Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations

A number of methods are used for mutational analysis of BRCA1, a large multi-exon gene. A comparison was made of five methods to detect mutations generating premature stop codons that are predicted to result in synthesis of a truncated protein in BRCA1. These included four DNA-based methods: two-dimensional gene scanning (TDGS), denaturing high performance liquid chromatography (DHPLC), enzymatic mutation detection (EMD), and single strand conformation polymorphism analysis (SSCP) and an RNA/DNA-based protein truncation test (PTT) with and without complementary 5' sequencing. DNA and RNA samples isolated from 21 coded lymphoblastoid cell line samples were tested. These specimens had previously been analyzed by direct automated DNA sequencing, considered to be the optimum method for mutation detection. The set of 21 cell lines included 14 samples with 13 unique frameshift or nonsense mutations, three samples with two unique splice site mutations, and four samples without deleterious mutations. The present study focused on the detection of protein-truncating mutations, those that have been reported most often to be disease-causing alterations that segregate with cancer in families. PTT with complementary 5' sequencing correctly identified all 15 deleterious mutations. Not surprisingly, the DNA-based techniques did not detect a deletion of exon 22. EMD and DHPLC identified all of the mutations with the exception of the exon 22 deletion. Two mutations were initially missed by TDGS, but could be detected after slight changes in the test design, and five truncating mutations were missed by SSCP. It will continue to be important to use complementary methods for mutational analysis.     

Optimum cycle frequencies in hand-rim wheelchair propulsion

Summary To study the effect of different cycle frequencies on cardio-respiratory responses and propulsion technique in hand-rim wheelchair propulsion, experienced wheelchair sportsmen (WS group; n=6) and non-wheel chair users (NW group; n=6) performed wheelchair exercise tests on a motor-driven treadmill. The WS group wheeled at velocities of 0.55, 0.83, 1.11 and 1.39 m · s^–1 and a slope of 2°. The NW group wheeled at 0.83, 1.11 and 1.39 m · s^–1 and a 1° slope. In each test, a 3-min period at a freely chosen cycle frequency (FCF: 100%) was followed by four 3-min blocks of paced cycle frequencies at 60%, 80%, 120% and 140% FCF. Effects of both cycle frequency and velocity on physiological and propulsion technique parameters were studied.Analysis of variance showed a significant effect (p<0.05) of cycle frequency on oxygen cost and gross mechanical efficiency in both the WS and NW group. This indicated the existence of an optimum cycle frequency which is close to the FCF at any given velocity. The optimum cycle frequency increased with velocity from 0.67 to 1.03 cps over the range studied (p< 0.05). Oxygen cost was 10% less at 100% FCF than at 60% or 140% FCF. Gross mechanical efficiency for the WS group at 100% FCF was 8.5%, 9.7%, 10.4% and 10.1%, respectively, at the four velocities. The similarity in the trend of oxygen cost and gross mechanical efficiency data in both the WS and NW groups suggests that an optimum cycle frequency is not merely a consequence of practice alone, but also reflects a physiologically determined optimum, dependent on muscle mechanics, e.g. velocity of contraction and power output of the muscles used.     

A real-time drawing study of melt-crystallized ultra-high molecular weight polyethylene. Comparison with conventional X-ray results

In the past morphological changes, caused by uni-axial drawing of flexible polymers have been studied mostly under conditions, quite different from the drawing conditions. This could give rise to certain artefacts, leading to mis-interpretations. Up to now very little was known about this possibility. Therefore, X-ray patterns obtained by conventional drawing studies and by real-time X-ray drawing studies are compared in this paper. It will be shown, that although some results on melt-crystallized polyethylene discussed here show indeed small differences, conventional X-ray studies can be used without any problem for qualitative studies. However, studies of deformation phenomena in elastic deformable regions as well as quantitative X-ray studies require real-time measurements.     

Vaginal carriage of enterotoxigenic Bacteroides fragilis in pregnant women.

Bacteroides fragilis is an anaerobic bacterial species that is involved in gynecological infections and pathology. The incidence of vaginal carriage is largely unknown, and in order to study this, 120 pregnant women attending a general hospital for delivery were examined. Cultures were positive for eight of these women (6.6%). Interestingly, potential clonal relatedness could be demonstrated among several of the nonenterotoxigenic B. fragilis strains. Among the strains, only one produced metalloprotease enterotoxin. The presence of the gene for the metalloprotease, giving rise to the pathogenic effect on cultured eukaryotic HT29/C1 cells, was confirmed by a newly designed specific PCR assay. The enterotoxigenic B. fragilis (ETBF) strain was analyzed with the help of arbitrarily primed PCR (AP-PCR) and PCR-mediated ribotyping. The ETBF strain was shown to be genetically different compared to several other strains obtained from diverse sources. Our data indicate a relatively high vaginal B. fragilis carriage rate among pregnant women in Warsaw, Poland. Although neither ETBF nor B. fragilis colonization presented a clinical problem, the possible genetic relatedness among the colonizing B. fragilis strains indicates the need for additional research in the field of ETBF transmission and molecular epidemiology.     

Clinical evaluation of pneumococcal meningitis in adults over a twelve-year period

A retrospective study was performed to review the clinical features and outcome of 39 episodes of pneumococcal meningitis in 36 adult patients over a 12-year period. Overall mortality was 33.3%. Only a few of the deaths were directly related to the central nervous system disease and most of them were due to cardiorespiratory failure. Univariate analysis showed that death was more likely to occur in patients with advanced age, an absence of neck stiffness, a high pulse rate, an associated pneumonia, internal complications, or a long duration of the disease (>7 days) before treatment was started. Patients who died had a higher erythrocyte sedimentation rate and serum bilirubin level and a lower serum sodium level than those who survived. Discriminant analysis showed the development of internal complications to be the strongest predictive factor of a poor outcome of illness. Two other important predictors of a poor outcome were the absence of neck stiffness and associated pneumonia. The history of a skull fracture or head surgery was significantly correlated with a better than average prognosis. The incidence of sequelae in survivors at the time of discharge amounted to 72%. None of the clinical features were significantly correlated with the development of sequelae, except a higher cerebrospinal fluid protein content.     

Chloroquine interaction with inflammatory human polymorphonuclear leucocytes

The molecularin vitro association of radiolabelled chloroquine (CQ) with both normal resting and inflammatory polymorphonuclear leucocytes (PMNs) was measured. For this purpose a suitable ligandassocation assay was developed to measure the cell association and the intracellular concentration of CQ. Under the influence of inflammatory stimuli PMNs display altered interaction with CQ. The intracellular concentration of CQ is reduced with 30 to 40% under inflammatory (disease) states when compared with non-inflammatory conditions. The mechanisms of CQ-PMN interaction associated with these altered intracellular concentrations of CQ are considered, with particular attention to the effects of rheumatic disease. Association experiments of CQ with PMNs performed in the presence of different established transport inhibitors showed that both diffusive uptake and carrier-mediated transport are involved in the cell accumulation of CQ in inflammatory PMNs. From these results, emphasis is given to three explanations for the decrease of the intracellular CQ concentration in inflamed PMNs:

1. the expansion of the PMN volume under inflammatory conditions;
2. the cytoplasmic or lysosomal pH changes and activation of the PMN Na⁺/H⁺ antiport by inflammatory stimuli; and
3. the exocytic release of the granules (degranulation).

Our data suggest that all these mechanisms, based on the events involved in inflammatory responses, may be involved in the decrease of the intracellular CQ concentration in inflammatory PMNs.     

Monitoring of antibiotic resistance in Shigellae isolated in the Netherlands 1984–1989

During surveillance of antimicrobial resistance inShigella strains isolated in the Netherlands from 1984 to 1989 and forwarded to the National Institute of Public Health and Environmental Protection for typing, sensitivity to twelve anti-microbial agents was assessed. High rates of resistance to the older drugs of choice in treating shigellosis were found, i.e. ampicillin and trimethoprim-sulfamethoxazole. Ampicillin resistance varied from 33 to 53% amongShigella flexneri strains and from 10 to 17% amongShigella sonnei strains. Trimethoprim and trimethoprim-sulfamethoxazole resistance increased from 8% to about 25% amongShigella flexneri and from 16 to 46% amongShigella sonnei isolates. All strains were susceptible to the newer quinolones, but five strains resistant to nalidixic acid showed decreased susceptibility to norfloxacin. Approximately 10% of the isolates were resistant to the combination of ampicillin, trimethoprim and sulfamethoxazole.     

Outcome of surgical treatment for early adenocarcinoma of the esophagus or gastro-esophageal junction

Adenocarcinoma of the esophagus, or GEJ, has a poor prognosis. Early lesions [i.e. high grade dysplasia (HGD) or T1-carcinoma] are potentially curable. Local endoscopic therapies are promising treatment options for superficial lesions; however, for deeper lesions, surgical resection is considered to be the treatment of choice. To contribute to therapeutic decision-making, we retrospectively analysed the outcome of transhiatal esophagectomy in 120 patients with pathologically proven HGD (n=13) or T1-adenocarcinoma (n=107) of the distal esophagus or gastro-esophageal junction (GEJ). Tumors were subdivided into six different depths of invasion (T1-mucosal m1-m3, T1-submucosal sm1-sm3), and the frequency of lymphatic dissemination and time to locoregional and/or distant recurrence were analysed. Only one of the 79 T1m1-3/sm1 tumors (1%) showed lymph node metastases as compared with 18 out of 41 T1sm2-3 tumors (44%). There was a significant difference in recurrence-free period between T1m1-m3/sm1 versus T1sm2-sm3 tumor patients (P log rank <0.0001), with 5-year recurrence-free percentages of 97% and 57%, respectively. In multivariate analysis including age, gender, tumor differentiation grade, N-stage and depth of invasion, only N-stage was an independent prognostic factor for recurrence-free period (hazard rate=5.9, 95% CI 1.7–20.7). However, if N-stage was excluded from analysis, only depth of invasion (T1sm2-3 versus T1m1-m3/sm1) was an independent prognostic factor for recurrence-free period (hazard rate=7.5, 95% CI 2.0–27.7). These data indicate that T1m1-m3/sm1 adenocarcinomas of esophagus or GEJ show a very low risk of lymphatic dissemination and are therefore eligible for local endoscopic therapy. After transhiatal surgical resection, almost half of the patients with T1sm2-sm3 lesions develop recurrent disease within 5 years, and therefore need additional therapy to improve survival.