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Biomedical subjects

B Kastner

Publications and source records attributed to B Kastner.

At least 19 recordsLinked to original sources
PubMed2007

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.

Allergens↗
PubMed2001

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.

Cryoelectron Microscopy↗
PubMed2001

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.

Acute Disease↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Adolescent↗
PubMed2001

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.

Fungal Proteins↗
PubMed2000

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.

Canada↗
PubMed2000

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.

Attitude of Health Personnel↗
PubMed2000

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.

Child↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed1999

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.

Anion Exchange Resins↗
PubMed1999

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.

Binding Sites↗
PubMed1999

Hyperphagia in neonates withdrawing from methadone.

AIMS: To examine whether hyperphagia is a clinically significant problem in infants born to women receiving methadone maintenance. METHODS: The volume of feeds, changes in infant body weight, as well as occurrence of adverse clinical effects in infants withdrawing from methadone were studied during the first month of life. A retrospective chart review was conducted for all infants at San Francisco General between 1992 and 1995, born to women receiving methadone maintenance during their pregnancy. Forty four infants were identified and the data obtained from hospital medical records. The daily oral intake of these infants was recorded during the first month of life. The incidence of hyperphagia (oral intake > 190 cc/kg/day) was measured. Associations between infant oral intake and maternal methadone dose were studied using correlation analysis as well as Anova for repeated measures. Adverse clinical symptoms were also recorded. A subset of premature infants was studied separately. RESULTS: The incidence of hyperphagia was 26% by day 8 and 56% by day 16 of life in the infants. Hyperphagia was not associated with maternal methadone dose or with infant withdrawal scores. Infants who were hyperphagic lost significantly more weight during the first week of life than those who were not. Despite significantly greater intake, the hyperphagic infants did not gain weight more rapidly during the first month of life compared with those infants with lower oral intake. Infants who were hyperphagic (maximum intake of 290 cc/kg/day) did not experience increased vomiting, aspiration, diarrhoea, or abdominal distention. CONCLUSIONS: Hyperphagia is commonly found in infants withdrawing from methadone and can be persistent in a significant number. Hyperphagia was not associated with either increased neonatal weight gain or with adverse gastrointestinal consequences. Hyperphagia may occur in infants withdrawing from methadone who have high metabolic demands due to clinical signs not controlled by opiate treatment.

Adult↗
PubMed1998

Access barriers to health care for Latino children.

BACKGROUND: Latinos will soon be the largest minority group in the United States, but too little is known about major access barriers to health care for this group and whether these barriers result in adverse consequences. OBJECTIVE: To identify important access barriers to health care for Latino children, as cited by parents. DESIGN: Cross-sectional survey of parents of all 203 children coming to the pediatric Latino clinic at an innercity hospital. Questions focused on barriers to health care experienced prior to receiving care at the Latino clinic. RESULTS: Parental ethnicity included Dominican (36%), Puerto Rican (34%), Central American (13%), and South American (11%). Only 42% of parents were American citizens, whereas 36% had green cards, and 13% had no documentation. Eight percent of parents and 65% of the children were born in the United States. Parents rated their ability to speak English as follows: very well/well, 27%; not very well, 46%; and not at all, 26%. The median annual household income was $11,000; 40% of parents never graduated from high school, and 49% headed single-parent households. Forty-three percent of the children were uninsured. A sick child was routinely brought to hospital clinics by 56% of parents, to the emergency department by 21%, and to neighborhood health centers by 21%. When asked to name the single greatest barrier to health care for their children, parents cited language problems (26%), long waiting time at the physician's office (15%), no medical insurance (13%), and difficulty paying medical bills (7%). When parents were asked if a particular barrier had ever caused them not to bring their children in, transportation was cited by 21%; not being able to afford health care, 18%; excessive waiting time in the clinic, 17%; no health insurance, 16%; and lack of cultural understanding by staff, 11%. Some parents who spoke little or no English reported that medical staff not speaking Spanish had led to adverse health consequences for their children, including poor medical care (8%), misdiagnosis (6%), and prescription of inappropriate medications (5%). Multivariate analyses of selected health outcomes using 7 independent variables showed that low family income was significantly associated with greater odds of a child's having suboptimal health status (odds ratio, 1.5; 95% confidence interval, 1.04-2.2) and an increased number of physician visits in the past year (P<.04), but reduced odds (odds ratio, 0.6; 95% confidence interval, 0.4-0.9) of the child's being brought to the emergency department for a routine sick visit. Children whose parents had resided in the United States for fewer than 8 years were at reduced odds (odds ratio, 0.5; 95% confidence interval, 0.2-0.9) for having spent a day or more in bed for illness in the past year. CONCLUSIONS: Parents identified language problems, cultural differences, poverty, lack of health insurance, transportation difficulties, and long waiting times as the major access barriers to health care for Latino children. Language problems can result in adverse health consequences for some children, including poor medical care, misdiagnosis, and inappropriate medication and hospitalization. Low family income is an important independent risk factor among Latino children for suboptimal health and high utilization of health services.

Adult↗
PubMed1998

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure.

The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing. The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis. To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts. Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes. Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins. Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases. The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes. Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs. In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs. Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

Amino Acid Sequence↗
PubMed1997

Electron microscopy of assembly intermediates of the snRNP core: morphological similarities between the RNA-free (E.F.G) protein heteromer and the intact snRNP core.

All four spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 contain a common structural element called the snRNP core. This core is assembled from the common snRNP proteins and the small nuclear RNA (snRNA). We have used electron microscopy to study the structure of two intermediates of the snRNP core assembly pathway: (1) the (E.F.G) protein complex, which contains only the smallest common proteins E, F and G; and (2) the subscore of U5 snRNP, in which the U5 RNA and the common proteins D1 and D2 are bound to the (E.F.G) protein complex. The general structure of the subscore was found to resemble that of the complete snRNP core, which contains the components of the subscore plus the common proteins B/B' and D3. Both the complete snRNP core and subscore particles are globular, with diameters of 7 to 8 nm. They show a characteristic accumulation of stain at the centre. However, some subscore images showed nicked outlines not seen with the complete snRNP cores. The (E.F.G) protein complex appeared as a ring, with an outer diameter of about 7 nm and a central hole 2 nm across. The molecular dimensions of the E, F and G proteins imply that the thickness of the (E.F.G) ring structure is only about 2 nm. Comparison of the (E.F.G) structure complex with the snRNP core and subcore structures implicates that a flat side of the ring-shaped (E.F.G) complex provides the assembly site(s) for the other components of the snRNP during core assembly: first for the D1 and D2 proteins (and probably the snRNA) during subscore formation, and then for the B/B' and D3 proteins in the completion of the snRNP core particle.

Centrifugation, Density Gradient↗
PubMed1995

Isolation and characterization of the small nucleolar ribonucleoprotein particle snR30 from Saccharomyces cerevisiae.

The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1. All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation. NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the small nucleolar ribonucleoprotein particle snR30 (snoRNP). In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein. Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure. The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients. Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide.

Base Sequence↗