Overexpression of cardiotrophin-1 and gp130 during experimental acute Chagasic cardiomyopathy

The purpose of this study was to examine how antigen retrieval affected the yield of immunogold labeling on epoxy sections based on embedding with different amounts of accelerator. The concentration of accelerator DMP-30 (tri(dimethyl amino methyl) phenol) was varied in the range of 0-8% in the processing of the tissue for epoxy embedding. Immunogold labeling was performed on epoxy sections and LR-White sections of fibrin clots and renal tissue with IgG-deposits, and the antibodies used were anti-fibrinogen anti-IgG and, respectively. For some of the sections antigen retrieval was performed by heating the sections in citrate buffer. In all cases, the yield of immunogold labeling increased following antigen retrieval. The increase (%) in the yield of immunogold labeling as a result of antigen retrieval was larger for epoxy sections than for LR-White sections. The immunolabeling on high-accelerator epoxy sections exposed to antigen retrieval was about 20% more intense than on untreated LR-White sections both for IgG and fibrinogen. In addition to breaking fixations bonds introduced by the chemical fixation, we believe that the antigen retrieval also breaks bonds between the epoxy resin and the embedded tissue. The combination of increased amount of accelerator during tissue processing for epoxy embedding and antigen retrieval by heating in citrate buffer is a potent method for increasing specific immunolabeling on epoxy sections.     

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects

Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such 'bypass Sec14p' mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in 'bypass Sec14p' mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for 'bypass Sec14p', and yeast operating under 'bypass Sec14p' conditions are ethanol-sensitive. These data suggest that PLD supports 'bypass Sec14p' by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.     

Thrombospondin-1 expression in human myometrium before and during pregnancy, before and during labor, and in human myometrial cells in culture

The activation of latent transforming growth factor beta (L-TGFbeta) is essential for the action of TGFbeta, which, in turn, is involved in the regulation of expression of some progesterone-responsive genes. One mechanism by which TGFbeta is activated involves thrombospondin (TSP), a protein that binds extracellular proteins. Immunoreactive TSP (irTSP) protein and TSP-1 mRNA in myometrial tissues of ovulatory and pregnant women were localized by immunohistochemistry and in situ hybridization. IrTSP and TSP-1 mRNA were randomly distributed in myometrial smooth muscle cells of some, but not all, tissues of pregnant women at term before labor; but in some areas of most of these tissues, irTSP was intense and commonly localized extracellularly. Intense irTSP and TSP-1 mRNA in myocytes were more common in myometrium during labor. In myometrium from ovulatory women (n = 26), irTSP was localized primarily in vascular smooth muscle cells and was detected occasionally in scattered myocytes. Little TSP-1 mRNA was demonstrable by in situ hybridization in vessels or myocytes of myometrial tissue from ovulatory women (n = 7). By Northern analysis of total RNA, TSP-1 mRNA was detected in myometrial tissue of pregnant women and in human myometrial smooth muscle cells in culture. The levels of TSP-1 mRNA in myometrial tissues of pregnant women during labor (n = 18) were greater than those in myometrium at > 37 wk gestation before labor began (n = 25, p < 0.001). The ratios of TSP-1 to glyceraldehyde 3-phosphate dehydrogenase mRNAs in 3 myometrial tissues during oxytocin-induced labor were not statistically different from those in myometrium during spontaneous labor but were greater than those in myometrium before labor (p < 0.05). The level of TSP-1 mRNA in confluent human myometrial cells in culture was relatively high, was increased by treatment with fetal bovine serum, and was decreased by treatment with platelet-derived growth factor or activators of adenylyl cyclase or protein kinase C. Myometrial cells in culture constitute a useful model for studying the regulation of TSP-1 gene expression in human myometrium.     

Factors affecting serum selenium and vitamin E concentrations in dairy cows

Supplementation of selenium and vitamin E to enhance disease resistance in dairy cattle has become common, particularly to prevent periparturient reproductive disorders and mastitis. To establish reference values for serum vitamin E and selenium concentrations in postparturient dairy cattle and to determine whether serum concentrations of these micronutrients varied with season and stage of lactation, cows from a stratified random sample of 50 herds were studied for 1 year. Blood samples were collected from each of the 50 study herds twice, from the 10 most recently parturient cows or from 10% of the herd, whichever was greatest. Mean concentration of vitamin E and selenium was 2.55 micrograms/ml and 78.12 ng/ml, respectively. Vitamin E concentrations were significantly (P < 0.05) higher during the summer and fall than during the winter and spring. Selenium concentrations were significantly (P < 0.05) lower during the summer and fall than during the winter and spring. Herd, season of blood sample collection, and time since parturition were significant (P < 0.02) in explaining variation in vitamin E and selenium concentrations.     

Mechanical response properties of nociceptors innervating feline hairy skin

1. The responses of feline cutaneous nociceptors were examined in vivo by systematically manipulating the intensive and spatial dimensions of mechanical stimulation. A computer-controlled motor was used to apply prescribed forces (5-90 g) to a nociceptor's receptive field, with flat-tipped, cylindrical probes of various sizes (contact areas: 0.1-5.0 mm2). The stimulating device and protocols were similar to those previously used to evaluate human perception, thus allowing for comparisons of the two data sets. 2. With a ramp-and-hold stimulus of controlled force, most nociceptors showed a slowly adapting (SA) response throughout the stimulus. In this way, nociceptors resembled low-threshold SA mechanoreceptors. However, in contrast to SA mechanoreceptors, nociceptors failed to exhibit an onset burst of activity associated with the stimulus ramp. Nineteen percent (6 of 31) of the nociceptors often showed the opposite trend during the stimulus, e.g., a gradually increasing firing rate. Most of these nociceptors (5 of 6) had particularly high mechanical thresholds. 3. With 30 stimuli repeated at short intervals (6-8 s), response rates tended to decrease across trials. This phenomenon was most evident with more intense stimuli. When two series of stimuli were separated by 4-5 min, there was no apparent trend of reduced responsiveness between series. 4. Overall, nociceptors responded in an orderly way to variations in force and probe size. For a given probe size, larger forces produced greater responses; for a given force, smaller probes produced greater responses. The relationship between probe size and force was best described as an even tradeoff between force and a linear dimension of the probe (i.e., probe perimeter), rather than the area of the probe. Thus a given pressure (force/area) did not evoke the same response from nociceptors as probe size was varied. 5. There were two significant differences in the mechanical responsiveness between A fiber and C fiber nociceptors. First, for a given set of stimuli, A fiber nociceptors exhibited a greater response rate than the C fiber nociceptors. Second, the A fiber nociceptors exhibited a greater differential response related to probe size than the C fiber nociceptors. On the basis of these two features, the A fiber nociceptors' response profiles showed a closer parallel with previously reported human pain thresholds than the C fiber nociceptors did. 6. When the nociceptors were subdivided as to their mechanical threshold, those with lower thresholds [mechanically sensitive afferents (MSAs)] showed a response saturation with the more intense stimuli. On average, the stimulus levels at which saturation occurred were close to human pain threshold. Those nociceptors with higher thresholds [mechanically insensitive afferents (MIAs)] did not show such saturation. Thus only the MIAs appeared to have the capacity to unambiguously encode mechanical stimulus intensities above pain threshold. The MSAs, on the other hand, exhibited their greatest dynamic response range near the threshold for nonpainful sharpness. Thus the group of afferents commonly defined as nociceptors exhibit a heterogeneity of mechanical response properties, which may serve functionally different roles for perception.     

A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize

We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported [Upton, C. and Buckley, J. T. (1995) Trends Biol. Sci. 20, 178-9] and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize. A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated. There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E. coli, the gene product was lipolytic. Arab-1 and genes for some of the other plant proteins appear to be differentially expressed. They may play a role in the regulation of lipid metabolism during plant development.     

Analytical performance of the Tandem-R free PSA immunoassay measuring free prostate-specific antigen

The analytical performance of the Tandem-R free PSA assay available from Hybritech Inc. was evaluated. Comparison of recoveries of purified free (unbound) prostate-specific antigen (PSA) diluted in female serum in the Tandem-R free PSA assay and the Tandem-R (total) PSA assay demonstrated a link in calibration between the assays and an accurate determination of percent free PSA. The cross-reactivity of the assay to purified PSA-alpha 1-antichymotrypsin was determined to be < 1%. The minimum-detectable concentration was < 0.05 microgram/L. The within-run and between-day CVs were < or = 5% for samples with > 0.3 microgram/L free PSA. Dilution and recovery showed no significant deviations from linearity across the assay range. The assay was insensitive to interference from blood components. The Tandem-R free PSA kit was shown to be an accurate, precise, and reliable assay for the measurement of free PSA.     

Syncope in children and adolescents

OBJECTIVES: The objectives of this study were to 1) define the incidence of syncope coming to medical attention among children and adolescents, 2) determine the outcome of syncope in these patients, and 3) determine changes over time in the evaluation and charges for evaluating this problem. BACKGROUND: Syncope occurs commonly in children and adolescents. However, the mid- and long-term outcome of children and adolescents who experience syncope is unknown. METHODS: Utilizing the Rochester Epidemiology Project, we determined the incidence, outcome and charges for medical evaluation for patients seeking medical attention for syncope during an early 5-year period (1950 to 1954) and a more recent 5-year period (1987 to 1991). RESULTS: The incidence of syncope coming to medical attention was 71.9 and 125.8/100,000 population for the early and more recent cohort, respectively. The incidence was higher for female than for male patients. The incidence peaked in 15- to 19-year old patients. Acute illness and noxious stimuli were associated with 24% and 23% of the episodes, respectively. Although long-term survival was not different from that of the general population, one child died suddenly, and another had hereditary prolonged QT interval syndrome. These were two of only six patients who had exertional syncope. Total charges for evaluation of syncope were similar in the two time periods. However, charges for testing procedures were greater for the more recent cohort. CONCLUSIONS: In general, syncope in children and adolescents is a benign event. Syncope occurring during exercise may identify patients with a potentially fatal condition. Detailed evaluation should be considered for patients who have syncope during exercise or who have a family history of syncope, sudden death, myocardial disease or arrhythmias. It may be prudent to obtain an electrocardiogram for all patients who seek medical attention for syncope.