Two distinct GUCY2C circuits with PMV (hypothalamic) and SN/VTA (midbrain) origin.

Guanylyl cyclase C (GUCY2C) is the afferent central receptor in the gut-brain endocrine axis regulated by the anorexigenic intestinal hormone uroguanylin. GUCY2C mRNA and protein are produced in the hypothalamus, a major center regulating appetite and metabolic homeostasis. Further, GUCY2C mRNA and protein are expressed in the ventral midbrain, a principal structure regulating hedonic reward from behaviors including eating. While GUCY2C is expressed in hypothalamus and midbrain, its precise neuroanatomical organization and relationship with circuits regulating satiety remain unknown. Here, we reveal that hypothalamic GUCY2C mRNA is confined to the ventral premammillary nucleus (PMV), while in midbrain it is produced by neurons in the ventral tegmental area (VTA) and substantia nigra (SN). GUCY2C in the PMV is produced by 46% of neurons expressing anorexigenic leptin receptors, while in the VTA/SN it is produced in most tyrosine hydroxylase-immunoreactive neurons. In contrast to mRNA, GUCY2C protein is widely distributed throughout the brain in canonical sites of PMV and VTA/SN axonal projections. Selective stereotaxic ablation of PMV or VTA/SN neurons eliminated GUCY2C only in their respective canonical projection sites. Conversely, specific anterograde tracer analyses of PMV or VTA/SN neurons confirmed distinct GUCY2C-immunoreactive axons projecting to those canonical locations. Together, these findings reveal two discrete neuronal circuits expressing GUCY2C originating in the PMV in the hypothalamus and in the VTA/SN in midbrain, which separately project to other sites throughout the brain. They suggest a structural basis for a role for the GUCY2C-uroguanylin gut-brain endocrine axis in regulating homeostatic and behavioral components contributing to satiety.

Progression of alpha-synuclein pathology in multiple system atrophy of the cerebellar type.

AIMS: The aim of this study was to identify early foci of α-synuclein (α-syn pathology) accumulation, subsequent progression and neurodegeneration in multiple system atrophy of the cerebellar type (MSA-C). METHODS: We analysed 70-µm-thick sections of 10 cases with MSA-C and 24 normal controls. RESULTS: MSA-C cases with the lowest burden of pathology showed α-syn glial cytoplasmic inclusions (GCIs) in the cerebellum as well as in medullary and pontine cerebellar projections. Cerebellar pathology was highly selective and severely involved subcortical white matter, whereas deep white matter and granular layer were only mildly affected and the molecular layer was spared. Loss of Purkinje cells increased with disease duration and was associated with neuronal and axonal abnormalities. Neocortex, basal ganglia and spinal cord became consecutively involved with the increasing burden of α-syn pathology, followed by hippocampus, amygdala, and, finally, the visual cortex. GCIs were associated with myelinated axons, and the severity of GCIs correlated with demyelination. CONCLUSIONS: Our findings indicate that cerebellar subcortical white matter and cerebellar brainstem projections are likely the earliest foci of α-syn pathology in MSA-C, followed by involvement of more widespread regions of the central nervous system and neurodegeneration with disease progression.

Comparison of manual versus automated data collection method for an evidence-based nursing practice study.

OBJECTIVE: The objective of this study was to investigate and improve the use of automated data collection procedures for nursing research and quality assurance. METHODS: A descriptive, correlational study analyzed 44 orthopedic surgical patients who were part of an evidence-based practice (EBP) project examining post-operative oxygen therapy at a Midwestern hospital. The automation work attempted to replicate a manually-collected data set from the EBP project. RESULTS: Automation was successful in replicating data collection for study data elements that were available in the clinical data repository. The automation procedures identified 32 "false negative" patients who met the inclusion criteria described in the EBP project but were not selected during the manual data collection. Automating data collection for certain data elements, such as oxygen saturation, proved challenging because of workflow and practice variations and the reliance on disparate sources for data abstraction. Automation also revealed instances of human error including computational and transcription errors as well as incomplete selection of eligible patients. CONCLUSION: Automated data collection for analysis of nursing-specific phenomenon is potentially superior to manual data collection methods. Creation of automated reports and analysis may require initial up-front investment with collaboration between clinicians, researchers and information technology specialists who can manage the ambiguities and challenges of research and quality assurance work in healthcare.

Serial modules in parallel: the psychological refractory period and perfect time-sharing.

The authors describe ACT-R/perceptual-motor (ACT-R/PM), an integrated theory of cognition, perception, and action that consists of the ACT-R production system and a set of perceptual-motor modules. Each module (including cognition) is essentially serial, but modules run in parallel with one another. ACT-R/PM can model simple dual tasks such as the psychological refractory period (PRP), including subtle results previously explained with executive process interactive control (EPIC, D. E. Meyer & D. E. Kieras, 1997a). The central difference between the theories is that EPIC's productions can fire in parallel, whereas in ACT-R/PM, they are serial. Results from three PRP-like experiments with more demanding cognitive requirements indicate that cognitive processing for the 2 tasks need not overlap. ACT-R's activation-based retrieval processes are critical in accounting for the timing of these tasks and for explaining the dual-task performance decrement.

Taking a computational approach to aging: the SPAN theory of working memory.

The decline of working memory capacity associated with normal adult aging is well-known. What is less well established is the cause of this decline. One prominent proposal is that working memory decline is caused by a reduction in basic information-processing speed, but this account has lacked a demonstration that general slowing is computationally sufficient to produce a decrease in working memory capacity. This article presents a production system theory of working memory (SPAN) based on established mechanisms: slowing, decay, and displacement. Models of 2 tasks--digit symbol and computation span--which have been prominent in research on slowing, are presented in detail. These models demonstrate that slowing is sufficient to produce differences in these tasks, and they provide a quantitative match to observed young-old differences as well. This advance for slowing theory also demonstrates the viability of computational tools in aging research.

Multiple-phase equilibration headspace analysis for the determination of N2O and N2 during bacterial denitrification.

A gas-handling manifold for the preparation, introduction and analysis by gas chromatography (GC) system of the gaseous products of denitrification is described. A procedure of multiple-phase equilibration is adopted which allows the quantitative determination of the total gas present in sample vials. Assumptions of solubility coefficients are not required as these are determined during the analysis. The method is particularly suited to gases of appreciable solubilities as a significant proportion of the gas will be found in the liquid phase. This method was used for the determination of the stoichiometry of denitrification, in washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, namely NO2-:N2 and N2O:N2, which were found to be 2:1 and 1:1, respectively.

Properties of monoclonal antibodies to the genus-specific antigen of Chlamydia and their use for antigen detection by reverse passive haemagglutination.

The chlamydial genus-specific antigen was extracted with phenol/chloroform/petroleum ether (PCP) from preparations of Chlamydia trachomatis and C. psittaci, and quantities measured using an assay for lipopolysaccharide (LPS). The LPS from C. trachomatis contained 2.2% (w/w) of ketodeoxyoctanoic acid. Five IgG monoclonal antibodies reacted in an ELISA with LPS from both species, the antigen being periodate-sensitive and heat-resistant, confirming that all antibodies were against the genus-specific antigen. All the antibodies bound to the PCP extract of C. trachomatis on an immunoblot, at a position corresponding to the periodate-Schiff-stained bands of both C. trachomatis extract and Salmonella Re-LPS. When linked to trypsin-treated sheep erthrocytes and used in reverse passive haemagglutination tests, all antibodies gave indicator cells capable of detecting chlamydial LPS or crude preparations of chlamydiae grown in McCoy cells, the sensitivity varying with the antibody used. The antibodies varied in IgG subclass (either IgG2a or IgG3), and in ability to precipitate in immunodiffusion tests. Two antibodies cross-reacted with one strain of Acinetobacter in ELISA and with Salmonella Re-LPS in both ELISA and immunodiffusion tests. The other three did not react in ELISA with Acinetobacter strains or Salmonella Re-LPS, and none of the five reacted with LPS of E. coli or Pseudomonas morsprunorum.

Demonstration of shared antigens in the genus Clostridium by an enzyme-linked immunosorbent assay.

Antigens prepared from several strains of each of 10 Clostridium species were used in an indirect enzyme-linked immunosorbent assay with antisera raised against whole cells of a representative strain from each of the 10 species killed by ultra-violet irradiation. With the exception of C. cadaveris, the antisera gave similar results with antigens prepared from all strains of the homologous species. Antigens prepared from 13 other clostridial species were then investigated in an ELISA system with the 10 representative antisera. The results showed many cross-reactions, particularly in the C. perfringens group, the C. difficile/sordelli group and the C. botulinum/novyi/sporogenes group.

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli.

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.

Clostridium difficile in association with sporadic diarrhoea.

A total of 154 patients admitted to an infectious diseases unit were included in a year's prospective survey of sporadic diarrhoeal disease. Stools from 19 of them yielded Clostridium difficile, generally on more than one occasion. Twelve of these patients were assessed as having a severe or moderately severe gastrointestinal illness: Cl difficile was the only pathogen isolated from 10 of them, and two had an associated salmonella infection. Seven had had a recent course of antibiotics, but five had not taken antibiotics. Faeces from seven patients with moderate or mild gastrointestinal illness yielded Cl difficile, and two of these patients also had an associated salmonella infection. Two patients in this group had no antibiotic history. From these findings, the occurrence of C difficile in faeces could not be described as antibiotic-associated. Faecal Cl difficile cytotoxin was detected in only six patients, and generally at low levels. In such patients a more relevant pathogenic index might take account of the numbers of Cl difficile present and of their toxigenic potential.

Differential processing of the two subunits of human choriogonadotropin (hCG) by granulosa cells. I. Preparation and characterization of selectively labeled hCG.

The alpha- and beta-subunits of hCG were radioiodinated and recombined with unlabeled complementary subunits. The resultant recombined hormones, selectively labeled in either the alpha- or beta-subunit, were separated from unrecombined subunit by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, extracted with Triton X-100, and characterized by binding analysis. The estimates of maximum binding (active fraction) of the two resultant selectively labeled, recombined hCG preparations, determined with excess receptor were 0.41 and 0.59. These values are similar to those obtained when hCG is labeled as an intact molecule. The specific activities of the recombined preparations were estimated by four different methods, and the resulting values were used in combination with the active fraction estimates to determine the concentrations of active free and bound hormone. Binding analyses were run using varying concentrations of both labeled and unlabeled hormone. Estimates of the equilibrium dissociation binding constant (Kd) and receptor capacity were calculated in three different ways. The mean estimates of capacity (52.6 and 52.7 fmol/mg tissue) and Kd (66.6 and 65.7 pM) for the two preparations were indistinguishable. Additionally, these values were similar to values reported previously for hCG radioiodinated as an intact molecule. The availability of well characterized, selectively labeled hCG preparations provides new tools for studying the mechanism of action and the target cell processing of the subunits of this hormone.

Differential processing of the two subunits of human choriogonadotropin by granulosa cells. II. In vivo studies.

Using a well characterized preparation of hCG, consisting of a mixture of hCG labeled in the alpha-subunit with 125I and hCG labeled in the beta-subunit with 131I (see preceding paper), the hormone-specific preferential retention by granulosa cells in vivo of the radiolabel originally associated with the beta-subunit of hCG has been confirmed and extended. Additional studies have shown that this retention is peculiar to the granulosa cells. Luteal and interstitial/thecal elements of the ovary failed to show preferential accumulation of the label originally associated with the beta-subunit. Measurement of both radioactivities in crude subfractions of the ovarian tissues revealed that granulosa cells retain the excess of beta-subunit label in a plasma membrane/vesicular component. No such preferential retention of label was seen in any of the subfractions obtained from luteal or interstitial/thecal tissues. The radiolabeled components associated with the granulosa cells were shown to be mainly macromolecular by their precipitability with 13% trichloroacetic acid. Luteal tissue degraded the components associated with each label more rapidly than granulosa cells. In contrast, interstitial/thecal tissue degraded very little of the bound labeled components. The differential processing of individual hCG subunits by granulosa cells was shown not to result from different kinetics of binding of serum-borne hormone by two methods. Thus, changes over time in the ability of circulating hormone to bind to LH receptor in vitro were shown not to be a function of the hCG subunit having the label. Moreover, blockade of further radiolabel uptake by injection of a large excess of unlabeled hCG 30 min after radiolabel administration did not alter the rise in the ratio of beta-subunit label to alpha-subunit label normally observed in granulosa cells. The ability of kidney tissue to accumulate and metabolize hCG also varied with the physiological state. Within the limitations of following the radioiodides added to proteins rather than the peptides themselves, these studies demonstrate that differences exist in the metabolism of hCG by the various target cells of the ovary and that changes in processing occur during luteinization.

Detection of Clostridium difficile toxin by counterimmunoelectrophoresis: a note of caution.

Recent methods for detection of Clostridium difficile toxin by counterimmunoelectrophoresis might lead to errors. False-positives may be attributable to soluble cell surface antigens reacting with impure antitoxin.

Immunological analysis of the EDTA-soluble antigens of Clostridium difficile and related species.

Antigens were extracted with EDTA from 32 strains representing 10 species of Clostridium. When these antigens were examined by an enzyme-linked immunosorbent assay, marked cross-reactions were observed between C. difficile, C. sordellii and C. bifermentans. The cross-reactive antigen, visualized by crossed immunoelectrophoresis, was carbohydrate.