Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus

The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 X 10(6), having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3' and 5' termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence ("positive" polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3'-G2-G1-5'. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed.     

Transformation and genomic restriction mapping of Rochalimaea spp.

Transformation procedures using electroporation were established for Rochalimaea quintana. Several cosmid/plasmids possessing the RK2 or RSF1010 origin of replication were successfully inserted. Plasmid retention and replication were verified by antibiotic resistance and Southern blot analysis. The highest level of transformation was obtained at a voltage field strength of 12.5 kV/cm with a pulse time of 10 miliseconds. Transformation efficiency was low (0.3%) with approximately 10(5) transformants/microgram of DNA. One construct, designated pAG10, reached sufficient levels in R. quintana to be isolated by density gradient centrifugation. Analysis of this plasmid after several cycles of growth in R. quintana revealed no obvious modifications. Physical maps of Rochalimaea spp. chromosomal DNA using pulse-field electrophoresis are being developed. Digestion of R. vinsonii chromosomal DNA with NotI or SfiI resulted in three and one fragments, respectively. When R. quintana was digested in a similar manner, both NotI and SfiI produced four fragments. Double digestion of R. quintana DNA with NotI and SfiI yield seven fragments ranging in size from 11 to 925 kb. Summing the fragments indicate an approximate genome size of 2.1 x 10(6) bp for R. vinsonii and 1.7 x 10(6) bp for R. quintana chromosomal DNA.     

Constitutive macropinocytosis allows TAP-dependent major histocompatibility compex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.     

Separation anxiety: the etiology of nondisjunction in flies and people

Two new studies examine the recombinational history of humanchromosomes that nondisjoin at the first meiotic division infemales. Our analysis of these studies suggests two possibleetiologies of nondisjunction in terms of well-understood propertiesof chromosome mechanics. For both the X chromosome and for chromosome21, 60–70% of nondisjoined chromosomes are derived fromchlasmate bivalents, many of which display unusual patternsof exchange. The patterns of exchange and nondisjunction observedfor human chromosome 21 parallel those exhibited by a mutationin Drosophila that impairs spindle assembly and function. Basedon these similarities, we propose that nondisjunction of chromosome21 in human females results from an age-dependent loss of spindle-formingability. The recombinational histories of nondisjoining humanX chromosomes are quite different from those of chromosome 21,but rather parallel those obtained for spontaneous nondisjunctionin Drosophila females. The data for X chromosome disjunctionin both species can be explained by a model in which nondisjunctionis the consequence of the age-dependent movement of transposableelements. According to this model, nondisjunction is explainedas the consequence of the repair of transposon-induced breaksin the DNA. Both models provide reasonable alternatives to biologicallyimplausible explanations such as the ‘production linehypothesis’.     

Using nonword repetition to distinguish genetic and environmental influences on early literacy development: a study of 6-year-old twins.

This study considered whether cognitive profile could distinguish groups of children where genes or environment played a major role in influencing reading level. Same-sex twin pairs from an epidemiological study were categorized according to parental report at 4 years of age into those with low language skills and a typically developing group. A total of 132 same-sex twin pairs from the low language group and 66 from the control group were assessed at 6 years of age, to investigate heritability of reading ability adjusted for nonverbal IQ. For pairs where both twins had normal scores on a nonword repetition test, heritability was zero, with environmental influences explaining all the variance. For pairs where one or both twins had low nonword repetition, the heritability estimate was 0.79 and the variance due to shared environment was zero. Future studies of genetics of reading development should treat those with poor nonword repetition skills as a separate subgroup.     

Role of the Bötzinger complex in fastigial nucleus-mediated respiratory responses

We have reported that the phrenic neurogram (PN) is modulated by stimulation of the fastigial nucleus (FN) of the cerebellum. The present study was undertaken to search for brainstem site(s) involved in the FN efferent pathway to modulate phrenic nerve activities. Experiments were performed on 35 anesthetized, paralyzed, and ventilated cats, using the PN as the index of the respiratory motor output. Results showed that bilateral electrolytic lesions of the red nucleus (RN), the paramedian reticular nucleus (PRN), or the pontine respiratory group (PRG) had little effect on the ability of FN stimulation to modulate the respiratory output. However, the modulation was abolished by bilateral electrolytic lesions of the B?tzinger complex (B?tC). Further studies showed that bilateral chemical inactivation of B?tC neurons produced by topical microinjection of kainic acid or cobalt chloride failed to abolish the modulation. We concluded that fibers of passage, not synapses or cell bodies in the B?tC, were involved in the modulatory effect of FN stimulation on the PN. The RN, PRN, and PRG appear not to be important in the neural circuitry responsible for the FN modulation of the phrenic activity.     

Premedical and medical school performance in predicting first-year residency performance

In the study reported here the authors examined the relationships among 40 measures of undergraduate college and medical school performance and competence in 18 medical care tasks during the first year of residency. A rating form was developed for the study to assess residents' competency in the medical care tasks and was sent to the directors of the residency programs entered by the graduates of a medical school. Stepwise multiple regression procedures were used to analyze the relationship between these ratings of residency performance and the residents' premedical and medical school performance and to identify the best predictors of residency performance for the 1982 and 1984 classes. A Rasch model analysis of the residency performance ratings indicated the ease or difficulty of each of the 18 tasks. The results provide information that would allow medical educators to use premedical and medical school performance to predict residents' competencies. The task of "clinically evaluates research and clinical data" was the most difficult for the graduates; that is, they were rated lower on it than on any other task. Two groups of measures of undergraduate and medical school performance were significantly related to performance in the residency: the Part II examination of the National Board of Medical Examiners (particularly the scores on the obstetrics-gynecology, medicine, surgery, and pediatrics subtests and the overall score) and the clerkships (particularly the third-year medicine clerkship, the fourth-year medicine clerkship, and the surgery clerkship).     

Evaluation of the 1990 Centers for Disease Control and Prevention smoke-free policy

PURPOSE. To determine the prevalence of tobacco use among Centers for Disease Control and Prevention (CDC)/Agency for Toxic Substances and Disease Registry (ATSDR) employees and the effect of the smoke-free policy on smoking behavior and air quality at work. DESIGN. A stratified telephone survey of 1181 CDC/ATSDR employees randomly selected from employee rosters. SETTING. CDC/ATSDR work sites in Atlanta, Georgia, and other major CDC locations throughout the United States and Puerto Rico. SUBJECTS. Randomly selected employees of CDC/ATSDR1, or about 22% of the total CDC/ATSDR population; 98% of eligible persons selected agreed to participate. MEASURES. Demographic and smoking history variables, attitudes toward and impact of the smoke-free policy on smoking behavior, and self-report changes in air quality were the measures used. RESULTS. Overall cigarette smoking prevalence was only 11.1%. One percent reported using chewing tobacco, 1.1% reported smoking a pipe, and 1.4% reported smoking cigars. Average self-reported, daily cigarette consumption significantly decreased after the smoking ban took effect. Overall, 90% of the employees supported the smoke-free policy, and 80% of the employees believed that smokers were complying with the smoke-free policy. Most employees believed that the air quality of work areas and nonwork areas (65% and 69%, respectively) had improved since the smoke-free policy was implemented. CONCLUSIONS. These findings are consistent with previous evaluations of smoke-free policies and suggest that most employees are generally supportive of workplace smoking restrictions. Such policies can also have a positive impact on smoking behavior and perceived air quality.     

Sinus of Valsalva aneurysm or fistula: management and outcome

BACKGROUND: Few large or long-term series exist regarding the management of patients with sinus of Valsalva aneurysms or fistulas (SVAFs). METHODS: Between 1956 and 1997, 129 patients presented with a ruptured (64 cases; 49.6%) or nonruptured (65 cases; 50.4%) SVAF. The patients included 88 men and 41 women, with a mean age of 39.1 years. Associated findings included a history of endocarditis (42 cases; 32.6%), a bicuspid aortic valve (21 cases; 16.3%), a ventricular septal defect (15 cases; 11.6%), and Marfan's syndrome (12 cases; 9.3%). Operative procedures included simple plication (61 cases; 47.3%), patch repair (52 cases; 40.3%), aortic root replacement (16 cases; 12.4%), and aortic valve replacement/repair (75 cases; 58.1%). RESULTS: There were five in-hospital deaths (3.9%): four due to preexisting sepsis and endocarditis and one that followed dehiscence of the repair in a patient with Marfan's syndrome. Two patients (1.6%) had strokes during the early postoperative period. The survivors were followed up for 661.1 patient-years (5.3 years/patient). The following late complications occurred: prosthetic valve malfunction (5 cases; 3.9%), prosthetic valve endocarditis (3 cases; 2.3%), SVAF recurrence (2 cases; 1.6%), thrombosis (1 case; 0.8%), and anticoagulation-related bleeding (1 case; 0.8%). CONCLUSIONS: Resection and repair of SVAF entails an acceptably low operative risk and yields long-term freedom from symptoms. Early, aggressive treatment is recommended to prevent endocarditis or lesional enlargement, which causes worse symptoms and necessitates more extensive repair.     

Molecular diagnosis of exocrine pancreatic cancer using a percutaneous technique

Background: The K-ras oncogene is activated by point mutations at codon 12 in most patients with exocrine pancreatic cancer. Mutant-enriched polymerase chain reaction (PCR) amplification can enhance the detection of mutated K-ras. This technique was applied to patients undergoing percutaneous fine-needle aspiration (FNA) biopsy of suspect pancreatic lesions. Methods: Twenty-five patients underwent percutaneous FNA of the pancreas for cytologic and molecular analysis. After preparing cytologic smears, the 22-gauge needle and syringe used for FNA were rinsed in RPMI-1640. The specimen was centrifuged, and DNA was extracted from the supernatant and subjected to mutant-enriched PCR using appropriate mismatched primers that introduce a BstNI restriction endonuclease cleavage site at codon 12 of wild-type, but not mutant, K-ras. After digestion with BstNI, the DNA was reamplified. To increase assay sensitivity, the final five PCR cycles were completed incorporating 5 μCi of (α-32P)dCTP. The DNA was then redigested and subjected to gel electrophoresis and autoradiography. Results: The median amount of DNA retrieved per specimen was 3.33 μg. Mutant K-ras was detected as a band of 143 bps; residual wild-type DNA was seen as a 114-bp fragment. Twenty-one of 25 specimens demonstrated mutated K-ras DNA. Two patients with nondiagnostic cytology results had mutated K-ras DNA; adenocarcinoma of pancreatic origin was confirmed in both cases after pancreatectomy. Conclusion: The molecular diagnosis of pancreatic cancer through identification of mutations in K-ras can be readily performed on specimens obtained by percutaneous FNA. As aggressive multimodality management of this disease becomes more common, pretreatment analysis of molecular determinants may have greater clinical significance. Presented at the 48th Cancer Symposium of The Society of Surgical Oncology, Boston, Massachusetts, March 23–26, 1995.