Linkage analysis of schizophrenia in African-American families.

While many studies have sought a window into the genetics of schizophrenia, few have focused on African-American families. An exception is the Project among African-Americans to Explore Risks for Schizophrenia (PAARTNERS), which seeks to identify novel and known risk variation for schizophrenia by genetic analyses of African-American families. We report a linkage study of diagnostic status in 217 African-American families using the Illumina Linkage Panel. Due to assumed incomplete and time-dependent penetrance, we performed linkage analysis using two different treatments of diagnosis: (1) treating both affected and unaffected individuals as informative for linkage (using the program SIBPAL) and (2) treating only affected individuals as informative (using the program MERLIN). We also explore three definitions of affected status: narrowly defined schizophrenia; one broadened to include schizoaffective disorder; and another including all diagnoses indicating psychosis. Several regions show a decrease in the evidence for linkage as the definition broadens 8q22.1 (rs911, 99.26 cM; SIBPAL p-value [p] goes from 0.006 to 0.02), 16q24.3 (rs1006547, 130.48 cM; p from 0.00095 to 0.0085), and 20q13.2 (rs1022689, 81.73 cM; p from 0.00015 to 0.032). One region shows a substantial increase in evidence for linkage, 11p15.2 (rs722317, 24.27 cM; p from 0.0022 to 0.0000003); MERLIN results support the significance of the SIBPAL results (p=0.00001). Our linkage results overlap two broad, previously-reported linkage regions: 8p23.3-p12 found in studies sampling largely families of European ancestry; and 11p11.2-q22.3 reported by a study of African-American families. These results should prove quite useful for uncovering loci affecting risk for schizophrenia.

Inhibition of the catalytic activity of cell adhesion kinase beta by protein-tyrosine phosphatase-PEST-mediated dephosphorylation.

Protein-tyrosine phosphatase (PTP)-PEST is a cytoplasmic tyrosine phosphatase that can bind and dephosphorylate the focal adhesion-associated proteins p130(CAS) and paxillin. Focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta)/PYK2/CADTK/RAFTK are protein-tyrosine kinases that can colocalize with, bind to, and induce tyrosine phosphorylation of p130(CAS) and paxillin. Thus, we considered the possibility that these kinases might be substrates for PTP-PEST. Using a combination of substrate-trapping assays and overexpression of PTP-PEST in mammalian cells, CAKbeta was found to be a substrate for PTP-PEST. Both the major autophosphorylation site of CAKbeta (Tyr(402)) and activation loop tyrosine residues, Tyr(579) and Tyr(580), were targeted for dephosphorylation by PTP-PEST. Dephosphorylation of CAKbeta by PTP-PEST dramatically inhibited CAKbeta kinase activity. In contrast, FAK was a poor substrate for PTP-PEST, and treatment with PTP-PEST had no effect on FAK kinase activity. Tyrosine phosphorylation of paxillin, which is greatly enhanced by CAKbeta overexpression, was dramatically reduced upon coexpression of PTP-PEST. Finally, endogenous PTP-PEST and endogenous CAKbeta were found to localize to similar cellular compartments in epithelial and smooth muscle cells. These results suggest that CAKbeta is a substrate of PTP-PEST and that FAK is a poor PTP-PEST substrate. Further, PTP-PEST can negatively regulate CAKbeta signaling by inhibiting the catalytic activity of the kinase.

Cleavage of membrane-associated ICAM-1 from astrocytes: involvement of a metalloprotease.

Disease of the central nervous system (CNS) with immune-mediated pathogenesis is frequently associated with enhanced expression of intercellular adhesion molecule-1 (ICAM-1) on resident glial cells, including astrocytes. Recently, a soluble form of ICAM-1 (sICAM-1) has been demonstrated within the CNS and cerebrospinal fluid (CSF), arising from an intrathecal source. In this study, we investigated the ability of TNF-alpha treated astrocytes to generate sICAM-1 from a population of membrane-associated ICAM-1. To determine the ability of ICAM-1 to be released from the cell surface, generating sICAM-1, cell cultures were treated with TNF-alpha for 21 h prior to cell surface protein iodination or biotinylation. We show that the membrane-associated form of ICAM-1 (approximately 90 KD) is converted to a soluble form (approximately 83 KD) in cell culture supernatants. The half-life of TNF-alpha induced membrane-associated ICAM-1 on rat astrocytes is approximately 5 h. The proteolytic cleavage process for the conversion of membrane-associated ICAM-1 to sICAM-1 was sensitive to Batimastat (BB94) and phosphoramidon, two inhibitors of metalloproteases, whereas inhibitors of serine-, cysteine-, aspartic-, and chymotrypsin-like proteases had no effect on this process. These results indicate that astrocytes can be induced to produce sICAM-1, and this process involves a metalloprotease that is induced/activated in a TNF-alpha-dependent fashion. It is proposed that astrocytes may be a source of intrathecal sICAM-1 under inflammatory conditions.

Pro-opiomelanocortin gene expression and protein processing in rat mononuclear leukocytes.

Although production of pro-opiomelanocortin (POMC) mRNA and POMC-derived peptides in extrapituitary tissues, including immune tissues, has been demonstrated, questions remain concerning the nature of POMC transcripts and peptide products. With regard to POMC gene expression in lymphocytes, the expression of full-length mRNA and POMC has been questioned. In the present report, we have tested for the existence of these molecules. Western blot analysis with an antibody against POMC-derived adrenocorticotropic hormone (ACTH) specifically identified identical immunoreactive (ir) species in both rat anterior pituitary (AP) and splenocyte cell extracts. The relative molecular weights were those expected for nonglycosylated ACTH, as well as its biosynthetic intermediate, and POMC. Mitogen stimulation of splenic mononuclear cells (MNC) enhanced the levels of these three molecular species. Primer extension analysis identified a band which migrated with a size equivalent to a full-length POMC transcript (approximately 816 nt) in both mitogen-stimulated MNC and AP mRNA. Macrophages produced POMC protein and mRNA among unstimulated splenocytes, while lymphocytes could be induced to produce POMC mRNA upon stimulation. 5' RACE-tailed PCR products were cloned and sequenced. A mRNA encoding all three POMC exons was identified in Concanavalin A (ConA)-stimulated MNC and was identical to that from the anterior pituitary. These results unequivocally demonstrate that mononuclear cells produce full-length POMC transcripts. Its regulation in lymphocytes is distinct from that in macrophages which constitutively produce POMC-derived peptides and mRNA. Also, the biosynthetic pathway of ACTH from POMC in splenic MNC stimulated with ConA appears to be identical to that in rat corticotrophs.

The kinetics of ACTH expression in rat leukocyte subpopulations.

The pro-opiomelanocortin (POMC) gene encodes a family of peptides originally identified in the pituitary gland. An important POMC-derived peptide hormone, corticotropin (ACTH), is also produced by leukocytes and modulates in vitro immune functions. The present investigation was undertaken to determine the kinetics and cellular distribution of ACTH immunoreactivity (ACTH-ir) in vitro in rat splenic leukocyte subpopulations. Cells were cultured with Concanavalin A (ConA), lipopolysaccharide (LPS), or media alone. ACTH-ir was identified with a specific antiserum raised against ACTH 1-24. Double indirect-immunofluorescence was done at 0, 21, and 48 h for B, t-helper (Th), and T-cytotoxic (CTL) cells. Initial kinetic studies demonstrated peak ACTH-ir in all cell types at 18-21 h for both ConA and LPS treatments. A few leukocytes (1-2%) expressed ACTH-ir at 0 h and these were found to be macrophages (MO). Lymphocyte ACTH-ir is 0% at 0 h and rises to 90 +/- 5% and 75 +/- 6% at 21 h with ConA and LPS, respectively. This sharply contrasts with 9 +/- 4% of each cell type positive in media alone at 21 h. The percent immunoreactivity among the three lymphocyte subpopulations did not significantly differ at any single treatment at a single time point. However, there were significant differences in the intensity levels among the subpopulations. At 48 h of ConA or LPS treatment only 10 +/- 4% of B, Th and Tc were positive, while none were positive in media alone. Stimulated peritoneal MO also increase positivity for ACTH-ir (85 +/- 5%). These results indicate that rat splenic B, CTL, and Th lymphocytes can be immunologically stimulated to express the peptide hormone ACTH and that basal ACTH expression in macrophages is distinct from that in lymphocytes. Thus, lymphocyte-derived ACTH may be a paracrine or autocrine regulator of immune function.

Application of a social skills training program in the modification of interpersonal deficits among retarded adults: a clinical replication.

Six mentally retarded adults, equally divided into two treatment groups, were provided with individualized social skills training programs. Treatment, evaluated via a multiple-baseline design strategy, was sequentially and cumulatively applied across target behaviors over a four-week intervention period. Behavioral observation probes and social validation measures served as the primary dependent variables. Results indicated that (a) treatment was effective for virtually all behaviors across all subjects, (b) improvements occurred for both training and generalization scenes, and (c) behavioral performance was maintained one month following the termination of treatment.