Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae

Caspases (aspartate-specific cysteine proteases) play a critical role in the execution of the mammalian apoptotic program. To address the regulation of human caspase activation, we used the yeast Saccharomyces cerevisiae, which is devoid of endogenous caspases. The apical procaspases, -8beta and -10, were efficiently processed and activated in yeast. Although protease activity, per se, was insufficient to drive cell death, caspase-10 activity had little effect on cell viability, whereas expression of caspase-8beta was cytotoxic. This lethal phenotype was abrogated by co-expression of the pan-caspase inhibitor, baculovirus p35, and by mutation of the active site cysteine of procaspase-8beta. In contrast, autoactivation of the executioner caspase-3 and -6 zymogens was not detected. Procaspase-3 activation required co-expression of procaspase-8 or -10. Surprisingly, activation of procaspase-6 required proteolytic activities other than caspase-8, -10, or -3. Caspase-8beta or -10 activity was insufficient to catalyze the maturation of procaspase-6. Moreover, a constitutively active caspase-3, although cytotoxic in its own right, was unable to induce the processing of wild-type procaspase-6 and vice versa. These results distinguish sequential modes of activation for different caspases in vivo and establish a yeast model system to examine the regulation of caspase cascades. Moreover, the distinct terminal phenotypes induced by various caspases attest to differences in the cellular targets of these apoptotic proteases, which may be defined using this system.     

Detection of rubella virus-specific immunoglobulin G in saliva by an amplification-based enzyme-linked immunosorbent assay using monoclonal antibody to fluorescein isothiocyanate

An immunoglobulin G (IgG)-capture enzyme-linked immunosorbent assay (ELISA) for rubella virus is described. The assay uses a fluorescein isothiocyanate (FITC)-anti-FITC amplification system. The detection limit of the ELISA was approximately 7 IU of rubella virus-specific IgG per ml of serum sample. For saliva samples the performances of the capture ELISA and previously described radioimmunoassay were assessed, and the results of those two assays were compared to the rubella virus-specific IgG result obtained by a commercial ELISA (Behring Enzygnost) with a panel of paired serum and saliva samples. This comparison showed that the capture ELISA with saliva was more sensitive than the radioimmunoassay and that the results correlated better with the serum IgG result than the results of the radioimmunoassay did, with an overall sensitivity of 82% and a rank correlation of 0.68, whereas the sensitivity and rank correlation for the radioimmunoassay were 74% and 0.45, respectively. For subjects of 10 years of age or younger, the ELISA with saliva had a sensitivity of 94% and a specificity of 100% compared to the results of the ELISA (Behring Enzygnost) for rubella virus-specific IgG with corresponding serum samples. The sensitivity was much lower for subjects ages 17 years or older. The assay may have wider epidemiological use with saliva specimens, particularly those from children.     

Bedside activated partial thromboplastin time monitoring: just a matter of time?

Hydraulic Engineering, a junior∕senior-level course, is typically taught in a lecture-based format. Lecturing as a singular teaching technique has repeatedly been shown to be ineffective. Lecturing does not advance problem-solving skills, does not require creative or critical thinking, and does not prepare students for the types of problems they will face as professional engineers. In this study, two teaching techniques, problem-based learning (PBL) and cooperative learning (CL), were used to enhance learning in the hydraulic engineering course. The goals of PBL are to provide the student with an active role in learning and to allow the student to take responsibility for learning. The goals of CL are to have students work in teams, thereby learning from both each other and the instructor, and to teach students to work together cooperatively in small groups. Methods of developing teams, projects, and other assignments were explored. The course was assessed midterm and at the end of the semester. As a result, some changes were made midsemester and other recommendations are made for the future.     

Testis-like steroidogenesis in the ovotestis of the European mole, Talpa europaea

A gene (AglyA) encoding serine hydroxymethyltransferase of Acinetobacter radioresistens CMC-1 was cloned and sequenced. Nucleotide sequence analysis of AglyA predicted a single open reading frame (ORF) of 1251 bp encoding a 417-amino acid polypeptide. Two putative MetR-like binding sites (5'-TGAAACATGAGCT) and (5'-TGAGCAAAGTTCA), centered at bp -123 and -95 relative to the +1 translation start site were found, which have six out of nine and eight out of nine nucleotides that match to the consensus sequence of Escherichia coli (5'-TGAANNT/ANNTTCA), respectively. The enzyme also showed a high level of homology to other sources of serine hydroxymethyltransferase proteins.     

Structural model of the catalytic domain of an enzyme with cell adhesion activity: human vascular adhesion protein-1 (HVAP-1) D4 domain is an amine oxidase

Human vascular adhesion protein-1 (HVAP-1) is a multifunctional protein having at least two different cellular roles, functioning both as a lymphocyte-endothelial cell adhesion protein and as an enzyme with monoamine oxidase activity. HVAP-1 is a 180 kDa homodimeric glycoprotein consisting of a membrane-spanning domain and three predicted extracellular copper-containing amine oxidase domains. In HVAP-1 the extracellular domains are composed of a large domain D4, containing the active site and forming the interface of the dimer, while the smaller D2 and D3 domains surround the D4 dimer near the entrance to the active site. The structural model of the catalytic D4 domain of HVAP-1 reveals that all components necessary for enzymatic monoamine oxidase activity are indeed present within the HVAP-1 and pinpoints residues that may be key to substrate entry through a channel to the active site and residues likely to be involved in substrate specificity as well as structural features critical to dimer formation. Proper glycosylation is required for the cell adhesion function of HVAP- 1 and the predicted location of the sugar units at the solvent-exposed surface suits this function well.      

Role of the C-terminal extension in the interaction of S100A1 with GFAP, tubulin, the S100A1- and S100B-inhibitory peptide, TRTK-12, and a peptide derived from p53, and the S100A1 inhibitory effect on GFAP polymerization

Controversy over the efficacy of many topical wound treatments, particularly growth factors, is common, with many clinical practitioners still confused as to the real value of these agents. A serious lack of knowledge appears to exist concerning the diffusion and distribution of topically applied solutes in wounds. Without this basic understanding there seems little chance of accurately predicting the therapeutic window of drugs targeted at cellular activities, such as division and chemotaxis, and processes, such as collagen lattice deposition and contraction, occurring below the surface of the granulating layer. This study was designed to determine the absorption and distribution of a number of radiolabeled solutes (water, sodium chloride, lidocaine) and growth factors (basic fibroblast growth factor, epidermal growth factor) applied topically to full-thickness excisional wounds in rats during the early (2 d), mid (7 d), and late (12 d) stages of repair. Results showed that water and sodium penetrated deepest into wound sites and that changes in water distribution and retention in the wound paralleled the healing process. Multiple stepwise regression showed that molecular weight and tissue depth, but not day of healing, were significant factors in predicting the concentration of each solute in wound and underlying tissue sites. This finding was consistent with a tissue diffusion model developed in this study. Basic fibroblast growth factor and epidermal growth factor only penetrated slightly into the upper granulating layers of the wound site, and calculation of therapeutic doses, based on the percentage of applied solute reaching the deeper granulating layers, is presented.     

Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR

Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.     

Nosocomial infection following cardiovascular surgery: comparison of two periods, 1987 vs. 1992

BACKGROUND: QT dispersion has been proposed as a simple, noninvasive measure for identifying patients at risk of postinfarction arrhythmia. It is assumed to reflect nonuniform ventricular repolarization, which, in turn, may result from regional differences in repolarization time as well as from localized activation delay. The aim of this study was to examine the relation between QT dispersion and intraventricular conduction abnormalities in patients with acute anterior wall myocardial infarction. METHODS AND RESULTS: Standard 12-lead electrocardiographic and 12-lead signal-averaged electrocardiographic recordings were performed in 25 patients with a first Q-wave anterior wall myocardial infarction. Measures calculated by using the 6 precordial (V1 through V6) leads for QT dispersion were (1) difference between maximum and minimum QT and QTc intervals and (2) standard deviation of QT and QTc intervals. Measures calculated from the signal-averaged electrocardiogram were (1) maximum filtered QRS duration; (2) mean; and (3) standard deviation of filtered QRS duration. No relation was found between any measure of filtered QRS duration and that of QT dispersion by using linear correlation analysis. Similarly, no significant association was demonstrated between the filtered QRS duration and corresponding QT interval measurements (total 131 leads). CONCLUSIONS: The lack of correlation between signal-averaged electrocardiogram indexes of slow intraventricular conduction and electrocardiogram variables of QT dispersion suggests an independent predictive value for the 2 methods in identifying patients at risk of postinfarction arrhythmia. This suggestion is further supported by the finding that altered activation sequence is an unlikely mechanism of QT dispersion in patients with acute myocardial infarction, as indicated by the lack of association between the filtered QRS duration and corresponding QT interval measurements.     

The mutagenicity of benzo[a]pyrene in mouse small intestine

OBJECTIVE: To determine the effects of controlled ovarian hyperstimulation (COH) on endometrial maturation. DESIGN: Prospective, before and after evaluation of midluteal endometrial biopsies in oocyte donor's spontaneous and subsequent COH cycles. SETTING: Tertiary academic medical center assisted reproductive technologies clinic. PATIENT(S): Nineteen oocyte donors. INTERVENTION(S): Exogenous gonadotropins, endometrial biopsies. MAIN OUTCOME MEASURE(S): Endometrial histology and an immunohistochemical marker of uterine receptivity, the alphavbeta3 vitronectin. RESULT(S): Glandular and stromal dyssynchrony was more common after COH in 16 (80%) of 20 cycles than 6 (30%) of 20 spontaneous cycles (P <.05). Glandular lag was more frequent in COH cycles and unaffected by progesterone administration. The beta3 subunit of the alphavbeta3 vitronectin receptor was present in 9 (45%) of 20 spontaneous and 2 (10%) of 20 COH cycles (P <.05). CONCLUSION(S): Exogenous gonadotropin use in healthy reproductive age women did not result in endometrial evidence of a luteal phase defect. A greater incidence of glandular-stromal dyssynchrony resulted from the use of exogenous gonadotropins. The presence of alphavbeta3 was noted in most endometrial specimens demonstrating in phase glandular maturation. We conclude that endometrial dyssynchrony that results from delayed glandular development most likely represents a normal histologic variant.