Role of active site tyrosine residues in catalysis by human glutathione reductase

Tyr114 and Tyr197 are highly conserved residues in the active site of human glutathione reductase, Tyr114 in the glutathione disulfide (GSSG) binding site and Tyr197 in the NADPH site. Mutation of either residue has profound effects on catalysis. Y197S and Y114L have 17% and 14% the activity of the wild-type enzyme, respectively. Mutation of Tyr197, in the NADPH site, leads to a decrease in Km for GSSG, and mutation of Tyr114, in the GSSG site, leads to a decrease in Km for NADPH. This behavior is predicted for enzymes operating by a ping-pong mechanism where both half-reactions partially limit turnover. Titration of the wild-type enzyme or Y114L with NADPH proceeds in two phases, Eox to EH2 and EH2 to EH2-NADPH. In contrast, Y197S reacts monophasically, showing that excess NADPH fails to enhance the absorbance of the thiolate-FAD charge-transfer complex, the predominant EH2 form of glutathione reductase. The reductive half-reactions of the wild-type enzyme and of Y114L are similar; FAD reduction is fast (approximately 500 s-1 at 4 degreesC) and thiolate-FAD charge-transfer complex formation has a rate of 100 s-1. In Y197S, these rates are only 78 and 5 s-1, respectively. The oxidative half-reaction, the rate of reoxidation of EH2 by GSSG, of the wild-type enzyme is approximately 4-fold faster than that of Y114L. These results are consistent with Tyr197 serving as a gate in the binding of NADPH, and they indicate that Tyr114 assists the acid catalyst His467'.     

Periodontal considerations in an overdenture population

Three variable microsatellite loci have been isolated from the American lobster, Homarus americanus. In a population sample from the Gulf of Maine, the effective numbers of alleles (Ne) for the two most variable loci were 16.33 and 13.19, respectively. Reduced variability at all three loci was seen in the European lobster, H. gammarus, for which the maximum Ne was 4.00. The reduction in variability in H. gammarus is consistent with a bottleneck event. Inheritance analysis using H. americanus demonstrated segregation of codominant alleles and the absence of linkage. Null alleles were observed at two loci in inheritance studies. This study demonstrates that microsatellite loci should be useful in studying the population structure of clawed lobsters.     

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.