Performance of the TechLab C. DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C. difficile Tox A/B II EIA kit, the Triage C. difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea

We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C. DIFF CHEK-60 (TL-GDH), in combination with the C. difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C. difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C. difficile. All C. difficile isolates were tested for the presence of the toxin genes by PCR. If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive. Eighty-seven of 93 and 79 of 93 C. difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively. No test was able to detect toxin in all samples with true-positive results. Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results. Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C. difficile-associated diarrhea, respectively, if used alone.     

Age-related changes in adrenergic neuronal function of rabbit vascular smooth muscle

The adrenergic control of vascular smooth muscle was compared in young and adult rabbits using a variety of in vitro techniques. Norepinephrine (NE) content and accumulation of ³H-NE were not different in blood vessels from the two age groups. In contrast, stimulation-evoked release of endogenous NE was reduced by 40–60% in vessels from the aged animals. Functional studies of smooth muscle contractions were carried out using isolated ring segments of the ear artery. There were no differences in the resting force-response relationship between vessels from young and adult rabbits. Maximum contractile responses to nerve stimulation, NE or KCl were not different in vessels from the two age groups, nor was the NE ED₅₀. However, blockade of the neuronal uptake system with desmethylimipramine produced a greater shift in the NE concentration-response curve in vessels from the young animals compared to the shift in vessels from adult animals. This observation reflects a decline in neuronal NE uptake with age. Although maximal contractile responses to transmural nerve stimulation at 16 Hz were unchanged, responses to stimulation at lower frequencies were reduced in vessels from adult rabbits, an effect which was also enhanced when an antagonist of neuronal uptake was present. Thus, there is a decline in function of adrenergic nerves in adult animals, reflected in a decrease in stimulation-evoked NE release and a decrease in norepinephrine uptake revealed by functional studies. These two effects tend to balance each other, so that there is a small decrease in contractile response to adrenergic nerve stimulation which is exacerbated when neuronal uptake mechanisms are blocked.     

Defects in regulation of apoptosis in caspase-2-deficient mice

During embryonic development, a large number of cells die naturally to shape the new organism. Members of the caspase family of proteases are essential intracellular death effectors. Herein, we generated caspase-2-deficient mice to evaluate the requirement for this enzyme in various paradigms of apoptosis. Excess numbers of germ cells were endowed in ovaries of mutant mice and the oocytes were found to be resistant to cell death following exposure to chemotherapeutic drugs. Apoptosis mediated by granzyme B and perforin was defective in caspase-2-deficient B lymphoblasts. In contrast, cell death of motor neurons during development was accelerated in caspase-2-deficient mice. In addition, caspase-2-deficient sympathetic neurons underwent apoptosis more effectively than wild-type neurons when deprived of NGF. Thus, caspase-2 acts both as a positive and negative cell death effector, depending upon cell lineage and stage of development.     

Reduced cell proliferation in fetal lung after maternal administration of pilocarpine. A scintillation autoradiographic study

Fetuses were obtained on the 28th gestational day from pregnant New Zealand white rabbits treated daily, on the 24th through the 27th gestational day, with pilocarpine HCl, 5 mg/kg in saline, or saline alone. Lung fragments from these fetuses were incubated for two hours in medium containing ³H-thymidine. Scintillation autoradiography of 1-μm-thick sections of these fetal lungs revealed that the lung tissue from pilocarpine-treated fetuses had significantly lower labelled cell indices for both alveolar epithelial cells and interstitial cells. These results indicate that pilocarpine treatment promotes differentiation of immature cells in the fetal lung at the expense of cell proliferation.     

Elevated abeta42 in skeletal muscle of Alzheimer disease patients suggests peripheral alterations of AbetaPP metabolism

The levels of amyloid-beta40 (Abeta40) and Abeta42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Abeta42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Abeta40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Abeta40 and muscle Abeta42 levels were correlated with each other (P < 0.001), 2) muscle Abeta40 levels were positively correlated with age (P = 0. 036), and 3) muscle Abeta42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Abeta peptide were discovered by mass spectrometry, revealing the presence of Abeta starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Abeta and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Abeta in the temporalis muscles of AD patients suggest that alterations in AbetaPP and Abeta metabolism may be manifested in peripheral tissues.     

Controlled elimination of intracellular H(2)O(2): regulation of peroxiredoxin, catalase, and glutathione peroxidase via post-translational modification

The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.     

Sequence and chromosomal localization of two PET genes required for cytochrome c oxidase assembly in Saccharomyces cerevisiae

Summary The nuclear genes PET117 and PET191 are required for the assembly of active cytochrome c oxidase in S. cerevisiae, yet their gene products are not subunits of the final assembled cytochrome c oxidase complex. Plasmids bearing PET117 or PET191 were isolated by their ability to complement the pet117-1 or pet191-1 mutations, respectively. By restriction mapping, subcloning, and deletion analysis of yeast DNA fragments that complement these mutations, the PET117 and PET191 genes were localized to smaller regions of DNA, which were then sequenced from both strands. The PET117 open reading frame is of 107 codons and the PET191 open reading frame is of 108 codons. Neither the PET191 nor PET117 DNA sequences have been reported previously, and the derived amino-acid sequences of the PET191 and PET117 open reading frames exhibit no significant primary amino-acid sequence similarity to other protein sequences available in the NBRF data base, or from translated Genbank sequences. By hybridization of PET117 or PET191 probes first to a chromosome blot and next to a library of physically mapped fragments of yeast genomic DNA, the map locations of the PET191 and PET117 genes were determined. PET117 is located on chromosome V near the HIS1 gene and PET191 is located on chromosome X near the CYC1 gene.     

A function of Fas-associated death domain protein in cell cycle progression localized to a single amino acid at its C-terminal region

FADD is an adaptor known to transmit apoptotic signals from members of the tumor necrosis factor receptor family. We show here that FADD has a domain implicated in cell proliferation. Mice bearing the Asp mutation in the serine 191 phosphorylation site are runted and anemic and display splenomegaly. Apoptosis is unimpaired in these mice, but they exhibit many immune developmental problems indicative of proliferative defects. Mutant FADD T cells are defective in cell cycle progression, suggesting that regulation of phosphorylation at serine 191 is essential for growth/proliferation. Remarkably, serine 191 is conserved among mammalian FADD proteins, but this C-terminal region is absent in lower organisms, suggesting that FADD acquired a domain during evolution, rendering it a "proliferation-apoptosis coupler" that balances cell proliferation and apoptosis.     

A simple breathing circuit to maintain isocapnia during measurements of the hypoxic ventilatory response

We report the development and testing of a simple breathing circuit that maintains isocapnia in human subjects during hypoxic hyperpnea. In addition, the circuit permits rapid switching between two gas mixtures with different partial pressures of oxygen. Eleven volunteers breathed repeated cycles of exposure to air (2 min of 21% O(2), balance N(2)) and hypoxia (2 min of 8.3+/-0.1% O(2), balance N(2)). Hypoxia induced significant increases in minute ventilation, breathing frequency and tidal volume (P < 0.05) that were consistent over repeated cycles of hypoxia (P > 0.1, one-way ANOVA). The system successfully maintained isocapnia in all subjects, with an average change in end-tidal CO(2) of only -0.2 mmHg during hyperventilation in hypoxia (range 0.4 to -0.8 mmHg). This system may be suitable for repeated tests of the hypoxic ventilatory response (HVR) and may prove useful for exploring intra- and inter-individual variability of HVR in humans.     

Factors influencing levels of research activity in clinical psychologists: a new model

There are concerns in the USA and UK about low levels of research activity amongst clinical psychologists emerging from doctoral level training. The authors explore existing evidence and theory pertaining to this phenomenon. Three relevant theoretical strands are identified, along with related empirical studies and commentaries in practitioner journals. These different sources are integrated to suggest a more complete model of factors influencing clinical psychologists' research intentions, and in turn research activity itself: vocational preferences, research training experienced during qualifying training, practice context, value placed on expected outcomes of doing research, perceived norms for clinical psychologists in relation to doing research, research self‐efficacy, professional identity and, most tentatively, sex role identity. Our new model points to training interventions, either pre‐ or post‐qualification, that may increase the likelihood of clinical psychologists carrying out clinical research that will contribute to their profession's knowledge base, beyond their time in qualifying training. Copyright © 2006 John Wiley & Sons, Ltd.