Osteocalcin, adipokines and their associations with glucose metabolism in type 1 diabetes.

To determine osteocalcin (OC) and adipokines in type 1 diabetes (T1D) and healthy controls, and to explore possible associations between glucose and bone metabolism, body composition and adipokines. Serum levels of total OC, undercarboxylated (UC-OC), leptin, adiponectin, and other parameters of glucose and bone metabolism were measured in 128 patients with T1D (mean duration 21.2years) and in 77 healthy controls, matched for gender, age, and body mass index (BMI). Partial correlations (adjusted for age and gender) with parameters of body composition (BMI, fat body mass [derived from bone mineral density scans]), glycaemic control (hemoglobin A1c (HbA1c), daily insulin dose in T1D), skeletal homeostasis (osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), all measured in serum), and serum insulin-like growth factor 1 (IGF-1) were also examined. Independent predictors of total and UC-OC were then explored. Total OC was lower in males with T1D (16.3±6.4 vs. 22.2±9.9ng/ml; p=0.001), whereas UC-OC did not show group differences. Adiponectin was higher in T1D patients, both for males and females (8.9±6.6 vs. 5.7±2.5µg/ml; p=0.004 and 13.8±6.4 vs. 8.8±4.0µg/ml; p<0.001). IGF-1 was lower only in females with T1D (146.6±68.8 vs. 203.0±74.4ng/ml; p<0.001). BMI and fat body mass were similar in T1D and controls. In T1D patients, total OC was inversely correlated with BMI and HbA1c, and UC-OC inversely correlated with HbA1c. In T1D patients, leptin positively correlated with BMI, fat body mass and daily insulin dose, while adiponectin inversely correlated with BMI and daily insulin dose. Multivariate regression modelling showed that determinants of higher total OC levels were male gender (p=0.04, ß-coefficient=2.865) and lower HbA1c (p=0.04, ß-coefficient=-0.117), whereas determinants of UC-OC levels were T1D (p=0.016, ß-coefficient=2.015), higher IGF-1 (p=0.004, ß-coefficient=0.011) and lower HbA1c (p=0.011, ß-coefficient=- 0.061). Total OC and UC-OC are associated with good glycaemic control in T1D, with gender-specific differences for total-OC. The association of leptin and adiponectin with glycaemic control, as observed in controls, does not seem to be a feature in T1D, although both adipokines appear to be related to the insulin demand. This article is part of a Special Issue entitled "Bone and diabetes".

Trabecular bone score in type 1 diabetes--a cross-sectional study.

UNLABELLED: Trabecular bone score (TBS) seems to provide additive value on BMD to identify individuals with prevalent fractures in T1D. TBS did not significantly differ between T1D patients and healthy controls, but TBS and HbA1c were independently associated with prevalent fractures in T1D. A TBS cutoff <1.42 reflected prevalent fractures with 91.7 % sensitivity and 43.2 % specificity. INTRODUCTION: Type 1 diabetes (T1D) increases the risk of osteoporotic fractures. TBS was recently proposed as an indirect measure of bone microarchitecture. This study aimed at investigating the TBS in T1D patients and healthy controls. Associations with prevalent fractures were tested. METHODS: One hundred nineteen T1D patients (59 males, 60 premenopausal females; mean age 43.4 ± 8.9 years) and 68 healthy controls matched for gender, age, and body mass index (BMI) were analyzed. The TBS was calculated in the lumbar region, based on two-dimensional (2D) projections of DXA assessments. RESULTS: TBS was 1.357 ± 0.129 in T1D patients and 1.389 ± 0.085 in controls (p = 0.075). T1D patients with prevalent fractures (n = 24) had a significantly lower TBS than T1D patients without fractures (1.309 ± 0.125 versus 1.370 ± 0.127, p = 0.04). The presence of fractures in T1D was associated with lower TBS (odds ratio = 0.024, 95 % confidence interval (CI) = 0.001-0.875; p = 0.042) but not with age or BMI. TBS and HbA1c were independently associated with fractures. The area-under-the curve (AUC) of TBS was similar to that of total hip BMD in discriminating T1D patients with or without prevalent fractures. In this set-up, a TBS cutoff <1.42 discriminated the presence of fractures with a sensitivity of 91.7 % and a specificity of 43.2 %. CONCLUSIONS: TBS values are lower in T1D patients with prevalent fractures, suggesting an alteration of bone strength in this subgroup of patients. Reliable TBS cutoffs for the prediction of fracture risk in T1D need to be determined in larger prospective studies.

High serum pentosidine but not esRAGE is associated with prevalent fractures in type 1 diabetes independent of bone mineral density and glycaemic control.

UNLABELLED: Fracture risk in type 1 diabetes (T1D) is supposed to be underestimated by bone mineral density (BMD). Individuals with T1D had more prevalent fractures in a cross-sectional study. Serum levels of pentosidine, an advanced glycation end product, and poor glycaemic control were associated with prevalent fractures independent of BMD. INTRODUCTION: Type 1 diabetes (T1D) is associated with increased fracture risk. Bone mineral density (BMD) underestimates the risk of fractures in some individuals. The accumulation of advanced glycation end products (AGEs) impairs bone matrix and reduces bone strength. METHODS: In a cross-sectional study, 128 men and premenopausal women with T1D were evaluated. We compared traditional risk factors for fractures, BMD, parameters of bone metabolism and AGEs in individuals with and without prevalent fractures. An independent association of serum AGE levels with prevalent fractures was investigated. RESULTS: Individuals with prevalent fractures exhibited a longer duration of T1D, higher HbA1c and more diabetic-related complications. BMD at the femoral neck (z-score -0.76 ± 0.94 vs. -0.23 ± 1.02; p = 0.031) and total hip (z-score -0.54 ± 0.93 vs. 0.11 ± 1.11; p = 0.017) was lower in those with prevalent fractures. Individuals with fractures had higher pentosidine levels (164.1 ± 53.6 vs. 133.2 ± 40.4; p = 0.002). The levels of N-ε-(carboxymethyl)-lysine (CML) and endogenous secretory receptor for AGEs (esRAGE) did not significantly differ. Multivariate logistic regression analysis adjusted for age, BMI, family history of fractures, smoking, vitamin D deficiency, BMD at lumbar spine, femoral neck and total hip identified pentosidine levels and HbA1c as independent factors associated with prevalent fractures (odds ratio 1.02, 95% CI 1.00-1.03/pmol/ml increase of pentosidine; p = 0.008 and odds ratio 1.93, 95% CI 1.16-3.20 per percentage increase of HbA1c; p = 0.011). CONCLUSIONS: The pentosidine levels but not BMD are independently associated with prevalent fractures. Impaired bone quality in T1D may result from increased AGE formation.

Glycaemic control is positively associated with prevalent fractures but not with bone mineral density in patients with Type 1 diabetes.

AIM: There are conflicting data regarding the risk of osteoporosis in patients with Type 1 diabetes. We investigated an association between diabetes, bone mineral density and prevalent fractures. METHODS: A single-centre, cross-sectional study of men and pre-menopausal women with Type 1 diabetes (n = 128) and a matched control group (n = 77) was conducted. The primary outcome measure was bone mineral density and secondary measures were markers of bone metabolism and prevalent fractures. RESULTS: Hip and total body bone mineral densities were significantly lower in women with diabetes compared with control subjects. In men, no difference in bone mineral density was found. A multivariate regression analysis in women with diabetes revealed higher BMI as the strongest predictor of higher total hip, femoral neck and total body bone mineral density, whereas previous fractures were inversely associated with total hip bone mineral density and C-terminal telopeptide of type I collagen with total body bone mineral density. Poor long-term glycaemic control was not associated with low bone mineral density. Fracture frequency was higher in patients with diabetes compared with control subjects (1.64 vs. 0.62 per 100 patient-years; P < 0.05). In a multivariable model, long-term HbA(1c) control was associated with increased clinical fracture prevalence (OR 1.92; 95% CI 1.09-2.75) in those with diabetes. CONCLUSIONS: Type 1 diabetes contributes to low bone mineral density in women. Previous fractures and low BMI were strong predictors of impaired bone mineral density and should therefore be considered in risk estimation. Fractures are more frequent in Type 1 diabetes. Long-term hyperglycaemia may account for impaired bone strength, independently from bone mineral density.

Reduction of uterus dose in clinical thoracic computed tomography.

PURPOSE: The aim of this study was to investigate the potential dose reduction in the uterus as a result of lead apron protection during thoracic CT scans. Moreover, the distribution of the radiation dose in the uterus was determined in order to obtain information about the ratio of internally and externally scattered radiation. MATERIALS AND METHODS: The uterus doses during thoracic CT were determined by measuring organ doses using an Alderson-RANDO®-Phantom and thermoluminescent dosimeters. A 0.25 mm lead equivalent protective apron was used to shield the abdominal area. Three measurement conditions were evaluated: without lead apron, covered with lead apron and wrapped with lead apron. The uterus dose with and without shielding describes the mean value and standard deviation of all examinations and all measurement points in the organ. RESULTS: The uterus dose by thoracic CT was measured to be approximately 66.5 ± 3.1 µGy. If the abdomen is covered with a 0.25 mm Pb equivalent lead apron in the front area and on both sides, the uterus dose is reduced to 49.4 ± 2.8 µGy (26% reduction, p < 0.001). If a lead apron is wrapped around the abdomen, providing 0.50 mm Pb shielding in the anterior section due to overlap, and 0.25 mm Pb in the posterior section and on both sides, the uterus dose is reduced even more to 43.8 ± 2.5 µGy (34% reduction, p < 0.001). The dose distribution when the lead apron covers the abdomen shows that the shielding is effective for the scatter radiation that comes from the anterior part. Moreover, the wrapped apron protects the uterus from all directions and is even more effective for dose reduction than the covering apron. CONCLUSION: Our findings demonstrate that protective aprons are an effective dose reduction technique without additional costs and little effect on patient examination time.

Theoretical and experimental investigations of Coulomb blockade in coupled quantum dot systems.

Two strongly coupled quantum dots are theoretically and experimentally investigated. In conductance measurements on a GaAs based low-dimensional system additional features to the Coulomb blockade have been detected at low temperatures. These regions of finite conductivity are compared with theoretical investigations of a strongly coupled quantum dot system and good agreement between the theoretical and the experimental results has been found.

Mediator release assays based on human or murine immunoglobulin E in allergen standardization.

BACKGROUND: The biological potency of allergens can be measured by provoking mediator release from effector cells. As established immunochemical methods in allergen standardization only determine inhibition potency or major allergen content, routine tests for biological potency may enhance standardization and batch control of allergen products. OBJECTIVE: The general performance and application potential of biological in vitro assays in batch control and standardization of allergens and as a tool for verifying activity and stability of allergen standards were analysed. METHODS: Allergen extracts of five clinically relevant allergens from three to five different manufacturers were investigated. A CAP-IgE-inhibition assay was compared with mediator release assay (MRA)s based on murine or human basophils. Rat basophilic leukaemia (RBL) cells were passively sensitized with pooled murine allergen-specific IgE-containing sera. Humanized RBL cells and human-stripped basophils were sensitized with pooled patient's sera, which were also used for the CAP-IgE-inhibition assay. Allergen specificity of the sera was determined by immunoblotting. RESULTS: A good batch-to-batch consistency was found with each assay among all manufacturers and allergens tested. Between different manufacturers, the products showed differences in activity and the various assays indicated an almost identical ranking. However, the biological assays revealed qualitative differences of biological activity or composition of allergen preparations undetectable by IgE-inhibition assay. CONCLUSIONS: MRAs provide refined information on allergen activity, either confirming the results of IgE-inhibition assay, or indicating differences requiring further investigation, and represent a highly sensitive novel tool in allergen standardization. By using permanently cultivated cell lines, repeated venepuncture to obtain human basophils is avoided. As in the RBL assay, the coefficient of variation for the release values were below 15% and for the ED50 below 25%, the assay is suitable to determine differences that are relevant for batch control purposes.

Metered-dose inhaler: the emergency department orphan.

OBJECTIVE: Physicians providing emergency department care to children primarily use nebulizers for the delivery of bronchodilators and these physicians have misconceptions regarding the advantages and disadvantages of using metered-dose inhalers (MDIs) with a spacer (MDI + S) for acute asthma exacerbations. DESIGN: Self-administered mail survey. SETTING: Emergency department. PARTICIPANTS: Emergency medicine section members of the American Academy of Pediatrics and Canadian Pediatric Society. INTERVENTIONS: Bronchodilator delivery methods in acute pediatric asthma. MAIN OUTCOME MEASURES: The 2 principal outcomes for bivariate analysis were self-reported nebulizer use in all patients and MDI + S use in patients with mild acute asthma. RESULTS: Of eligible physicians, 333 (51%) of 567 responded. The majority were dual trained in pediatrics and pediatric emergency medicine (72%) and practiced full time (83%) in an urban (83%) pediatric emergency department (80%). The most commonly cited advantages of MDIs were their cost (33%) and speed of use (28%). The most commonly cited disadvantages were patient or parent dissatisfaction (24%) and relative ease of nebulizer use (23%). Only 10% to 21% of participants used MDIs in the emergency department and reserved this delivery method for children with mild asthma exacerbations. There were no significant associations between selected respondent demographic variables and the use of MDIs. CONCLUSIONS: Misconceptions regarding the efficacy and safety of MDI + S for the treatment of acute asthma exacerbations exist but are limited to a minority of surveyed emergency medicine physicians caring for children. Nebulizers remain the preferred method of routine bronchodilator delivery by physicians providing care to pediatric asthmatics in the emergency department.

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.

Prophylaxis against possible human immunodeficiency virus exposure after nonoccupational needlestick injuries or sexual assaults in children and adolescents.

BACKGROUND: Nonoccupational human immunodeficiency virus (HIV) postexposure prophylaxis (PEP) for adults has been described, although the Centers for Disease Control and Prevention, Atlanta, Ga, offer no specific recommendations. There is limited information about its use in children and adolescents. OBJECTIVE: To describe the current practices of physicians in pediatric infectious disease (PID) and pediatric emergency medicine (PEM) departments regarding nonoccupational HIV PEP for children and adolescents. DESIGN: Survey. PARTICIPANTS: Directors of all PID and PEM departments with fellowship programs in the United States and Canada between July and November 1998. MAIN OUTCOME MEASURES: General questions regarding HIV PEP and questions concerning 2 scenarios (5-year-old with a needlestick injury and a 15-year-old after sexual assault). RESULTS: The return rate was 67 (78%) of 86 for PID and 36 (75%) of 48 for PEM physicians. Fewer than 20% of physicians reported institutional policies for nonoccupational HIV PEP; 33% had ever initiated nonoccupational HIV PEP. In both scenarios, PID physicians were more likely than PEM physicians to recommend or offer HIV PEP in the first 24 hours after the incident (55 [83%] of 66 vs 20 [56%] of 36 for needlestick injuries [odds ratio, 4.0; 95% confidence interval, 1.6-10.1] and 47 [72%] of 65 vs 16 [50%] of 32 for sexual assault [odds ratio, 2.6; 95% confidence interval, 1.1-6.3]). Seven different antiretroviral agents in single, dual, or triple drug regimens administered for 2 to 12 weeks were suggested. CONCLUSIONS: Although few physicians reported institutional policies, and only one third had ever initiated HIV PEP, many would offer or recommend HIV PEP for children and adolescents within 24 hours after possible HIV exposure. A wide variation of regimens have been suggested. There is a need for a national consensus for nonoccupational HIV PEP.

Dental concerns unrelated to trauma in the pediatric emergency department: barriers to care.

OBJECTIVES: To describe patients with nontraumatic dental problems treated in our pediatric emergency department (PED) and to determine if barriers to access prompted seeking care in the PED rather than from a dentist or dental clinic. DESIGN: Questionnaire administered to a convenience sample of patients with nontraumatic dental complaints. SETTING: An urban PED. MAIN OUTCOME MEASURES: Insurance status, primary medical and dental care, duration of symptoms, diagnosis, and reason for seeking care in the PED. RESULTS: Two hundred patients were enrolled. Median age was 17 years (range, 1-22 years). Forty-five percent were African American. Forty-nine percent had Medicaid. Fifty percent identified a regular dentist, whereas 71% had a primary care physician. Thirty-four percent of patients 4 years and older had not seen a dentist in more than a year. Children younger than 13 years were more likely than teenagers to identify a regular dentist (odds ratio [OR] = 2.8; 95% confidence interval [CI], 1.3-6.1). Those with a regular medical provider were more likely to have a regular dentist (OR = 7.7; 95% CI, 3.4-18). The most common reasons for not going to a dentist were as follows: dentist closed, 34%; lack of dental insurance or money, 17%; and lack of a dentist, 16%. Patients with symptoms for more than 72 hours were more likely to cite lack of a dentist as their reason for coming to the PED (OR = 7.4; 95% CI, 1.9-33). CONCLUSIONS: Many pediatric patients do not have regular dental care, and this is associated with a lack of primary medical care. Access barriers to acute dental care include lack of insurance or funds, lack of a dentist, and limited hours of dental care sites. Improved insurance reimbursement, active enrollment of adolescents into preventive dental care, and expansion of provider hours may limit PED dental visits and improve the health of patients.

Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle.

In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of small nuclear ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6 and U5) follow. Here we describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 A resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs, supporting a proposed model of circular Sm protein arrangement. By taking earlier biochemical results into account, we were able to assign the remaining density of the map to the other known components of U1 snRNP, deriving a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP.

A comparison of suburban and urban daytime telephone triage calls.

OBJECTIVE: To compare daytime nurse telephone triage calls received by a suburban practice with those received by a hospital-based, inner-city pediatric practice. METHODS: A research assistant, listening simultaneously with triage personnel, prospectively coded all calls received by the nurse triage telephone offices in 2 pediatric practices. Calls were coded the first full, nonholiday week of 3 consecutive summer and 3 consecutive winter months, alternating morning and afternoon sessions. One practice was suburban and had almost all commercially insured patients; the other was hospital-based, in an inner-city, and consisted mostly of patients with Medicaid coverage or no health insurance. RESULTS: A total of 901 calls were triaged in the suburban practice (SP) and 768 in the urban practice (UP). The chief complaints of calls regarding medical problems were similar at both sites. Difficulties with language were noted less often in SP compared with UP (1% vs 17%). The reason for the calls differed by site: medical problem relating to illness or injury (SP 55% vs UP 40%); social issue (SP 1% vs UP 9%); documentation request (SP 2% vs UP 7%); request for laboratory work (SP.3% vs UP 4%); and well child advice (SP 9% vs UP 5%). The disposition of calls also differed by site: telephone advice was offered significantly more in SP than in UP (32% vs 20%); fewer calls required the need for the medical record in SP than in UP (2% vs 12%). CONCLUSIONS: Calls received by a daytime nurse telephone triage office in an affluent SP and a UP are similar in regard to medical problems. Training programs can feasibly prepare physicians and nurses for both kinds of practice settings. The urban site received more calls affected by language and social issues. This could have administrative implications for staffing ratios, language skills of staff and knowledge of available support services.

The teaching of cultural issues in U.S. and Canadian medical schools.

PURPOSE: Despite the importance of culture in health care and the rapid growth of ethnic diversity in the United States and Canada, little is known about the teaching of cultural issues in medical schools. The study goals, therefore, were to determine the number of U.S. and Canadian medical schools that have courses on cultural issues, and to examine the format, content, and timing of those courses. METHOD: The authors contacted the deans of students and/ or directors of courses on cultural issues at all 126 U.S. and all 16 Canadian medical schools. Using a cross-sectional telephone survey, they asked whether each school had a course on cultural sensitivity or multicultural issues and, if so, whether it was separate or contained within a larger course, when in the curriculum the course was taught, and which ethnic groups the course addressed. RESULTS: The response rates were 94% for both U.S. (118) and Canadian (15) schools. Very few schools (U.S. = 8%; and Canada = 0%) had separate courses specifically addressing cultural issues. Schools in both countries usually addressed cultural issues in one to three lectures as part of larger, mostly preclinical courses. Significantly more Canadian than U.S. schools provided no instruction on cultural issues (27% versus 8%; p = .04). Few schools taught about the specific cultural issues of the largest minority groups in their geographic areas: only 28% and 26% of U.S. schools taught about African American and Latino issues, respectively, and only two thirds of Canadian schools taught about either Asian or Native Canadian issues. Only 35% of U.S. schools addressed the cultural issues of the largest minority groups in their particular states. CONCLUSIONS: Most U.S. and Canadian medical schools provide inadequate instruction about cultural issues, especially the specific cultural aspects of large minority groups.

Pediatricians' attitudes, beliefs, and practices regarding clinical practice guidelines: a national survey.

BACKGROUND: Clinical practice guidelines are increasingly being used for a wide variety of medical conditions, but not enough is known about physicians' attitudes and beliefs about guidelines, how often and under what circumstances they are used, and factors associated with their acceptance. OBJECTIVE: To determine practice guideline attitudes, beliefs, practices, and factors associated with use among a representative national sample of general pediatricians. STUDY DESIGN: Cross-sectional mail survey. SUBJECTS: Random sample of general pediatrician members of the American Academy of Pediatrics residing in all 50 states and Puerto Rico. SURVEY INSTRUMENT: Twenty-four multiple-choice, Likert scale, yes-no, and open-ended questions about pediatric clinical practice guidelines. RESULTS: From 1088 respondents, 461 specialists were excluded; the remaining 627 general pediatricians were mostly male (61%), white (81%), and in group practice (62%) in a suburban location (48%). Practice guidelines are used by 35% of pediatricians, in part by 44%, and not at all by 21%. Over 100 different practice guidelines are used, most commonly for asthma (77%), hyperbilirubinemia (27%), and otitis media (19%). Common reasons for use of practice guidelines include standardization of care (17%) and helpfulness (10%). Commonly cited problems with practice guidelines include failure to allow for clinical judgment (54%), use in litigation (16%), and limitation of autonomy (5%). In multivariate analysis, the odds of practice guideline use were greater among pediatricians in health maintenance organization practices (odds ratio [OR]: 9.1; 95% confidence interval [CI]: 1.2-68.0) and those who were nonwhite (OR: 2.3; 95% CI: 1.1-4.8), but lower in those with more weekly patient visits (OR:.7; 95% CI:.5-.9). Features most likely to lead to practice guideline use include simplicity (16%), feasibility (12%), and evidence of improved outcomes (10%). Most pediatricians agree that practice guidelines improve outcomes (89%), are motivated by a desire to improve quality (94%), and should not be used in litigation (82%) or disciplinary actions (77%), nor be motivated by a desire to reduce costs (73%). CONCLUSIONS: Most general pediatricians use practice guidelines, but no specific guidelines, except those for asthma, are used by >27% of pediatricians. The results of this study suggest that practice guidelines are most likely to be followed if they are simple, flexible, rigorously tested, not used punitively, and are motivated by desires to improve quality, not reduce costs.

A doughnut-shaped heteromer of human Sm-like proteins binds to the 3'-end of U6 snRNA, thereby facilitating U4/U6 duplex formation in vitro.

We describe the isolation and molecular characterization of seven distinct proteins present in human [U4/U6.U5] tri-snRNPs. These proteins exhibit clear homology to the Sm proteins and are thus denoted LSm (like Sm) proteins. Purified LSm proteins form a heteromer that is stable even in the absence of RNA and exhibits a doughnut shape under the electron microscope, with striking similarity to the Sm core RNP structure. The purified LSm heteromer binds specifically to U6 snRNA, requiring the 3'-terminal U-tract for complex formation. The 3'-end of U6 snRNA was also co-precipitated with LSm proteins after digestion of isolated tri-snRNPs with RNaseT(1). Importantly, the LSm proteins did not bind to the U-rich Sm sites of intact U1, U2, U4 or U5 snRNAs, indicating that they can only interact with a 3'-terminal U-tract. Finally, we show that the LSm proteins facilitate the formation of U4/U6 RNA duplices in vitro, suggesting that the LSm proteins may play a role in U4/U6 snRNP formation.

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.

Complex formation among rat pancreatic secretory proteins under mild alkaline pH conditions.

Previous in vitro studies have demonstrated that enzyme proteins liberated from isolated zymogen granules of the rat pancreas aggregate already at neutral or slightly basic pH and form small particles which in the acidic pH range progressively condense into dense cores of about the size of zymogen granules. To characterize the protein composition of the original particles in more detail non-denaturing agarose gel electrophoresis was employed. Five major protein complexes were identified which upon separation of individual complexes in 1-D or 2-D gel electrophoresis were shown to be composed of a distinct set of known enzymes and several unknown proteins. Complexes 1-4 quickly dissociated when enzyme activation was induced by enterokinase, but complex 5 was resistant even to this treatment. All 5 complexes revealed a distinct fine structure when eluted from the gels and studied in negative staining electron microscopy. These findings suggest that pancreatic zymogens form complexes already in the lumen of the rough endoplasmic reticulum and are transported as such to the Golgi complex where they aggregate into granule cores due to the internal acidic pH. Complex formation may thus facilitate zymogen sorting within the rough endoplasmic reticulum and may prevent premature enzyme activation within cellular compartments.

Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.