Requirement of cysteine-rich repeats of the Fas receptor for binding by the Fas ligand

The Fas receptor is a member of a family of cell death receptors, including tumor necrosis factor receptor I (TNFR I), death receptor 3 and 4 (DR3 and DR4), and cytopathic avian receptor 1 (CAR1). The Fas receptor is composed of several discrete domains, including three cysteine-rich domains (CRDs), a transmembrane domain, and an intracellular domain responsible for transmitting an apoptotic signal. While the mechanism of Fas-mediated cell death has become elucidated, the requirements for Fas ligand binding to the receptor have not been fully defined. Using a series of chimeric Fc-receptor fusion proteins between the human Fas receptor and TNFR I, each cysteine-rich domain of Fas was found to be required for interaction with the Fas ligand. Interestingly, TNFR I CRD1 could partially substitute for the Fas CRD1. The importance of this domain was underscored by the analysis of a Fas extracellular mutation (C66R), which resulted in a complete loss of ligand binding. This mutation was cloned from a human patient suffering from Canale-Smith syndrome, which is characterized by autoimmunity resembling that observed in the lpr and lprcg mice. The localization of essential ligand binding domains in the Fas receptor correlated exactly with the ability of the Fas receptor fusion proteins to prevent cell death mediated by the Fas ligand.     

Continued development of the Nimbus/University of Pittsburgh (UOP) axial flow left ventricular assist system

Nimbus and the University of Pittsburgh (UOP) have continued the development of a totally implanted axial flow blood pump under the National Institutes of Health (NIH) Innovative Ventricular Assist System (IVAS) program. This 62 cc device has an overall length of 84 mm and an outer diameter of 34.5 mm. The inner diameter of the blood pump is 12 mm. It is being designed to be a totally implanted permanent device. A key achievement during the past year was the completion of the Model 2 pump design. Ten of these pumps have been fabricated and are being used to conduct in vitro and in vivo experiments to evaluate the performance of different materials and hydraulic components. Efforts for optimizing the closed loop speed control have continued using mathematical modeling, computer simulations, and in vitro and in vivo testing. New hydraulic blade designs have been tested using computational fluid dynamics (CFD) and flow visualization. A second generation motor was designed with improved efficiency. To support the new motor, a new motor controller fabricated as a surface mount PC board has been completed. The program is now operating under a formal QA system.     

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca2+-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension-Ca2+ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca2+ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca2+-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca2+ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.     

Accelerated inflammatory bowel disease of TCR-alpha-deficient mice persistently infected with Cryptosporidium parvum

TCR-alpha-deficient mice spontaneously develop inflammatory bowel disease (IBD) at 8-9 mo old. This study characterizes an accelerated form of IBD induced by Cryptosporidium parvum infection. Cryptosporidium parvum-infected TCR-alpha-deficient mice developed IBD as early as 4 wk old when challenged at 1 wk old. The lesions of this accelerated IBD resembled the lesions of spontaneous IBD in TCR-alpha-deficient mice and consisted of a mononuclear cell infiltrate within the intestinal lamina propria and an increased proliferation of enterocytes. The mononuclear cells within the lamina propria consisted of B cells and gamma delta T cells. The distal ileum, cecum, and colon were grossly thickened due to a hyperplastic mucosa and edematous submucosa. The mechanism by which C. parvum infection accelerates development of IBD is presently unclear.     

New ribosome-inactivating proteins with polynucleotide:adenosine glycosidase and antiviral activities from Basella rubra L. and bougainvillea spectabilis Willd

New single-chain (type 1) ribosome-inactivating proteins (RIPs) were isolated from the seeds of Basella rubra L. (two proteins) and from the leaves of Bougainvillea spectabilis Willd. (one protein). These RIPs inhibit protein synthesis both in a cell-free system, with an IC50 (concentration causing 50% inhibition) in the 10(-10) M range, and by various cell lines, with IC50S in the 10(-8)-10(-6) M range. All three RIPs released adenine not only from rat liver ribosomes but also from Escherichia coli rRNA, polyadenylic acid, herring sperm DNA, and artichoke mottled crinkle virus (AMCV) genomic RNA, thus being polynucleotide:adenosine glycosidases. The proteins from Basella rubra had toxicity to mice similar to that of most type 1 RIPs (Barbieri et al., 1993, Biochim Biophys Acta 1154: 237-282) with an LD50 (concentration that is 50% lethal) < or = 8 mg.kg-1 body weight, whilst the RIP from Bougainvillea spectabilis had an LD50 > 32 mg.kg-1. The N-terminal sequence of the two RIPs from Basella rubra had 80-93% identity, whereas it differed from the sequence of the RIP from Bougainvillea spectabilis. When tested with antibodies against various RIPs, the RIPs from Basella gave some cross-reactivity with sera against dianthin 32, and weak cross-reactivity with momordin I and momorcochin-S, whilst the RIP from Bougainvillea did not cross-react with any antiserum tested. An RIP from Basella rubra and one from Bougainvillea spectabilis were tested for antiviral activity, and both inhibited infection of Nicotiana benthamiana by AMCV.     

The puncture and catheterization of the peripheral vessels using ultrasonic scanning

1% acetylcholine (5.5 10 M--acetylcholine chloride) applied directly to the cortex of conscious non-immobilized rabbits was established to produce neuroglial reaction in motor area. Following 30 min long application the number of astrocytes was significantly increased. Application lasting for 2 hrs resulted in a reverse effect. The above-mentioned changes were observed in surface and deep cortical layers. They were functional and could be attributed to the cell tinctorial properties.     

Anatomic basis of continuous axillary block of the brachial plexus with thoracic reservoir

Although continuous axillary block is effective for periods of up to four to six weeks, pain clinic patients with severe chronic pain in the upper extremities, mainly resulting from neuropathic disease, can require continuous drug delivery by catheter-reservoir for up to three months or longer. We studied possible locations for implanting reservoirs or catheters from the perivascular axillary space in fresh cadavers, checking for possible vascular or nerve damage after applying the usual technique for reservoir implantation. The ideal location for the reservoir is the subcutaneous tissue of the homolateral infraclavicular space of the anterior side of the thorax. The customary procedures for inserting the catheter along the subcutaneous route that extends from the perivascular axillary space to the reservoir carry no risk of damaging vascular or nerve structures.     

Detection of the local H+ gradients on the internal mitochondrial membrane

Respiration-dependent responses of a pH probe (fluorescein isothiocyanate, FITC), covalently bound to the membrane proteins of mitochondria and submitochondrial particles (SMP) have been studied. A spectral shift indicating FITC deprotonation was observed when respiration was activated in coupled mitochondria. Such a response was increased by valinomycin and reduced by uncoupler. Some FITC deprotonation was detected in the presence of excess of an uncoupler, but the response was smaller and insensitive to valinomycin. FITC deprotonation was also observed in submitochondrial particles after succinate addition. In this case it was not affected by uncoupler. Increase in the buffer concentration was found to (i) decrease the FITC response and (ii) increase the rate of uncoupled respiration in both mitochondria and submitochondrial particles. The results are consistent with the assumption that respiration initiates appearance of local H+ activity gradients on the inner side of the internal mitochondrial membrane during the steady-state H+ pumping. We suggest that the formation of this gradient is due to kinetic barrier to proton transfer from the bulk phase to the respiratory proton pump vicinity.     

Noncanonical conformation of nucleic acids: structure with slipped-loops, occurring in DNA oligonucleotides

A HPLC method was developed and validated for the quantitation of 9-cis-retinoic acid (ALRT1057) and its major metabolite, 4-oxo-9-cis-retinoic acid (LG100182) in human plasma. Samples were buffered and extracted with methyl-tert-butyl-ether. The analytes and an I.S. were separated on a C18 HPLC column using a shallow gradient of 70-89% organic solvent. The analytes were quantitated by UV detection at 348 nm. Selectivity against endogenous compounds and potential metabolites (retinol, all trans-, 13-cis-, and 4-hydroxy-9-cis-retinoic acid) was demonstrated. The run time was 29 min. The standard curve was linear from 2.5 to 450 ng ml-1. Interassay precision for both analytes in quality control samples was less than 5.0% RSD. Accuracy was within 11.0% RE for both compounds. Analyte stability during sample storage, extraction processing, and chromatography was established. Method ruggedness was tested by two analysts and on two HPLC systems. This method has been applied to the quantitation of clinical samples.     

Vaginal-sigmoid fistula caused by diverticular disease

A case of a 66 years old patient suffering from a sigmoido-vaginal fistula and diverticulosis, previously treated with a total laparohysterectomy, is reported. Retrograde studies demonstrated the presence of fistula, whereas colonoscopy and barium enema failed. After laparotomy and an appropriate bowel preparation, surgery was restricted to the bowel resection and anastomosis, whereas the vaginal defect was not closed.