Comparative distribution of VIP in the central nervous system of various species measured by a new radioimmunoassay

Vasoactive intestinal polypeptide (VIP) occurs in high concentrations throughout the gut and the nervous system. The presence of VIP has been shown in a number of species, mainly by immunohistochemistry. The aim of the present study was to develop a new, highly specific VIP radioimmunoassay to investigate the distribution of VIP in the central nervous system of various vertebrate and invertebrate species. Different areas of the brain and spinal cord were removed from rats, chickens, turtles, frogs and fishes. The cerebral ganglia and the ventral ganglionic chain were investigated in the earthworm. The tissue samples were processed for VIP radioimmunoassay. Our results show that the antiserum used in the radioimmunoassay turned to be C-terminal specific, without significant affinity to other members of the VIP peptide family. Detection limit of the assay was 0.1 fmol/ml. Highest concentrations were found in the turtle diencephalon, followed by other brain areas in the turtle and rat. All other brain areas in the examined species contained significant levels of VIP. Immunoreactivity was also shown in the cerebral and ventral ganglia of the earthworm. In summary, our results show comparative quantitative distribution in representative species of the phylogenetic line, using the same experimental conditions.     

Influence of pinealectomy on levels of PACAP and cAMP in the chicken brain

One of the recently found functions of pituitary adenylate cyclase activating polypeptide (PACAP) is the modulation of circadian rhythms. Widespread distribution of PACAP-containing neurons and receptors has been shown in the chicken. Recently, we have demonstrated that PACAP levels oscillate in a circadian manner in the chicken brain. Daily variation in PACAP levels might be influenced by several regulatory mechanisms. Among the structures that may regulate PACAP levels, one candidate is the pineal gland. Therefore, in the present study, we investigated the effect of pinealectomy on the levels of PACAP in the chicken brain. Animals were kept under 12:12-h light-dark schedule. Pinealectomy was performed at 3 weeks of age; sham-operated animals were used as controls. The animals were sacrificed at 15 and 24 h 1 week after pinealectomy. The brainstem and diencephalon were removed, and tissue samples were processed for PACAP and cAMP radioimmunoassay (RIA).PACAP and cAMP levels showed nighttime elevations in both the sham-operated and pinealectomized animals, except for the PACAP content in the diencephalon of pinealectomized chicken. PACAP levels of pinealectomized animals were significantly higher in the diencephalon and brainstem as compared to the control animals at both time-points. Levels of cAMP correlated well with levels of PACAP. The present results provide evidence that the pineal gland has an inhibitory impact on PACAP-neurons in the chicken brainstem and diencephalon.     

Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease

Reaction patterns of the 7th Human Leukocyte Differentiation Antigen Workshop blind panel adhesion molecules were studied on CD3/CD4, CD3/CD8, CD3/TCR gamma delta double positive T cells from peripheral blood of patients with chronic graft versus host disease (n = 8) and healthy controls (n = 4). Reactivity of 14 adhesion antibodies was tested by three-colour immunophenotyping. The mean proportion of CD3+ T cells (69 +/- 19%). CD3/CD8++ (31 +/- 13%) and CD3/TCR gamma delta++ (4 +/- 2%) T sub-populations of patients were comparable with the healthy controls. However, the mean percentage of CD3/CD4++ T cell subset in patients (14 +/- 12%) proved to be significantly decreased in comparison with the normal control value (34 +/- 16%) presumably due to secondary immunodeficiency. The workshop antibodies proved to be reactive with three T cell subsets expressing the examined antigens. Based on the results of the adhesion molecule workshop new CD categories have been introduced: CD156b as a transmembrane protein, CD167a as an epithelial tyrosin kinase receptor, CD168 as a receptor for hyaluronan mediated motility (RHAMM) and CD171 as a co-stimulatory adhesion molecule. There were significant differences in the expression of the CD167a and CD156b antigens on the CD3/CD4++ subset between the samples of patients compared with the controls characterizing the CD4+ T lymphocyte subpopulation in chronic graft versus host disease.     

Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells

Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects.     

Prevalence of cagA and vacA genes in isolates from patients with Helicobacter pylori-associated gastroduodenal diseases in Recife, Pernambuco, Brazil

Geographical differences in the prevalence of Helicobacter pylori genes and their association with disease severity have been identified. This study analyzes the prevalences of the cagA gene and alleles of the vacA gene in H. pylori-associated gastroduodenal diseases in isolates from Recife, PE, Brazil. Gastric biopsy of 61 H. pylori-positive patients were submitted to DNA extraction and gene amplification by polymerase chain reaction. Among the 61 patients, 21 suffered from duodenal ulcer (DU) and 40 from gastritis (GT). The prevalence of H. pylori strains harbouring the cagA gene was higher in the DU group (90.5%) than in the GT group (60%) (p=0.02). The vacA gene was amplified in 56 out of 61 biopsies, of which 43 (76.8%) contained bacteria carrying the s1 allele and 13 (23.2%) the s2. However, the prevalence of the vacA s1 genotyping was the same in either DU or GT group. The majority of the s1-typed strains, 39 (90.7%) out of 43, were subtype s1b. In resume there was a strong association between the H. pylori cagA+ gene and DU. However, there were no differences between the DU and GT groups in relation to the vacA s1 and s2 alleles distribution, albeit the subtype s1b was predominant.     

Oesophageal acid stimulation in humans: does it alter baroreflex function?

The aim of this study was to investigate if oesophagel acid stimulation (Bernstein test) had an influence on heart rate and blood prsure variability and baroreflex gain. We compared the cardiovascular responses in 10 patients with established gastro-esophageal reflux disease (Group 1) and 10 control subjects (Group 2) during esophageal saline and 0.1 mol/l hydrochloric acid instillation. Indices of heart rate and blood pressure variability and baroreflex gain (derived from linear spontaneous sequences and cross spectral analysis) were calculated. In Group 1 the standard deviation of RR intervals (SDRR: 46 ms vs 51 ms, p=0.030) and the root mean square of successive differences (RMSSD: 24 ms vs. 26 ms p=0.027) were significantly lower during acid infusions, than during saline. We found no significant difference in minimum, maximum and mean RR intervals and systolic blood pressures and in the percentage of RR intervals, which differed from adjacent cycles by more than 50 ms (PNN50). The power spectra of RR intervals in the high frequency band tended to be lower during acid infusion (p=0.055). There was no significant difference in blood pressure spectra, neither in low nor in high frequency band. In Group 2 there was no significant difference between any parameters measured during acid and saline. The baroreflex gain was not changed during the studied conditions in any group. Neither increased vagal tone, nor increased vagal variability occurred and the baroreflex gain was not altered during oesophageal acid simulation.     

Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane

Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.