Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Validation of the Applied Biosystems Prism 377 automated sequencer for the forensic short tandem repeat analysis

The Applied Biosystems (ABI) Prism 377 DNA sequencer has been evaluated in an attempt to increase the throughput of samples for short tandem repeat (STR) analysis, in both forensic casework and the UK National Criminal Intelligence DNA Database. The gel system assessed consisted of 0.2 mm, 4% acrylamide 6 M urea gels, with a well-to-read distance of 36 cm. Gels were run at a constant voltage of 3 kV and constant temperature of 51 degrees C. The run time of our second generation multiplex (SGM) STR system was achieved in less than 2 h. Rigorous validation has been performed on the instrument hardware and software. Complete resolution of 1 base differences was obtained, up to and beyond 350 bases; sizing precision across gels was more than 2-fold higher than the 373A and the sensitivity was increased by one third.     

Phenotypic analysis of donor cells infiltrating the small intestinal epithelium and spleen during graft-versus-host disease

A pyriform sinus fistula can cause acute thyroiditis or recurrent infection in the neck. This fistula is believed to be a remnant of the branchial apparatus, although its origin has yet to be pinpointed. The spatial distribution of C cells in the thyroid gland was mapped by immunohistologic method in four patients with a pyriform sinus fistula. The C cells were identified immunohistologically with anticalcitonin antibody. The stained calcitonin-positive cells also crossreacted with the antibodies to carcinoembryonic antigen, chromogranin A, and neuron-specific enolase. The C cells were mainly distributed near the end of the fistula, and in three patients their concentration per unit volume of thyroid tissue was found to be inversely proportional to the distance from the end of the fistulas. Comparison of distant locations of the left-sided thyroid lobe in patients and the same region in control subjects showed a similar number of C cells. Thus this limited distribution of C cells suggested that the pyriform sinus fistula was either a remnant of the ultimobranchial body, the result of disturbed migration of the C cell in the fetus, or both.     

Using health status surveys in medical consultations

The goal of this research was to identify patients' preferences for physician inquiry into various aspects of health status and to examine whether the preconsultation availability of health status data (collected from the SF-36 Health Status Questionnaire) influenced the physician's conduct during the consultation. Results from 58 prenatal patient visits yielded the following findings. First, patients expressed strong preferences for physicians to ask about the patient's perceptions of health in general and about physical dimensions of health status such as pain, vitality, and role limitations due to physical functioning. Patients also were more satisfied when doctors were perceived as having asked about these issues. Second, patients varied considerably in their preferences for physician inquiries into psychosocial issues such as social functioning, mental health, and role limitations due to emotional problems. Approximately half the patients wanted these matters discussed, whereas the remainder either did not care or preferred that doctors not ask about these topics. Third, the preconsultation availability of health status information had little effect on the degree to which physicians asked about the patient's health-related quality of life. Clinical implications are discussed.     

Defining the positive tilt test: a study of healthy adults with moderate acute blood loss

STUDY OBJECTIVES: To define a set of orthostatic vital signs that minimize the frequency of false-positives among healthy individuals while maximizing sensitivity in detecting acute moderate blood loss and to determine the sensitivity and specificity of this optimized tilt test in detecting acute moderate blood loss. DESIGN AND INTERVENTION: Postural vital signs were recorded in a standardized manner before and after 450-mL phlebotomy. Paired comparisons were done for a variety of criteria for a positive tilt test using receiver-operating characteristic curves. SETTING AND TYPE OF PARTICIPANTS: Three hundred forty-five healthy euvolemic adult volunteer blood donors were tested at three community blood donation centers over a one-year period. Subjects were prospectively divided into group 1 (less than age 65; 301) and group 2 (age 65 or older; 44). MEASUREMENTS AND MAIN RESULTS: For each combination of pulse and blood pressure in group 1, a change in pulse alone had the same or higher sensitivity with at least the same specificity. Pulse alone was similarly superior in group 2 compared with previously published combinations of pulse and blood pressure. Even the optimized tilt test had limited sensitivity in detecting acute moderate blood loss with high specificity. CONCLUSION: In applying the tilt test to young adults without cardiovascular disease, pulse measurement usually is all that is necessary.     

The refinement of retinotectal projection on tectal whole mount during the regeneration of the goldfish optic nerve labeled with DiI anterogradely

Radiographic computed tomography and magnetic resonance imaging are the procedures of choice in the diagnosis of patients with suspected pituitary gland abnormalities. Several cases are presented where the diagnostic value of such procedures in patients with diseases affecting the pituitary gland is demonstrated.     

Effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections in young children in Brazil

A beneficial effect of periodic vitamin A supplementation on childhood mortality has been demonstrated, but the effect on morbidity is less clear. We investigated the effect of vitamin A supplementation on diarrhoea and acute lower-respiratory-tract infections (ALRI) in children from northeastern Brazil in a randomised, double-blind, placebo-controlled community trial. 1240 children aged 6-48 months were assigned vitamin A or placebo every 4 months for 1 year. They were followed up at home three times a week, and data about the occurrence and severity of diarrhoea and ALRI were collected. Any child with cough and respiratory rate above 40 breaths per min was visited by a paediatrician. The overall incidence of diarrhoea episodes was significantly lower in the vitamin-A-supplemented group than in the placebo group (18.42 vs 19.58 x 10(-3) child-days; rate ratio 0.94 [95% Cl 0.90-0.98]). The benefit of supplementation was greater as regards severe episodes of diarrhoea; the incidence was 20% lower in the vitamin A group than in the placebo group (rate ratio 0.80 [0.65-0.98]). With the standard definition of diarrhoea (> or = 3 liquid or semi-liquid stools in 24 h) the effect of vitamin A on mean daily prevalence did not reach significance, but as the definition of diarrhoea was made more stringent (increasing number of stools per day), a significant benefit became apparent, reaching for diarrhoea with 6 or more liquid or semi-liquid stools in 24 h a 23% lower prevalence. We found no effect of vitamin A supplementation on the incidence of ALRI. The reduction in severity of diarrhoea may be the most important factor in the lowering of mortality by vitamin A supplementation.     

Reactivity of trypsin in reverse micelles: pH-effects on the W0 versus enzyme activity profiles

We have adapted the new MxA gene-induction bioassay to measure neutralizing antibodies to interferon-beta1b (IFN-beta1b, the active ingredient in Betaseron) in sera from patients treated with Betaseron. This antibody assay has been validated to quantify neutralizing titers of 1:20 and above, with a precision of +/- 0.20 in log10. We have used this MxA gene-induction antibody assay to reinvestigate serum samples from multiple sclerosis (MS) patients treated with Betaseron. The titers measured were closely comparable to those obtained in antiviral assays. Data obtained by both methods show that neutralizing antibodies may appear and subsequently disappear over time in the sera of some patients treated with Betaseron. Sera from some patients contain binding antibodies to IFN-beta1b. It was shown that binding antibody titers do not correlate quantitatively or qualitatively with neutralizing antibody titers, and indeed, a number of patients develop high levels of binding antibodies but never form measurable levels of neutralizing antibodies.