Linkage exclusion and mutational analysis of the noggin gene in patients with fibrodysplasia ossificans progressiva (FOP)

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare and disabling genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic endochondral ossification in predictable anatomical patterns. Although elevated levels of bone morphogenetic protein 4 (BMP4) occur in lymphoblastoid cells and in lesional cells of patients with FOP, mutations have not been identified in the BMP4 gene, suggesting that the mutation in FOP may reside in a BMP4-interacting factor or in another component of the BMP4 pathway. A powerful antagonist of BMP4 is the secreted polypeptide noggin. A recent case report described a heterozygous 42-bp deletion in the protein-coding region of the noggin gene in a patient with FOP. In order to determine if noggin mutations are a widespread finding in FOP, we examined 31 families with 1 or more FOP patients. Linkage analysis with an array of highly polymorphic microsatellite markers closely linked to the noggin gene was performed in four classically-affected multigenerational FOP families and excluded linkage of the noggin locus to FOP (the multipoint lod score was -2 or less throughout the entire range of markers). We sequenced the noggin gene in affected members of all four families, as well as in 18 patients with sporadic FOP, and failed to detect any mutations. Single-strand conformation polymorphism (SSCP) analysis of 4 of these patients plus an additional 9 patients also failed to reveal any mutations. Among the samples analyzed by SSCP and DNA sequencing was an independently obtained DNA sample from the identical FOP patient previously described with the 42-bp noggin deletion; no mutation was detected. Examination of the DNA sequences of 20 cloned noggin PCR products, undertaken to evaluate the possibility of a somatic mutation in the noggin gene which could be carried by a small subset of white blood cells, also failed to detect the presence of the reported 42-bp deletion. We conclude that mutations in the coding region of noggin are not associated with FOP.     

Filamentous phage morphogenetic signal sequence and orientation of DNA in the virion and gene-V protein complex

The circular single-stranded viral DNA (ssDNA) of filamentous phage is oriented in the virus with a hairpin forming sequence called mos at the leading end of the virion-the end that emerges from the cell first. In the experiments that originally defined mos it was also found to enhance virion production 100-fold, though in other circumstances it has no such effect. Using a new electron-microscopic method, we have ascertained the orientation of ssDNA in phage having mutations involving mos and other viral functions, and also in the intracellular precursor to the virion-a rod-shaped complex between the ssDNA and the phage-encoded gene-V protein, pV. The results show (1) that the ssDNA is oriented in the complex as in the virion, with mos at one end; (2) that orientation is maintained even if assembly is not mediated by the complex; (3) that orientation is manifested in polyphage--abnormal particles in which many unit-length ssDNA molecules are sheathed in a single, extremely long capsid; (4) that orientation is imposed by mos itself (or something very nearby) since it disappears in a mos deletion; and (5) that a 229-base segment including the minus strand of mos is also effective at imposing orientation. On the basis of our findings, we speculate that mos determines orientation in two stages, one imposing an axis on the ssDNA loop during formation of the pV/ssDNA complex, the other imposing a direction on the loop during initiation of particle assembly. The sequence requirements for the two stages may be different.     

Clinical evaluation of three urine screening tests

Evaluations of screening tests for bacteriuria have traditionally compared the test results with those of quantitative urine cultures. However, many patients with symptomatic urinary tract infections can have less than 10(5) CFU/ml in their urine. Therefore, the results of urine culture and three screening tests (Bac-T-Screen, Chemstrip LN [which tests for leukocyte esterase and nitrate reductase], and Gram stain) were correlated with the clinical classification of urinary tract infection. The Bac-T-Screen test detected 98, 93, and 100% of the infections classified as probable, possible, and asymptomatic, respectively. In contrast, the Gram stain, leukocyte esterase, and nitrate reductase tests were all insensitive screening tests for infection. Additionally, only 45% of the patients with probable infections had greater than or equal to 10(5) CFU/ml. Thus, the majority of infected patients would not have been detected if quantitative urine cultures were used alone.     

Sensory and autonomic innervation of the rat eyelid: Neuronal origins and peptide phenotypes

Neuronal origins, peptide phenotypes and target distributions were determined for sensory and autonomic nerves projecting to the eyelid. The retrograde tracer, Fluoro-Ruby, was injected into the superior tarsal muscle and meibomian gland of Sprague-Dawley rats. Labelled neurons were observed within the pterygopalatine (31 ± 6 of a total of 8238 ± 1610 ganglion neurons), trigeminal (173 ± 43 of 62 082 ± 5869) and superior cervical ganglia (184 ± 35 of 21 900 ± 1741). Immunostaining revealed vasoactive intestinal polypeptide immunoreactivity (VIP-ir) in nearly all Fluoro-Ruby-labelled pterygopalatine ganglion neurons (86 ± 5%) but only rarely in trigeminal (0.3 ± 0.3%) or superior cervical (1.4 ± 1.4%) ganglion neurons. Calcitonin gene-related peptide (CGRP)-ir was not observed in pterygopalatine or superior cervical ganglion somata, but was present in 24 ± 4% of trigeminal neurons. Bright dopamine β-hydroxylase (DBH) immunofluorescence was observed in the majority of eyelid-projecting neurons within the superior cervical ganglia (65 ± 5%) and lighter staining was detected in pterygopalatine neurons (63 ± 3%), but no DBH-ir was observed in trigeminal neurons. Examination of eyelid sections revealed dense VIP-ir innervation of meibomian gland acini and vasculature and modest distribution within tarsal muscle. CGRP-ir fibers surrounded ductal and vascular elements of the meibomian gland and the perimeter of tarsal muscle. DBH-ir fibers were associated with meibomian gland blood vessels and acini, and were more densely distributed within tarsal muscle. This study provides evidence for prominent meibomian gland innervation by parasympathetic pterygopalatine ganglion VIP-ir neurons, with more restricted innervation by sensory trigeminal CGRP-ir and sympathetic neurons. Tarsal muscle receives abundant sympathetic innervation, as well as moderate parasympathetic and sensory CGRP-ir projections. The eyelid contains substantial non-CGRP-ir sensory innervation, the targets of which remain undetermined. The distribution of identified autonomic and sensory fibers is consistent with the idea that meibomian gland function, as well as that of the tarsal muscle, is regulated by peripheral innervation.     

Methods for obtaining and analyzing unattended polysomnography data for a multicenter study. Sleep Heart Health Research Group

This paper reviews the data collection, processing, and analysis approaches developed to obtain comprehensive unattended polysomnographic data for the Sleep Heart Health Study, a multicenter study of the cardiovascular consequences of sleep-disordered breathing. Protocols were developed and implemented to standardize in-home data collection procedures and to perform centralized sleep scoring. Of 7027 studies performed on 6697 participants, 5534 studies were determined to be technically acceptable (failure rate 5.3%). Quality grades varied over time, reflecting the influences of variable technician experience, and equipment aging and modifications. Eighty-seven percent of studies were judged to be of "good" quality or better, and 75% were judged to be of sufficient quality to provide reliable sleep staging and arousal data. Poor submental EMG (electromyogram) accounted for the largest proportion of poor signal grades (9% of studies had <2 hours artifact free EMG signal). These data suggest that with rigorous training and clear protocols for data collection and processing, good-quality multichannel polysomnography data can be obtained for a majority of unattended studies performed in a research setting. Data most susceptible to poor signal quality are sleep staging and arousal data that require clear EEG (electroencephalograph) and EMG signals.     

Comparison of agar-based media for primary isolation of glycopeptide-resistant enterococci

Objective: To compare four vancomycin-containing agar media for the isolation of glycopeptide-resistant enterococci (GRE) from clinical fecal specimens: kanamycin-aesculin-azide (KAA) agar; bile-aesculin-polymixin (BAP) agar; aztreonam-amphotericin blood (CBAA) agar; and neomycin blood (CBN) agar.
Methods: Fecal specimens from 125 patients were inoculated onto each medium. Media were examined for enterococci after incubation for up to 48 h. Enterococci were identified to species level, and glycopeptide phenotypes were determined by measuring minimum inhibitory concentrations of vancomycin and teicoplanin.
Results: GRE were isolated from 44/125 samples. Enterococcus faecalis and Enterococcus faecium isolates, expressing glycopeptide resistance of the VanA or VanB phenotypes, were recovered from 27/33 (82%) specimens on BAP medium, 26/33 (79%) on KAA medium, and 21/33 (64%) on CBN and CBAA media. Enterococcus gallinarum and Enterococcus casseliflavus isolates expressing low-level glycopeptide resistance (VanC phenotype) were recovered from 14/15 (93%) specimens on CBAA medium, 7/15 (47%) on KAA and CBN media, and 6/15 (40%) on BAP medium.
Conclusions: The media tested in this study, with the exception of CBN medium, detected at least 75% of patients colonized by GRE. Further development of BAP, CBAA and KAA media is warranted to improve growth and selectivity.     

Surgery-attached psychogeriatric nurses: an evaluation of psychiatric nurses in the primary care team

Aspects of the work of five community psychiatric nurses are examined by means of a questionnaire completed by the nurses for a sample of 50 patients. Information sharing and a close working relationship with the general practitioner, skills relating to assessment and psychological support of patients, organization of care by other agencies, a detailed knowledge of community agencies, and a capacity for interdisciplinary working were all found to be important. Tasks relating to body care were reported in only a small number of cases. The nurses saw themselves as advising on the planning of patient management and the selection of medication in a substantial minority of cases. The original brief had been to care for psychogeriatric patients but a third of the patients fell into a younger age group. These findings are discussed in connection with their implications for the future training of nurses and the relationship between primary care teams and local psychiatric services.