Scale for assessing perceptual anomalies. Validation of a Spanish version of the SIAPA scale in a sample of Cuban schizophrenic patients.

INTRODUCTION: Perceptual-attentional disorders other than hallucinations in schizophrenic patients have been studied little. In this work, the results of the Spanish version of the SIAPA scale to detect perceptual-attentional anomalies to real stimuli other than hallucinations in a sample of schizophrenic patients in a community study in Cuba are presented. METHODS: 329 subjects were studied: 129 schizophrenic patients and 200 controls. Patients were diagnosed by psychiatrists according to DSM-IV criteria. The SIAPA and PANSS scales were used for the study. Cronbach's alpha coefficient was determined to analyze internal consistency. Reliability, validity of current criterion and structural validity were measured. Comparisons between groups were made using the ANOVA. RESULTS: Schizophrenic patients had more perceptual anomalies than healthy controls. Auditory and visual perceptual anomalies were more frequent. The scale showed high internal consistency (Cronbach's alpha: 0.84). Using a PANSS scale cut-off score of 60, validity had a sensitivity of 56 % and specificity of 79 %. All modalities of SIAPA scales showed good kappa coefficients (0.72-0.85). CONCLUSIONS: This scale showed similar internal validity and test-retest reliability to those reported in the English version. The results showed that this scale can differentiate the presence of perceptual anomalies in schizophrenic patients from healthy controls. Therefore, we suggest that the SIAPA scale may be useful for assessing perceptual anomalies in clinical researching for cognitive impairment evaluations.

Local action of estrogen and thyroid hormone on vasoactive intestinal peptide (VIP) and galanin gene expression in the rat anterior pituitary.

Neuropeptides act within the pituitary as autocrine or paracrine factors, modulating the synthesis and release of primary pituitary hormones, and possibly regulating cell proliferation and/or plasticity. Manipulation of the endocrine status of rats produces dramatic long-term changes in the pituitary expression of several peptides, including the neuropeptides galanin and vasoactive intestinal peptide (VIP). Whether or not these changes are caused indirectly by hypothalamic factors, or by hormone actions directly in the pituitary, has been only partially addressed. To determine if estrogen or thyroid hormone can act directly within the pituitary to regulate VIP and galanin gene expression, cultured female rat pituitary cells were treated with 10 nM 1,17 beta-estradiol (E2) or triiodothyronine (T(3)). E2 treatment for three days resulted in an approximate 5-fold and 7-fold increase in VIP and galanin mRNA, respectively. In contrast, T(3) treatment reduced the mRNA levels of these neuropeptides to approximately 40% and 30% of control values. A time course study indicated that the actions of estrogen on VIP and galanin mRNA, and of thyroid hormone on VIP mRNA were readily apparent after 24h. The rat pituitary tumor cell line RC-4B/C was found to express easily detectable levels of galanin but not VIP mRNA. Galanin gene expression in these cells was moderately increased by E2 and decreased by T(3). Transfection of a series of luciferase plasmids containing 5 kb to 131 bp of the bovine galanin promoter fused to luciferase revealed cell-type specific enhancer sequences located between -452 and -131 bp of the galanin gene transcription start site. However, transfected plasmids were minimally responsive to E2 and T(3) treatment. Overall the results suggest that E2 and T(3) exert significant local actions in the pituitary on VIP and galanin gene expression. The bovine galanin gene fragment used in these studies contains a potential pituitary cell-type specific enhancer, but appears to lack strong E2-and T(3)-responsive sequences.

Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica.

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.

Targeting of embryonic and postnatal autonomic and enteric neurons with a vasoactive intestinal peptide transgene.

The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest-derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5' flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the beta-galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP-expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and beta-galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS.

Retinoic acid regulation of the VIP and PACAP autocrine ligand and receptor system in human neuroblastoma cell lines.

Neuroendocrine tumors, neuroblastoma in particular, commonly express the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) and their receptors. Retinoic acid (RA) has been shown to induce differentiation of neuroblastoma cell lines, possibly by augmenting or interfering with neuropeptide autocrine loops. We sought to determine which receptor gene subtypes are expressed in selected human neuroblastoma cell lines (SH-SY5Y, IMR-32, and LA-N-5), and the effect of RA on the VIP/PACAP ligand/receptor system. Expression of both PACAP1 and VIP1/PACAP2 receptor genes was detected by Northern analysis, which characteristically encode Type I (PACAP-preferring), and Type II (bivalent VIP/PACAP) receptors, respectively. Binding experiments carried out on IMR-32 cells, using 125I VIP and 125I PACAP-27 as tracers, corroborated that both receptor subtypes were expressed. In contrast to RA upregulation of VIP binding (confirmed here in IMR-32 cells), levels of both receptor mRNAs were reduced after RA treatment. VIP mRNA in each cell line was increased by RA, whereas PACAP mRNA, detected in IMR-32 cells only, was reduced. The studies indicate that several components of the VIP/PACAP autocrine system are regulated in neuroblastoma cell lines during RA differentiation.

Embryonic expression of vasoactive intestinal peptide (VIP) and VIP receptor genes.

Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.

High levels of vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor mRNA expression in primary and tumor lymphoid cells.

Neuropeptides exert a variety of putative immunomodulatory actions. Despite the molecular cloning of multiple forms of receptors for several neuropeptides with putative immunomodulatory effects, including vasoactive intestinal peptide (VIP), the related peptide pituitary adenylate cyclase-activating peptide (PACAP), the opiate peptides, tachykinins, somatostatin and corticotropin-releasing factor, it has not been reported that any of the receptor genes are expressed at significant levels in cells of the immune system. The low level of expression of these receptors and lack of knowledge concerning receptor subtype has impeded progress in understanding how neuropeptides regulate immune function. For example, it is not understood why VIP produces immunomodulatory effects at concentrations far below its receptor-binding affinity. Receptors for VIP and PACAP have recently been cloned. We show here by Northern blot analysis that the VIP/PACAP1 receptor mRNA is present in total RNA prepared from mouse spleen B- and T-lymphocytes. The VIP/PACAP1 receptor mRNA was also present in human peripheral blood lymphocytes, and in a B-lymphocyte and a myelocytic cell line. The mRNA for a second form of the receptor, the VIP/PACAP2 receptor, was not expressed at detectable levels in normal cells, but was detected in several human T-cell lines and a murine mast cell line. The results indicate that VIP/PACAP1 and perhaps VIP/PACAP2 receptors mediate the diverse effects of VIP and PACAP on immune cells.

Differential expression of VIP/PACAP receptor genes in breast, intestinal, and pancreatic cell lines.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are structurally-related neuropeptides that function as trophic factors in addition to their more classical roles as neurotransmitters. Binding and molecular cloning studies have shown that their actions are mediated by receptors encoded by at least three different genes. VIP binding has been demonstrated on many tumor types, and radiolabeled VIP has recently been used as a novel method to localize intestinal tumors in humans and their sites of metastasis. To determine the receptor subtype and level of gene expression, we screened breast, intestinal, and pancreatic, cell lines by Northern blot analysis. Breast lines expressed VIP/PACAP1 receptor mRNA levels comparable to intestinal lines, in agreement with the studies showing particularly high VIP binding in these tumors and their derived cell lines. Pancreatic cell lines expressed mRNA for several receptor types. This extends the potential utility of VIP and PACAP in the localization of tumors, and because VIP and PACAP may regulate the growth rate of some tumors by autocrine or other mechanisms, the identification of receptor subtypes on these lines sets the stage for studies in which the activity of these individual receptors in growth and other processes can be investigated.

High conservation of upstream regulatory sequences on the human and mouse vasoactive intestinal peptide (VIP) genes.

The neuropeptide vasoactive intestinal peptide (VIP) gene is subject to complex transcriptional regulation resulting in expression of the encoded peptides in distinct subpopulations of neurons in most structures of the nervous system, and tissue-specific changes in expression in response to a variety of hormone and environmental factors. This diverse regulation allows the encoded peptides to carry out putative neurotransmitter, neuromodulator, trophic, neuroendocrine, and immune functions. Despite the potential significance of the processes governing its expression, only the human gene has been studied in any depth, and only a single regulatory element has been identified, a cAMP-responsive sequence less than 100 bp upstream from the transcriptional start site. Because tissue-specific patterns of VIP expression are remarkably well conserved between rodents and humans, we isolated the mouse VIP gene and compared 5' flanking sequences with that of the human gene to identify homologous regions which might be involved in regulation common to both species. Of significant interest is a 210 bp region located more than 1.1 kb upstream from the transcription start site that is 91% conserved between the two species. Of additional interest is a 34 bp perfect dCA.dTG repeat present only on the mouse gene which may be capable of forming Z-DNA.

Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice.

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.