surgical resection, whereas in one case the aneurysm of the ductus arteriosus regressed spontaneously. In the third case the patient died of associated lesions. In a review of the literature, 61 previous cases of ductus aneurysm in infants less than 2 months were found. Ductus arteriosus aneurysm may be diagnosed by echocardiography both pre- and postnatally. A ductus aneurysm should be monitored closely by echocardiography. Since a high incidence of complications is reported, surgical resection is indicated if the aneurysm does not regress within a few days.


Endocarditis with acute mitral regurgitation caused by Fusobacterium necrophorum. Infective endocarditis is uncommon in young children, especially in the absence of structural heart disease. We report the case of a 2-year-old boy who presented with acute rupture of the mitral valve chordae 6 weeks after an episode of Fusobacterium necrophorum septicemia. His heart had been structurally normal before. Mitral valve replacement was successfully performed. This is the first recorded case of endocarditis in a child caused by necrobacillosis.


Necrotizing arteritis in uncorrected tetralogy of Fallot with pulmonary artery. A 10-year-old girl with uncorrected tetralogy of Fallot with pulmonary atresia presented with fevers of unknown origin and left lung infiltrates. At autopsy, necrotizing vascular changes resembling those of severe pulmonary hypertension (grade VI in the Health-Edwards classification) were confined to the left lung. Pulmonary blood flow and pressure were greater in the left lung and were provided by an enlarged collateral artery arising directly from the descending thoracic aorta. To our knowledge, this is the first report of necrotizing arteritis of the pulmonary arteries in uncorrected tetralogy of Fallot with pulmonary atresia.


Intracardiac calcifications in a case of pseudohypoparathyroidism type Ia (PHP-Ia). In a 10-year-old boy with pseudohypoparathyroidism type Ia (PHP-Ia) echocardiography detected calcifications within the ventricular septum. This is thought to be the first report of intracardiac calcifications in PHP. Echocardiography should be done routinely in all patients with this rare disorder.


Occupational asthma. 'My job is making me sick!'. Future well-organized epidemiologic studies may enhance our understanding of the risk factors, prognosis, and natural course of asthma caused by a variety of inciting agents found in the workplace. Until such studies are completed, employees should be educated regarding exposure to hazardous chemicals, and strict safety measures should be enforced to prevent large spills of chemicals and other unnecessary risks.


Anticoagulant therapy for atrial fibrillation. Recommendations from major studies. The role of antithrombotic therapy in reducing thromboembolic complications in patients with chronic atrial fibrillation has been clarified by the results of four major randomized and placebo-controlled trials. Patients with rheumatic heart disease complicated by atrial fibrillation should receive long-term warfarin therapy to reduce the risk of stroke unless an absolute contraindication exists. Patients with nonrheumatic atrial fibrillation should also be treated with low-dose warfarin therapy, especially if high-risk features for thromboembolism exist. In patients who have contraindications to warfarin therapy and in young patients with lone atrial fibrillation or paroxysmal atrial fibrillation, therapy with 325 mg of aspirin a day is preferred. Ongoing trials directly comparing aspirin and warfarin will provide additional insight into the optimal role of these antithrombotic agents in patients with atrial fibrillation.


Resurgence of rheumatic fever in the United States. The changing picture of a preventable illness. The recent resurgence of rheumatic fever reported in eight locations in the United States after years of decline has several noteworthy characteristics. Most patients were children of families in high- to middle-income brackets with ready access to medical care. In four of the outbreaks, the majority of patients were adults, who were more likely than children to have arthritis and less likely to have Sydenham's chorea. Many patients had no clinical history of streptococcal pharyngitis. Rheumatic fever developed in some patients despite antibiotic treatment for streptococcal pharyngitis. Analysis of the outbreaks supports the following conclusions: No population is exempt from rheumatic fever. Physicians should be diligent in performing throat cultures in cases of suspected streptococcal pharyngitis. The efficacy of orally administered penicillin in preventing rheumatic fever should be reexamined, and oral antibiotics that are potentially more effective should be sought.


Depression in physically ill patients. Don't dismiss it as 'understandable'. Depression in the physically ill is common and may even be caused by certain physical disorders (eg, hypothyroidism, pancreatic cancer) or the use of some types of drugs. It should not be dismissed because it is "understandable" in particular situations, but rather, it should be differentiated from overlapping symptoms of the physical disorder and treated. The effect of psychosocial factors should be carefully considered.


Prophylaxis for malaria. Helping world travelers come home healthy. Malaria is largely preventable, so travelers should be taught general protective measures and given appropriate chemoprophylaxis before they leave on their trip. Chloroquine phosphate (Aralen) is still the drug of choice in locations where malaria remains chloroquine-sensitive. However, chloroquine-resistant areas infested with Plasmodium falciparum are becoming more numerous. In such areas, mefloquine hydrochloride (Lariam), doxycycline, or proguanil (Paludrine) (obtainable outside the United States) may be used. A single dose of pyrimethamine-sulfadoxine (Fansidar) may be used to treat presumptive malarial infection if medical care is not immediately available. For prevention of relapse of Plasmodium vivax and Plasmodium ovale infection, primaquine phosphate is recommended for the final 2 weeks of chemoprophylaxis on return from a malarious area.


Preventing postoperative medical complications. How primary care physicians can help. Just as your concern for your patients' welfare does not stop at the operating room door, neither does your opportunity to improve their prognosis. Drs Elnicki and Shockcor believe that primary care physicians have a unique role in total patient care. In this article, they discuss prevention of major postoperative complications and recommend ways primary care physicians can help optimize their patients' perioperative status.


Medical complications of substance abuse. Substance abuse is involved in many instances of intentional and unintentional injury. It can also cause medical complications that affect various organ systems--among them, the cardiac, vascular, neurologic, pulmonary, gastrointestinal, immunologic, and reproductive systems. Even though there is pressure to create a new medical specialty to specifically address substance-abuse issues, the truth is that any physician, regardless of specialty, may encounter patients with substance-abuse problems. Alcoholism and drug abuse, with their associated psychosocial and clinical ramifications and complications, cut across all specialty fields. Consequently, all physicians need to be familiar with the spectrum of clinical problems associated with substance abuse and comfortable with addressing these problems prudently and promptly.


The ABZzzzs of snoring.

The ABZzzzs of snoring. Snorers sleep through their obnoxiously loud breathing, but other members of their household often do not. Their bed partner and family members may grow so tired of the noise that they insist the offender seek medical attention. Physiologic problems such as obesity, narrowing of the airway, or inadequate oropharyngeal muscle tone often can be identified as the cause of snoring. Whenever possible, the underlying disorder should be treated.


Common thyroid disorders in the elderly. Thyroid disease in the elderly is common but often has an insidious onset with symptoms that mimic those of normal aging. Understanding the significance of thyroid function test results requires an appreciation of the normal physiologic variations of aging and the complicating effects of diseases and medications; only then can one accurately discriminate between various disease processes, order additional appropriate tests, and deliver rational management. The increasing prevalence of thyroid disorders with age, coupled with the current and future dramatic growth of the elderly population, demands that primary care providers be vigilant to the likelihood of thyroid disease and informed as to its most proper, expedient, and cost-effective diagnosis and management.


The role of barium enema in detecting colorectal disease. A radiologist's perspective. The single-contrast barium enema examination remains useful for patients with acute diseases such as bowel obstruction, diverticulitis, appendicitis, and fistulas. It is also the procedure of choice for those patients who are too elderly, debilitated, or ill to cooperate with the maneuvers necessary for a double-contrast examination. The double-contrast technique is more sensitive than the single-contrast technique for detection of polyps, early inflammatory bowel disease, and lesions of the rectum. In the older population, there has been an increase in the incidence of colonic polyps and carcinomas in the right side of the colon. This emphasizes the need to examine the entire colon in these patients. The double-contrast barium enema is a safe, accurate, and cost-effective tool for accomplishing this. It is also recommended as the initial procedure in the examination of patients with positive results on fecal occult blood testing.


Autologous transfusion. A safe alternative for the 1990s. Autologous transfusion is a safe and affordable alternative to the traditional use of homologous blood. Many persons who would not ordinarily meet the criteria for homologous or directed donation can be autologous donors. The use of autologous blood ensures that the patient will not contract AIDS or hepatitis from transfused blood products. Many community hospitals have autologous blood programs, which allow them to draw, store, and reinfuse blood all under one roof.


A diagnostic approach to 'difficult' asthma. When patients with asthma do not respond adequately to standard treatment, they are said to have "difficult" asthma. These patients require a lot of healthcare resources, are usually receiving long-term oral corticosteroid therapy, and are significantly sicker than other asthma patients, with attendant increases in morbidity and mortality. In these patients, the management areas summarized in table 1 should be fully explored to determine whether asthma is the correct diagnosis and whether the best care is being given.


Correct use of metered-dose inhalers and spacer devices. In addition to prescribing an appropriate drug regimen, physicians must carefully educate asthmatic patients. For 10 million such patients in the United States as well as millions more around the world, successful education is critical to quality of life and can save lives. Responsible patients who understand that asthma attacks are preventable and who use their medications correctly usually have a gratifying response to treatment. Of special importance is the optimal use of aerosolized drugs, most often given by a metered-dose inhaler plus spacer device.


Pharmacology and clinical use of a new group of antiarrhythmic drugs: derivatives of tricyclic nitrogen-containing systems. This paper is concerned with a new group of antiarrhythmic drugs: derivatives of phenothiazine and dibenzazepine which belong to tricyclic nitrogen-containing systems. It has been discovered that the transition from omega-aminoalkyl to omega-aminoacyl phenothiazine derivatives results in a decrease in psychotropic activity and an increase in cardiovascular, particularly antiarrhythmic, action. Based on structural and antiarrhythmic activity studies, the new and effective drugs ethmozine, ethacizine and bonnecor have been selected and extensively studied. Data on spectra and mechanisms of their action, obtained in different arrhythmia models, have been confirmed by clinical studies. Correlation between pharmacological properties and electrophysiological mechanisms, and differences in action of the drugs studied on brain and heart plasma membrane receptors may serve as a starting point for the further directed search for new antiarrhythmic drugs among tricyclic nitrogen-containing structures.


Effects of slow release bezafibrate on the lipid pattern and on blood glucose of type 2 diabetic patients with hyperlipidaemia. Ninety-eight type 2 diabetic patients with hyperlipidaemia in stable metabolic control with diet alone (41) or diet plus hypoglycaemic agents (57) were divided into two groups: group 1 was put on treatment with slow release bezafibrate 400 mg a day, while group 2 was considered as control. In group 1, after 1 month of bezafibrate, serum triglycerides fell by 47% and cholesterol by 13%. HDL cholesterol showed a non-significant trend toward an increase. Fasting blood glucose significantly decreased by 6%, fructosamine and glycated haemoglobin by 5%. During OGTT, the area under the curve of both serum C-peptide and blood glucose showed a trend toward a decrease after bezafibrate. However, the difference did not reach statistical significance. Thirty-six patients continued the treatment with the drug for 4 months and 23 for 8 months, without further changes of the lipid pattern and glycaemic control. In the control group no significant variation of the lipid levels occurred and diabetic control slightly worsened during the study. Bezafibrate has been proved to be effective in the treatment of hyperlipidaemia in type 2 diabetic patients. The drug seems moreover to improve glycaemic control. The mechanism by which bezafibrate produces this latter effect remains to be elucidated, though an increase of peripheral insulin sensitivity might be suggested.


Effect of furazolidone on tissue sulphydryl groups, ascorbic acid, and lipid peroxide levels in the rat, and the influence of dimethylsulphoxide thereon. The concentrations of sulphydryl groups (SH), ascorbic acid (AA) and lipid peroxide (LP) were measured in the liver, kidney and brain of rats 24 h after treatment with furazolidone at single oral doses of 75, 150 and 300 mg/kg. The drug produced significant reductions in the concentrations of SH, and significant increases in the concentrations of LP, in the tissues studied. The level of AA was significantly decreased in the livers (but not the kidneys or brains) of rats treated with furazolidone at a dose of 300 mg/kg. The concentrations of AA in the tissues of rats treated with lower doses were unaffected. In another experiment, furazolidone was given orally to rats at a dose of 300 mg/kg, together with a subcutaneous injection of dimethylsulphoxide (DMSO) at a dose of 8 g/kg, and SH measured in the liver 24 h later. DMSO alone had no significant effect on SH concentration. However, when DMSO was given concomitantly with furazolidone, SH concentration was significantly less depressed than when furazolidone was given alone. It is concluded that DMSO protected tissues against toxicity induced by furazolidone.


Reduced L-arginine/lysine and L-arginine/aspartic acid ratios in aorta from Watanabe heritable hyperlipidaemic (WHHL) rabbit. L-Arginine is the endogenous source of nitric oxide in the endothelial cells of blood vessels. In thoracic aorta from Watanabe heritable hyperlipidaemic (WHHL) rabbits, the L-arginine/lysine and L-arginine/aspartic acid ratios were reduced in comparison to New Zealand rabbit aorta. This result indicates that a variation in aortic arginine/lysine or arginine/aspartic acid ratios could be related to the hyperlipidaemic status.


Hypolipidaemic activity in rodents of boron analogs of phosphonoacetates and cyanoborane adducts of dialkyl aminomethylphosphonates. Boron analogues of phosphonoacetates proved to be potent hypolipidaemic agents in rodents, lowering both serum cholesterol and triglyceride levels. (C2H5O)3PBH2COOCH3 proved to be the most effective agent in mice, lowering serum cholesterol 46% and serum triglycerides 54% after 16 days. (C2H5O)3PBH2COOH and Na+H+(C2H5O)2(-O)PBH2COO- caused greater than a 40% reduction in lipids. The cyanoborane adducts of aminomethylphosphonates were generally less effective; (C6H5O)2P(O)CH2NH2BH2CN was the most effective, lowering serum cholesterol 32% and serum triglycerides 43% after 16 days. The phosphonoacetates appeared to lower lipid concentrations by several mechanisms. First, they lowered the de novo synthesis of cholesterol and triglycerides in the liver. Second, they accelerated the excretion of lipids into the bile and faeces. Thirdly, they modulated LDL and HDL-cholesterol contents in a manner which suggests they reduced the deposition of lipids in peripheral tissues, and accelerated the movement of cholesterol from tissues (e.g. plaques) to the liver for excretion into the bile.


Inhibition of aromatic amino acid decarboxylase by a group of non-steroidal anti-inflammatory drugs. In vitro inhibition of aromatic amino acid decarboxylase (AAD) by eight nonsteroidal anti-inflammatory drugs (NSAIDs) was studied. The most potent inhibitors were fenamates (IC50 for tolfenamate was 4.4 x 10(-5) M), while fluorinated biphenylic compounds (flobufen and 2',4'-difluorobiphenyl-4-yl acetate) and propionic acid derivatives (ibuprofen, ketoprofen, fenoprofen) were one order weaker. The in vitro inhibition of AAD by the compounds studied is probably not strong enough to contribute to their in vivo anti-inflammatory effects.


Influence of indomethacin on histamine- and acetylcholine-induced responses in the intact and denuded epithelium of guinea pig tracheal smooth muscle. We have investigated the effect of indomethacin on histamine- and acetylcholine (ACh)-induced responses in the intact and denuded epithelium of guinea pig isolated tracheal smooth muscle. Epithelium removal resulted in increased responsiveness to ACh and histamine. Indomethacin (2.8 microM) enhanced the sensitivity of both intact and denuded preparations to histamine and ACh. These findings suggest that the tracheal epithelium of guinea pig plays a protective role against bronchoconstrictors, such as ACh and histamine. Furthermore, indomethacin-mediated hyperresponsiveness caused by these agonists in epithelium denuded preparations might be a reflection of removal of prostaglandin (PG) biosynthesis. A similar process of interaction in indomethacin-treated asthmatic patients (with damaged airway epithelium) might take place. The significance of these findings is discussed.


Field potential analysis in the freely moving rat during the action of cyclandelate or flunarizine. Cyclandelate and flunarizine, two vasoactive Ca2+ channel or Ca2+ overload blockers have been compared with respect to their in vivo action on field potentials recorded from the depths of the brain in freely moving rats. Whereas cyclandelate showed a dose dependent rapid onset of action in the range of 15 to 120 mg/kg i.p., flunarizine only induced weak effects very slowly, not reaching statistical relevance before the fourth hour after the injection (0.1 to 1.6 mg/kg). Even then no clear dose dependence could be recognized for flunarizine. With respect to the frequency content of the recorded signals a rather close similarity between both drugs could be seen. Comparison of the drug effects to our reference data base of more than 80 compounds revealed a close relationship to memantine, an antiparkinson drug, suspected to act on the NMDA (N-methyl-d-aspartate) receptor-ionophor complex controlling Ca2+ fluxes. There is some indication that cyclandelate might also act in a similar way at the molecular level.


[The beginnings of child analysis].

[The beginnings of child analysis]. The notions hitherto existing of the beginnings of child analysis have to be rectified. The meaning of childhood neuroses within the scope of scientific theory, i.e. the sexual genesis of the neuroses can no longer be maintained. The approach of distinguishing between the latent period and the Oedipus complex as the two phases of sexual development can nowadays be considered to be refuted. The personality of the first children's analyst--Hermine Hug-Hellmuth--needs to be redescribed. She fell victim to her father-transference and deceased murdered by her nephew with the help of whom she had satisfied her own scientific ambitions.


[Family pattern with reference to self organization from the psychoanalytic systematic viewpoint]. Families in crisis may develop a variety of dysfunctional patterns of self-organisation. They prove consisting of elements as defined by psychoanalysis, family dynamics and systemic-constructivistic thinking. Such patterns are: "Innerfamiliar polarisation" (produced by a family specific "Sprachregelung") the "fight of shadow families", "competition for the same place in the generational net", "transjection" and "magic threshold". Those patterns will be demonstrated by the example of an anorectic family, treated with a psychodynamic concept. The knowledge of these patterns proved to be useful for different kinds of family therapy. Some theoretical questions as analogies from social and physiological (formal) genetics and modalities of transgenerational transmission of family patterns will be discussed shortly.


Validity of health-related quality of life subscales. This report assesses the validity of subscales from the Quality of Life Index (QLI). Subscale validity is tested across different samples of patients with cancer and other diseases. The results of the factor analyses of different versions of the QLI tested in six studies confirm four important health-related quality of life dimensions. These dimensions are psychological well-being, physical well-being, symptoms/side effects, and nutrition. Psychological well-being is represented by a satisfying life, having a good (general) quality of life, having fun, feeling happy and enjoying life. Physical well-being is defined by tiring easily (fatigue), ability to work/carry out usual tasks, strength, and feeling healthy. The symptoms/side effects dimension is characterized by nausea, vomiting, pain distress, and perhaps, by pain amount and pain frequency. Finally, the nutrition dimension is represented by able to eat/eat sufficiently, eating pleasure/appetite, worry about weight, and possibly also taste changes. Good construct validity can be attributed to these health-related quality of life subscales.


[Problems in diagnosis of borderline disorders in childhood]. While, for adults, there exists an instrument for diagnosing borderline-disorders (DSM-III-R, ICD 10), this is not the case for children and adolescents. Several attempts of classifying borderline-disorders are discussed (Bemporad et al., Pine, Vela et al.). Exemplified by a case study of the treatment of a ten-year-old borderline girl originally diagnosed as "hysteria", eight criteria are listed and explained that seem relevant for diagnostic purposes: (1) heredity and affective disorders, (2) diseases and traumata during the first year of life, (3) development in early childhood, (4) character and intensity of anxiety, (5) self-destructiveness, (6) defensive mechanisms, (7) object relationships, (8) countertransference. Attention is drawn to a large-scale empirical study in which, by means of an elaborate questionnaire, child psychiatrists and therapists are asked to characterize symptoms, personality traits and aetiological factors of young borderline patients. This will hopefully solve some problems of differential diagnosis discussed in the article.


Hydrophobicity and structural classes in proteins. The bulk hydrophobic character for the 20 natural amino acid residues, has been obtained from a database of 60 protein structures, grouped in the four structural classes alpha alpha, beta beta, alpha + beta and alpha/beta. The hydrophobicity coefficients thus obtained are compared with Ponnuswamy's original values using scales normalized to average = 0.0 and standard deviation = 1.0. Even though most of the amino acid residues do not change their hydropathic character in the different structural classes, their behaviour suggests the convenience that averaging methods should only consider proteins of the same structural class and that this information should be included in the secondary structure methods.


[The importance of the transitional object for the psychological development of the child]. This study addresses the question whether children taken for treatment to a psychiatric hospital for children and adolescents differ from other, not noticeably disturbed children in their use of a transitional object. Parents of mentally disturbed children between six and eight years of age were interviewed about the transitional objects used by their children. The control group was made up of children of corresponding ages from an ordinary nursery school. Based on the extant literature, the concept of "transitional object" is described and defined. Twenty two research hypotheses were formed and subjected to statistical tests. As the main result concerning the research question it emerged that the children suffering from psychiatrical disturbances needed a transitional object mainly in their fourth year of life, but to some extent even in their fifth and sixth year whereas the "healthy" children in the control group without noticeable disturbances used a transitional object mainly in their first and second years. It could be shown with statistical significance that psychiatrically disturbed children had a transitional object at a later age and for a longer period of time than healthy, undisturbed children.


IPSA-Inductive Protein Structure Analysis. The Inductive Structure Protein Analysis (IPSA) project presents a new method for investigating protein structure. IPSA includes the creation of a new database which was designed specifically for the analysis of protein structure by statistics and machine learning. The Protein Representation Language (PRL) database includes explicit and symbolic representations of geometrical, topological and chemophysical information about secondary structures and the relationships between secondary structures. The IPSA methodology consists of: the use of PRL information to produce a new database of examples of secondary structures which associate together (examples of possible super-secondary structures); then the use of a variety of clustering techniques to produce a consensus clustering of these examples (super-secondary structures); these super-secondary structures are finally examined to uncover any biological features of significance. We have applied this method to find simple super-secondary structures consisting of pairs of alpha-helices. We found four well-defined super-secondary structures, one formed exclusively by long range interactions, and another in association with an additional element of secondary structure (alpha t alpha-motif). Examinations were carried out using homologous pairs and conformational fits which confirm our clustering.


[Gene library of Dunaliella salina, prepared in a cosmid vector of new design]. The gene library (56,160 clones) of the halotolerant alga Dunaliella salina was obtained using a novel cosmid vector pBbv-cosII. The mean size of the insertions is 35-45 kb. Hybridization screening demonstrated that both nuclear and chloroplast DNA-containing clones are presented in the library.


Molecular modeling studies in the complex between cyclophilin and cyclosporin A. The structure of the complex between cyclophilin and cyclosporin A is predicted by combining X-ray crystallographic and NMR spectroscopic data using molecular modeling. The drug was placed at the receptor site using a directed docking procedure in which an impulse is imparted to a pre-oriented ligand along an established path. Both ligand and receptor atoms are flexible during the procedure. Two conformers of the MeBMT side chain are shown to result in similar ligand-receptor interaction energies. The models for the drug-receptor complex appear consistent with known experimental data and provide a significant opportunity for the design of compounds with enhanced therapeutic value.


Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue. The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 A resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is close to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.


Relative importance of dimensions in the assessment of health-related quality of life for patients with hypertension. There are many different approaches to quality of life measurement. Most of these methods consider several dimensions of health status. However, few of the current methods evaluate the relative importance of the various dimensions. We suggest that the relative importance dimension is of central importance in the development of a general theoretical conceptualization of health outcome. Further, we offer a General Health Policy Model that can be used to evaluate outcomes in cardiovascular care and to compare the cost/utility of cardiovascular interventions to investments in other aspects of health care. Employing the relative importance concept requires several important assumptions and methodological challenges. For example, we must select between competing measurement methodologies and we must decide whose values to use in the models. As new research addresses these questions, we expect greater use of the models in patient decision making and in health policy research.


Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus. The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.


Multidimensional assessment of quality of life in clinical studies. Four types of issues in conducting a multidimensional assessment of quality of life in clinical studies are presented: practical issues, measurement issues, data quality issues, and data analysis issues. The various issues are described, and ways of dealing with the issues are discussed. Examples are used from the literature and also from the author's multivariate, longitudinal study on quality of life in cardiac transplant patients. Consideration of these issues and knowing options for dealing with them before the study is conducted is imperative. With a carefully thought out approach to better planning of the study, the clinician/investigator can conduct a scientifically sound study with credible results that will provide meaningful direction for improving the quality of life of clinical populations in the future.


Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket. Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate. In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128. Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA). An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate. Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain. The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering). Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)


Conceptualizations of quality of life in cardiovascular research. The purpose of this article is to examine how quality of life has been conceptualized in studies of patients with cardiovascular disorders. To accomplish this purpose the instruments that were used as measures of quality of life in 22 published studies were examined. The majority of conceptualizations could be grouped into five broad categories: social utility, happiness/affect, satisfaction, achievement of personal goals, and normal life. Most of the studies used more than one category to measure quality of life.


The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease. Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.


The integration of health quality of life in clinical research: experiences from cardiovascular clinical trials. Health Quality of Life (HQL) assessment in clinical trial and epidemiological research is necessary to determine the efficacy of treatments and interventions. A definition of HQL is presented with the following dimensions of HQL identified: social, physical, and neuropsychological functioning; emotional stability; personal productivity; and intimacy. A strategy for selecting HQL dimensions and measures is discussed and includes consideration of the study population, the intervention(s), and the clinical trial design. Examples from NHLBI-sponsored clinical trials illustrate how HQL measures are selected as well as the goals that HQL assessment can address. That is, the authors discuss how HQL data can be used to characterize the study populations, determine the effects of HQL on critical outcomes, and assess how treatment affects overall HQL and specific dimensions of HQL.


Permuteins of interleukin 1 beta--a simplified approach for the construction of permutated proteins having new termini. A technique for the rapid and simple generation of permutated versions of the interleukin-1 beta (IL-1 beta) gene is described. In this method, the human IL-1 beta cDNA is twice amplified by the polymerase chain reaction (PCR) and the resulting DNA fragments are ligated in tandem. Between the two genes, the DNA sequence encodes a short four amino acid loop to link the native N- and C-terminal ends of the IL-1 beta protein. By using PCR amplification from this starting template, a new version of the IL-1 beta cDNA was obtained that encodes a permutated form of the IL-1 beta protein where the new N- and C-terminal amino acids correspond to residues 65 and 64 of the native IL-1 beta sequence, respectively. The name 'permutein' is proposed to describe proteins generated by this technology. The molecular profile (IL-1 receptor binding, biologic activity and solution properties) of the IL-1 permutein produced by this technology, permutein 65/64, is shown to be identical to that of native IL-1 beta. The approach should be useful to define further the structural features of this protein that are important for its function.


Bipartite organization of the Bacillus subtilis endo-beta-1,4-glucanase revealed by C-terminal mutations. The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-beta-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate. CD spectra indicated that the bulk of the alpha-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and approximately 4- to 5-fold higher with carboxymethylcellulose (soluble cellulose). These results along with data which show that EG470 binding capacity to microcrystalline cellulose is approximately 11 times more than that of EG300, demonstrate the importance of residues 330-499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330-499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine-326 and serine-321, respectively. Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids.


Mutant forms of beta-galactosidase with an altered requirement for magnesium ions. By random approaches we have previously isolated many variants of Escherichia coli beta-galactosidase within a short contiguous tract near the N-terminus (residues 8-12 of wild-type enzyme), some of which have increased stability towards heat and denaturants. The activity of these mutants was originally analysed and quantitated in situ in activity gels without the addition of magnesium ions to the buffer system. We now show that the improved stability is only observable under such conditions of limiting magnesium ion concentrations or in the presence of appropriate concentrations of a metal chelator. In the presence of EDTA, purified preparations of one of these mutant enzymes were much more resistant to denaturants than wild-type, but this differential was completely nullified in the presence of 1 mM Mg2+. However, the stability of this mutant enzyme in EDTA was lower than that shown by it, or the wild-type enzyme, in the presence of magnesium ions. In addition, certain alterations within another N-terminal tract (residues 27-31 of wild-type) resulted in enzymes with greater dependence on Mg2+ than natural beta-galactosidase. We conclude that a small number of residue changes in a large protein can profoundly modulate the requirement for metal ion stabilization, allowing partial abrogation of this need in certain cases. Thus, some enzymes which require divalent metal ions for structural purposes only may be engineered towards metal independence.


Complex additivity of the effects of suppressor mutations in differing protein environments. In the alpha-complementation of beta-galactosidase, a defective beta-galactosidase protein interacts with an autologous peptide fragment (alpha-peptide) to restore enzymatic activity. Within a specific site of a defective alpha-peptide we have previously isolated a large number of mutations, many of which suppress the functional defect. The alpha-peptide was originally defective due to both insertional and substitutional sequence alterations near its N-terminus, which provided an increase in the sensitivity of detection of (suppressor) secondary mutations which conferred improved function. We have now studied the effects of the suppressor mutations when the primary deleterious mutations are sequentially reversed. This was done in intact beta-galactosidase, as we have shown that mutations in the alpha-peptide have related functional effects in the whole protein. Evidence was obtained showing that the effects of at least some suppressor mutations were not simply additive when the mutations are placed into the original wild-type protein environment. One suppressor appeared to function less effectively in the normal environment, while another when tested in the same manner functioned at a relatively increased level. This failure to show simple additivity may be attributable to the physical proximity of the original defective mutations and the introduced suppressors. Nevertheless, even in such cases it may be feasible to use a defective protein as a sensitive starting point for the identification of mutations which improve the wild-type protein.


Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli. For the first time the pro-form of a recombinant cysteine proteinase has been expressed at a high level in Escherichia coli. This inactive precursor can subsequently be processed to yield active enzyme. Sufficient protein can be produced using this system for X-ray crystallographic structure studies of engineered proteinases. A cDNA clone encoding propapain, a precursor of the papaya proteinase, papain, was expressed in E. coli using a T7 polymerase expression system. Insoluble recombinant protein was solubilized in 6 M guanidine hydrochloride and 10 mM dithiothreitol, at pH 8.6. A protein-glutathione mixed disulphide was formed by dilution into oxidized glutathione and 6 M GuHCl, also at pH 8.6. Final refolding and disulphide bond formation was induced by dilution into 3 mM cysteine at pH 8.6. Renatured propapain was processed to active papain at pH 4.0 in the presence of excess cysteine. Final processing could be inhibited by the specific cysteine proteinase inhibitors E64 and leupeptin, but not by pepstatin, PMSF or EDTA. This indicates that final processing was due to a cysteine proteinase and suggests that an autocatalytic event is required for papain maturation.


Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter. 5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the product of the Escherichia coli aroA gene, has been overproduced in E. coli BL21(lambda DE3) under the control of the T7 gene 10 promoter and ribosome binding site, to a level of approximately 50% of total cell protein. EPSP synthase is the primary target of the post-emergence herbicide, glyphosate, commonly known as Roundup. A simple two step purification is described, which results in 99% pure homogeneous protein (as determined by PAGE). The integrity of the protein has been compared with previously characterized protein from E. coli AB2829(pKD501) by determination of its kinetic parameters, N-terminal protein and DNA sequences, amino acid analysis and 13C-NMR spectroscopy. This new overproducing strain readily provides the gram quantities of highly pure protein required for NMR studies of the active site and the development of novel time-resolved solid-state NMR techniques currently underway in this laboratory.


NMR docking of a substrate into the X-ray structure of staphylococcal nuclease. The conformation of the staphylococcal nuclease-bound metal-dTdA complex, previously determined by NMR methods [Weber, D.J., Mullen, G.P., Mildvan, A.S. (1991) Biochemistry 30:7425-7437] was docked into the X-ray structure of the enzyme-Ca(2+)-3',5'-pdTp complex [Loll, P.J., Lattman, E.E. (1989) Proteins: Struct., Funct., Genet. 5:183-201] by superimposing the metal ions, taking into account intermolecular nuclear Overhauser effects from assigned aromatic proton resonances of Tyr-85, Tyr-113, and Tyr-115 to proton resonances of the leaving dA moiety of dTdA, and energy minimization to relieve small overlaps. The proton resonances of the Phe, Tyr, and Trp residues of the enzyme in the ternary enzyme-La(3+)-dTdA complex were sequence specifically assigned by 2D phase-sensitive NOESY, with and without deuteration of the aromatic protons of the Tyr residues, and by 2D heteronuclear multiple quantum correlation (HMQC) spectroscopy and 3D NOESY-HMQC spectroscopy with 15N labeling. While resonances of most Phe, Tyr and Trp residues were unshifted by the substrate dTdA from those found in the enzyme-La(3+)-3',5'-pdTp complex and the enzyme-Ca(2+)-3',5'-pdTp complex, proton resonances of Tyr-85, Tyr-113, Tyr-115, and Phe-34 were shifted by 0.08 to 0.33 ppm and the 15N resonance of Tyr-113 was shifted by 2.1 ppm by the presence of substrate. The optimized position of enzyme-bound dTdA shows the 5'-dA leaving group to partially overlap the inhibitor, 3',5'-pdTp (in the X-ray structure). The 3'-TMP moiety of dTdA points toward the solvent in a channel defined by Ile-18, Asp-19, Thr-22, Lys-45, and His-46. The phosphate of dTdA is coordinated by the metal, and an adjacent inner sphere water ligand is positioned to donate a hydrogen bond to the general base Glu-43 and to attack the phosphorus with inversion. Arg-35 and Arg-87 donate monodentate hydrogen bonds to different phosphate oxygens of dTdA, with Arg-87 positioned to protonate the leaving 5'-oxygen of dA, thus clarifying the mechanism of hydrolysis. Model building of an additional 5'-dGMP onto the 3'-oxygen of dA placed this third nucleotide onto a surface cleft near residues Glu-80, Asp-83, Lys-84, and Tyr-115 with its 3'-OH group accessible to the solvent, thus defining the size of the substrate binding site as accommodating a trinucleotide.


Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts. Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.


NMR restraint analysis of transforming growth factor alpha: a key component for NMR structure refinement. Structures of the protein, transforming growth factor alpha (TGF-alpha), have been derived from NMR data using distance geometry and subsequent energy refinement. Analysis of the sequential NOE distance bounds using a template algorithm provides a check for consistency in the calculation of bounds, stereospecific assignment of prochiral centers, and secondary structure assignment. Application of the template algorithm to the long range NOEs found within the NMR data sets collected at pH 6.3 and pH 3.4 is used to assess the confidence levels for the accuracy of the structures obtained from modeling. The method also provides critical insight in differentiating regions of the structure that are well defined from those that are not. Use of the restraint analysis protocol is shown to be a powerful adjunct to currently used methods for the assignment of protein structures from NMR data.


Modeling microdomains: the surface area of globin helices. The accessible surface areas of 53 high-resolution globin helices are correlated with molecular weight. The linear fit is assessed for statistical accuracy using a boot-strap analysis, and by comparison to the areas of 13 ideal polyalanine alpha-helices. The accessible area of the unfolded helices is compared with the folded values before helix-helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion-collision model of protein folding.


Amino acid substitution analysis of E. coli thymidylate synthase: the study of a highly conserved region at the N-terminus. Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a beta-strand. We find that residues surrounding a beta-bulge structure within the beta-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested.


Pancreatic spasmolytic polypeptide: crystallization, circular dichroism analysis, and preliminary X-ray diffraction studies. Pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas has been crystallized by the hanging drop vapor diffusion method. Crystals suitable for X-ray diffraction analysis were grown at pH 4.7 from a solution of 6% saturated ammonium sulfate. The space group is orthorhombic I222 or I2(1)2(1)2(1) with unit cell parameters a = 54.38 A, b = 72.29 A, and c = 180.85 A. There are three molecules of PSP per asymmetric unit and a water content of 46.9%. The crystals diffracts to an estimated resolution of 2.7 A. The far-UV CD spectrum of PSP shows some exceptional features which cannot be accounted for thoroughly in terms of standard secondary structures commonly seen in protein CD spectroscopy. With this limitation, the secondary structure analysis predicts 15% alpha-helix, between 10 and 20% antiparallel beta-strand, 10% parallel beta-strand, 15% turn, and 25 to 40% of other structures.


Isolation and characterization of a full-length cDNA coding for an adipose differentiation-related protein. We have previously isolated from a 1246 adipocyte cDNA library a cDNA clone called 154, corresponding to a mRNA that increases abundantly at a very early time during the differentiation of 1246 adipocytes and in adipocyte precursors in primary culture. We show here that the mRNA encoded by this cDNA is expressed abundantly and preferentially in mouse fat pads. A full-length cDNA for clone 154 was isolated by the RACE (rapid amplification of cDNA ends) protocol. Sequence analysis of this cDNA indicates that it encodes a protein of the 425 amino acids [tentatively named adipose differentiation-related protein (ADRP)] that does not have any similarity with sequences contained in the GenBank DNA and Protein Identification Resource protein data bases. Immunoblot of 1246 cell extracts with an antibody raised against the expressed ADRP shows that the 1246 cells contain a 50-kDa protein, the production of which increases as the cells differentiate. Localization of ADRP in 1246 cells indicates that ADRP is absent from nuclear and cytosolic fractions and is found as a membrane-associated protein. These results demonstrate that adipocyte differentiation is accompanied by early expression of a mRNA encoding a membrane-associated adipose differentiation related protein that is adipose tissue specific in vivo.


In vivo self-association of the Drosophila rel-protein dorsal. The Drosophila morphogen dorsal, KBF1, NF-kappa B, and the proto-oncogene c-rel belong to the rel family of transcription factors whose function is regulated post-translationally by selective nuclear import. In the early Drosophila embryo, dorsal protein is proposed to be retained in the cytoplasm through its interaction with cactus protein. The maternal dorsal group genes constitute a signal transduction pathway, which results in targeting cytoplasmic dorsal protein into the nuclei of the syncytial blastoderm embryo, in a ventral-to-dorsal gradient. The asymmetric transcriptional regulation of zygotic genes along the dorsoventral axis by the dorsal morphogen gradient establishes embryonic dorsoventral polarity. In the lymphocytes, the functional equivalent of cactus is I kappa B, which appears to retain NF-kappa B in the cytoplasm. This retention is relieved by extracellular signals in tissue culture. NF-kappa B and rel proteins each are known to function as oligomeric complexes. Here we present genetic and biochemical evidence for the existence and functional importance of an oligomeric dorsal complex in vivo.


Mutational spectrum at the Hprt locus in splenic T cells of B6C3F1 mice exposed to N-ethyl-N-nitrosourea. We have determined the mutational spectrum of N-ethyl-N-nitrosourea (ENU) in exon 3 of the hypoxanthine (guanine) phosphoribosyltransferase gene (Hprt) in splenic T cells following in vivo exposure of male B6C3F1 mice (5-7 weeks old) to ENU. Hprt- mutants were isolated by culturing splenic T cells in microtiter dishes containing medium supplemented with interleukin 2, concanavalin A, and 6-thioguanine. DNA was extracted from 6-thioguanine-resistant colonies and amplified by the polymerase chain reaction (PCR) using primers flanking Hprt exon 3. Identification of mutant sequences and purification of mutant DNA from contaminating wild-type Hprt DNA was accomplished by denaturing-gradient gel electrophoresis. Purified mutant DNA was then sequenced. Treatment of mice with ENU at 40 mg/kg of body weight produced a Hprt- mutant frequency of 7.3 x 10(-5) in splenic T cells, approximately 35-fold above background levels. Sixty-nine of the 521 Hprt- mutants analyzed contained mutations in exon 3 (13%). Transversions and transitions at A.T base pairs dominated the spectrum; 62 of the 69 exon 3 mutations were at A.T base pairs (14 different sites). Thirteen of 14 thymine bases undergoing mutation (61 of 62 mutations at A.T bases) were located on the nontranscribed strand of exon 3. The majority of the remaining mutations (6 of 69) were transitions at a single G.C base pair. These results suggest the importance of thymidine alkylation in ENU-induced mutagenesis in vivo. The mouse Hprt- T-cell cloning/sequencing assay described here may represent a useful system for studying the molecular mechanism of chemically induced mutation occurring in vivo in an endogenous gene.


A monomeric von Willebrand factor fragment, Leu-504--Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX [corrected]. von Willebrand factor interaction with glycoprotein Ib alpha (GPIb alpha) plays a critical role in the initial phase of platelet adhesion at high shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474--Pro-488 (peptide 153) and Ser-692--Pro-708 (peptide 154), inhibit von Willebrand factor--GPIb alpha interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504--Lys-728 [corrected], with a single intrachain disulfide bond linking residues Cys-509--Cys-695 and examined its ability to inhibit von Willebrand factor--GPIb alpha interactions and platelet adhesion at high shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetin-induced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an IC50 = 0.011-0.260 microM, significantly lower than the IC50 of peptide 153 or 154, IC50 = 86-700 microM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (IC50 greater than 500 microM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 microM and at 7.6 microM inhibited greater than 80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.


Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation. We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg. Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition. Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification. Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes. Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction. Mimicked-occupancy receptors exhibited transmembrane signaling without ligand. Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not. Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli. Signaling phenotypes were strikingly segregated by residue position. Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half. These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor. Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1'. Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s).


Phosphorylation of the retinoblastoma protein by cdk2. The retinoblastoma gene product (the RB protein) is phosphorylated in a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing of activation of cdk2 in the cell cycle was similar to that of the onset of phosphorylation of the RB protein. The kinase coprecipitated with the RB protein also exhibited a similar substrate specificity to cdk2 and a similar time course of activation during the cell cycle. We further showed that cdk2 formed a complex with the RB protein in vitro and that its formation was not competitively inhibited by the simian virus 40 large T antigen. These observations suggest that cdk2 or a cdk2-related protein is involved in the cell cycle-dependent phosphorylation of the RB protein.


Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells. Stable transformants of mammalian cells from gene transfer often show extreme variability in expression of the introduced transgene. This occurs from the highly variable number of copies integrated into the genome and from position effects on gene expression due to random integration. We have eliminated both of these constraints on predictable gene expression by use of a lox recombination vector. The positive selection vector system is designed to directly select Cre-mediated DNA integration at a lox target previously placed into the genome of cultured mammalian cells. Proper targeting activates expression of a defective lox-neomycin phosphotransferase (neo) fusion gene target. With CHO cell lines containing this target, almost all of the selected transformants (54 of 56 independent G418-resistant colonies) were simple single-copy integrants of the targeting DNA. To monitor gene expression at a single chromosomal site, we used a beta-actin promoter-lacZ reporter construct. Independent G418-resistant colonies from site-specific integration of the reporter gene all showed nearly identical levels of beta-galactosidase activity when the reporter construct integrated at a particular chromosomal position. The same construct integrated at a second chromosomal position exhibited a slightly different level of activity, characteristic of that second position. These results show that Cre-mediated site-specific integration can facilitate the construction of isogenic cell lines and thereby permit reproducible gene expression in stably transformed cell lines.


Hagfish humoral defense protein exhibits structural and functional homology with mammalian complement components. A genomic clone and cDNA fragment encoding a portion of a humoral recognition molecule from the hagfish were isolated and sequenced. The serum protein has previously been described as having structural features that are immunoglobulin-like. Amino acid sequence obtained from the 77-kDa H1 heavy chain facilitated the isolation of a genomic clone containing at least two coding regions. Through use of primers derived from the genomic sequences, a 231-base-pair cDNA fragment was obtained by PCR from liver RNA. Comparison of the deduced 120-amino acid sequence from the N terminus of H1 with known protein sequences revealed substantial sequence similarity with the beta chain of the murine fourth complement component C4 and with the related third and fifth complement molecules C5 and C3 and the major histocompatibility complex-encoded sex-limited protein. Observation of structural and functional similarities associated with the sequence similarity indicate that these molecules share an evolutionary relationship: the polypeptide chain structure of hagfish complement-like protein (CLP) resembles that of C4; CLP contains a hidden thioester group on the 70-kDa chain; CLP binds to streptococcal cells and enhances the phagocytosis of yeast by hagfish leukocytes. These data suggest that CLP forms part of a non-clonally-derived complement-related humoral defense system in the hagfish.


Cationic lipids direct a viral glycoprotein into the class I major histocompatibility complex antigen-presentation pathway. Recombinant glycoprotein B (gB) of herpes simplex virus (HSV) was processed and presented by class I major histocompatibility complex (MHC) molecules after delivery into cells by using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP), a commercially available cationic lipid used for DNA transfection. Cells treated with DOTAP-associated gB were susceptible to lysis by class I MHC-restricted, HSV-specific cytotoxic T lymphocytes (CTL), and the treated cells restimulated memory gB-specific CTL activity in spleen cells from HSV-infected mice. gB-specific CTL responses were detected in mice immunized with recombinant gB and DOTAP but not in those receiving gB emulsified in complete Freund's adjuvant. Thus, cationic lipids may facilitate induction of CD8+ T-cell responses in vaccinations with recombinant antigens, and they may serve as readily available reagents for dissecting class I MHC immunity to viruses and other intracellular pathogens.