Identification of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs

At fertilization, sea urchin eggs undergo a series of activation events, including a Ca2+ action potential, Ca2+ release from the endoplasmic reticulum, an increase in intracellular pH, sperm pronuclear formation, MAP kinase dephosphorylation, and DNA synthesis. To examine which of these events might be initiated by activation of phospholipase Cgamma (PLCgamma), which produces the second messengers inositol trisphosphate (IP3) and diacylglycerol, we used recombinant SH2 domains of PLCgamma as specific inhibitors. Sea urchin eggs were co-injected with a GST fusion protein composed of the two tandem SH2 domains of bovine PLCgamma and (1) Ca2+ green dextran to monitor intracellular free Ca2+, (2) BCECF dextran to monitor intracellular pH, (3) Oregon Green dUTP to monitor DNA synthesis, or (4) fluorescein 70-kDa dextran to monitor nuclear envelope formation. Microinjection of the tandem SH2 domains of PLCgamma produced a concentration-dependent inhibition of Ca2+ release and also inhibited cortical granule exocytosis, cytoplasmic alkalinization, MAP kinase dephosphorylation, DNA synthesis, and cleavage after fertilization. However, the Ca2+ action potential, sperm entry, and sperm pronuclear formation were not prevented by injection of the PLCgammaSH2 domain protein. Microinjection of a control protein, the tandem SH2 domains of the phosphatase SHP2, had no effect on Ca2+ release, cortical granule exocytosis, DNA synthesis, or cleavage. Specificity of the inhibitory action of the PLCgammaSH2 domains was further indicated by the finding that microinjection of PLCgammaSH2 domains that had been point mutated at a critical arginine did not inhibit Ca release at fertilization. Additionally, Ca2+ release in response to microinjection of IP3, cholera toxin, cADP ribose, or cGMP was not inhibited by the PLCgammaSH2 fusion protein. These results indicate that PLCgamma plays a key role in several fertilization events in sea urchin eggs, including Ca2+ release and DNA synthesis, but that the action potential, sperm entry, and male pronuclear formation can occur in the absence of PLCgamma activation or Ca2+ increase.     

Comparison of screening methods in the detection of bladder cancer

PURPOSE: We prospectively evaluate and compare the sensitivity and specificity of urine cytology, BTA stat, NMP22, fibrin/fibrinogen degradation products (FDP), telomerase, chemiluminescent hemoglobin and hemoglobin dipstick to detect bladder cancer. MATERIALS AND METHODS: Single voided specimens were obtained from 57 patients with bladder cancer, and 139 without evidence of bladder malignancy on cystoscopy or a negative biopsy of indeterminate lesions. A cytology report was available for 125 patients and interpreted independently. BTA stat, NMP22 and FDP were analyzed according to manufacturer specifications. The telomerase assay was performed on cells collected from urine by centrifugation in preparation for polymerase chain reaction based amplification using the telomeric repeat amplification protocol assay. The chemiluminescent screening assay for hemoglobin in urine uses the pseudoperoxidase activity of hemoglobin on hydrogen peroxide and subsequent oxidation of 7-dimethylaminonaphthalene-1,2-dicarbonic acid hydrazide to generate chemiluminescence emission. Hemoglobin dipstick was interpreted as positive if the hemoglobin content in the urine was trace or greater. RESULTS: Overall sensitivity with urine cytology, BTA stat, NMP22, FDP, telomerase, chemiluminescent hemoglobin and the hemoglobin dipstick was 44, 74, 53, 52, 70, 67 and 47%, respectively. Specificity with cytology, telomerase and FDP was high (95, 99 and 91%, respectively) but BTA stat, NMP22 (optimized), chemiluminescent hemoglobin (optimized) and the hemoglobin dipstick demonstrated lower specificity of 73, 60, 63 and 84%, respectively. Stepwise logistic regression analysis revealed that for all tumors, and within each tumor grade and stage telomerase had the strongest association with bladder cancer among all tests (69% overall concordance). Telomerase was also positive in 91% of the patients (10 of 11) with carcinoma in situ. CONCLUSIONS: Urinary telomerase had the highest combination of sensitivity and specificity (70 and 99%, respectively) for bladder cancer screening in these patients. It was the strongest predictor with superior accuracy in patients with grade 1 and noninvasive tumors (pTa), and extremely useful in patients with carcinoma in situ. Telomerase appears to be promising and outperformed cytology, BTA stat, NMP22, FDP, chemiluminescent hemoglobin and hemoglobin dipstick in the prediction of bladder cancer.     

Direct alloreactivity by human cytotoxic T lymphocytes can Be inhibited by altered peptide ligand antagonism

Alloreactive T lymphocytes that respond directly to foreign major histocompatibility complex (MHC) molecules and bound peptide are known to be central mediators of graft-versus-host disease (GVHD) and allograft rejection. We have recently identified a peptide from the human protein, cytochrome P450 (isotypes IIC9, 10, or 18), that is recognized in association with human leukocyte antigen (HLA) B*3501 by alloreactive cytotoxic T lymphocytes (CTLs). These CTLs with this specificity were isolated from several unrelated individuals and were found to express a common T-cell receptor (TCR). Synthetic analogs of the cytochrome P450 peptide were generated by introducing single amino acid substitutions at putative TCR contact positions. Four altered peptide ligands were powerful competitive antagonists of these CTL clones, reducing lysis levels of target cells expressing the alloantigen HLA B*3501 by over 80%. This first demonstration that it is possible to suppress CTL alloreactivity with structural variants of allodeterminants raises the prospect that such TCR antagonists could be exploited within the clinical arena to specifically modulate GVHD and allograft rejection.     

Usefulness of cholangioscopy in patients with focal stricture of the intrahepatic duct unrelated to intrahepatic stones

BACKGROUND: Intrahepatic duct strictures are usually caused by intrahepatic duct stones and cholangitis. However, focal strictures of the intrahepatic duct unrelated to intrahepatic stones often pose diagnostic problems. This study was undertaken to prospectively evaluate the usefulness of percutaneous transhepatic cholangioscopy in patients with focal intrahepatic duct stricture and no evidence of a stone. METHODS: Seventeen patients with focal strictures of the intrahepatic duct without any evidence of a stone were included. Percutaneous transhepatic cholangioscopic examination including procurement of biopsy specimens was performed after percutaneous transhepatic biliary drainage. RESULTS: A histopathologic diagnosis was obtained in all patients (9 adenocarcinomas, 1 squamous cell carcinoma, 2 hepatocellular carcinomas, 2 adenomas, and 3 benign strictures). Of the 9 patients with bile duct adenocarcinoma, 8 underwent surgery and a curative resection was possible in 7 patients (88%). Five patients (63%) had early-stage bile duct cancer in which cancer invasion was limited to the mucosa or fibromuscular layer and there was no evidence of lymph node metastasis. CONCLUSIONS: Percutaneous transhepatic cholangioscopy in patients with focal stricture of the intrahepatic duct unrelated to choledocholithiasis is useful for diagnosis including the detection of early bile duct cancer.     

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.