Candidate-gene association analysis of response to risperidone in African-American and white patients with schizophrenia.

Clinical trial data were evaluated for the association between 22 single-nucleotide polymorphisms (SNPs) and response in acutely ill patients diagnosed with schizophrenia, schizoaffective disorder or schizophreniform disorder, who were treated with oral risperidone. All patients in the exploratory (78 African Americans) and validation (65 whites) data sets received risperidone 2-6 mg per day over 2-12 weeks. Two SNPs were found to have significant associations with response to risperidone over 2-12 weeks in both African-American and white patients and had a consistent direction of effect in both cohorts. Metabotropic glutamate receptor (GRM3) SNP, rs724226, was associated with a change in the positive and negative syndrome scale (PANSS) total response. Catechol-O-methyltransferase (COMT) SNP, rs165599, was moderately associated with a change in the PANSS Negative score. The greater prevalence of poor-responder GRM3 and COMT alleles in white versus African-American patients might have a clinical significance in evaluating the ethnic-specific response to risperidone.

The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Cysteine residues in the zinc finger and amino acids adjacent to the finger are necessary for DNA binding by the LAC9 regulatory protein of Kluyveromyces lactis.

LAC9 is a positive regulatory protein that controls transcription of the lactose-galactose regulon in Kluyveromyces lactis. LAC9 is homologous to the GAL4 protein of Saccharomyces cerevisiae. Both proteins have a single "zinc finger" which plays a role in DNA binding. We previously hypothesized (L. V. Wray, M. M. Witte, R. C. Dickson, and M. I. Riley, Mol. Cell. Biol. 7:1111-1121, 1987) that the DNA-binding domain of the LAC9 protein consisted of the zinc finger as well as a region of amino acids on the carboxyl-terminal side of the zinc finger. In this study we used oligonucleotide-directed mutagenesis to introduce 13 single-amino-acid changes into the proposed DNA-binding domain of the LAC9 protein. Variant LAC9 proteins carrying an amino acid substitution in any one of the four highly conserved Cys residues of the zinc finger had reduced DNA-binding activity, suggesting that each Cys is necessary for DNA binding. Three of four variant LAC9 proteins with amino acid substitutions located on the carboxyl-terminal side of the zinc finger had reduced DNA-binding activity. These results support our hypothesis that the DNA-binding domain of the LAC9 protein is composed of the zinc finger and the adjacent region on the carboxyl side of the zinc finger, a region that has the potential to form an alpha-helix. Finally, LAC9 proteins containing His residues substituted for the conserved Cys residues also had reduced DNA-binding activity, indicating that His residues are not equivalent to Cys residues, as had been previously thought.

Characterization of a positive regulatory gene, LAC9, that controls induction of the lactose-galactose regulon of Kluyveromyces lactis: structural and functional relationships to GAL4 of Saccharomyces cerevisiae.

Lactose or galactose induces the expression of the lactose-galactose regulon in Kluyveromyces lactis. We show here that the regulon is not induced in strains defective in LAC9. We demonstrate that this gene codes for a regulatory protein that acts in a positive manner to induce transcription. The LAC9 gene was isolated by complementation of a lac9 defective strain. DNA sequence analysis of the gene gave a deduced protein of 865 amino acids. Comparison of this sequence with that of the GAL4 protein of Saccharomyces cerevisiae revealed three regions of homology. One region of about 90 amino acid occurs at the amino terminus, which is known to mediate binding of GAL4 protein to upstream activator sequences. We speculate that a portion of this region, adjacent to the "metal-binding finger," specifies DNA binding. We discuss possible functions of the two other regions of homology. The functional implications of these structural similarities were examined. When LAC9 was introduced into a gal4 defective strain of S. cerevisiae it complemented the mutation and activated the galactose-melibiose regulon. However, LAC9 did not simply mimic GAL4. Unlike normal S. cerevisiae carrying GAL4, the strain carrying LAC9 gave constitutive expression of GAL1 and MEL1, two genes in the regulon. The strain did show glucose repression of the regulon, but repression was less severe with LAC9 than with GAL4. We discuss the implications of these results and how they may facilitate our understanding of the LAC9 and GAL4 regulatory proteins.

Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements.

The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells.

Regulation of differentiation, proliferation and cancer suppressor activity.

This review suggests that carcinogenesis is linked to defects in linkages between cellular differentiation and regulation of cell proliferation. These linkages are discussed in terms of terminal and nonterminal states of differentiation and their relationship to the control of proliferation. The ability of differentiation to regulate proliferation potential proteins and cancer suppressor genes is also discussed, because these mechanisms may be important for cancer prevention and therapy.

Homodimerization and intermolecular tyrosine phosphorylation of the Tyk-2 tyrosine kinase.

The Jak kinases and Stat transcription factors play a major role in signaling of various cytokines including IFN alpha. In this report we show a ligand-independent interaction between Tyk-2 and Jak-1 kinases. We also demonstrate that the Tyk-2 kinase forms a homodimer that has the ability to undergo intermolecular tyrosine phosphorylation. The formation of the Tyk-2 homodimer is independent of both tyrosine phosphorylation and the presence of the tyrosine kinase domain.

Sex determination of dried blood stains using the polymerase chain reaction (PCR) with homologous X-Y primers of the zinc finger protein gene.

The ability to "sex" unknown dried bloodstains is frequently of evidentiary value in forensic casework. Chelex-extracted DNA from 115 specimens (105 dried blood standards and 10 casework samples) was amplified for specific ZFX and ZFY regions of the X and Y chromosomes and subsequent restriction enzyme digestion. The polymerase chain reaction (PCR) yielded a 209 base pair (bp) product containing a polymorphic position with a Y chromosome portion including an additional Hae III restriction site while the X chromosome portion contains only one. The digested PCR product was separated by polyacrylamide gel electrophoresis (PAGE) and detected by silver staining. Female samples exhibit bands of 172 bp and 37 bp only while male samples (XY) exhibit 2 additional bands appearing as an 88/84 bp doublet. Initially the 105 known bloodstains were typed according to the procedure discussed here and correct gender determination was achieved for all samples therefore establishing the reliability of this method. The 10 casework samples yielded the expected results as well. This assay demonstrates potential in both presumptive and confirmatory capacities.

Long-term caloric restriction reduces metabolic rate and heart rate under cool and thermoneutral conditions in FBNF1 rats.

The long-term metabolic and cardiovascular responses to caloric restriction (CR) are poorly understood. We examined the responses to one year of CR in FBNF1 rats housed in cool (COOL; T(a)=15 °C) or thermoneutral (TMN; T(a)=30 °C) conditions. Rats were acclimated to COOL or TMN for 2 months, instrumented for cardiovascular telemetry and studied in calorimeters. Baseline caloric intake, oxygen consumption (VO(2)), mean arterial blood pressure (MAP), and heart rate (HR) were determined prior to assignment to ad lib (AL) or CR groups (30-40% CR) within each T(a) (n = 8). Groups of rats were studied after 10 weeks CR, one year CR, and after 4 days of re-feeding. Both 10 weeks and one year of CR reduced HR and VO(2) irrespective of T(a). Evaluation of the relationship between metabolic organ mass (liver, heart, brain, and kidney mass) and energy expenditure revealed a clear shift induced by CR to reduce expenditure per unit metabolic mass in both COOL and TMN groups. Re-feeding resulted in prompt elevations of HR and VO(2) to levels observed in control rats. These findings are consistent with the hypothesis that long term CR produces sustained reductions in metabolic rate and heart rate in rats.

Assessing the infrequent oral supplementation of olanzapine long-acting injection in the treatment of schizophrenia.

OBJECTIVE: Adding another antipsychotic to a treatment regimen was previously used in evaluating the medication's efficacy. Supplementation of depot antipsychotics with oral antipsychotics is particularly meaningful because depot formulations are typically chosen for patients struggling with adherence to oral antipsychotics. This post-hoc analysis assessed supplementation of olanzapine long-acting injection (olanzapine-LAI) with oral olanzapine. SUBJECTS AND METHODS: We used 12 months of data from an open-label, single-arm extension study of patients with schizophrenia or schizoaffective disorder (N=931) treated with olanzapine-LAI. The prevalence, duration, time to first supplementation, and best predictors of oral supplementation were assessed. RESULTS: Oral supplementation occurred in 21% of patients for a median of 31 days with mean modal dose of 10.8 mg/day. Mean time to first supplementation was shorter for patients who were at least moderately ill at baseline compared to less ill patients (47 vs. 97 days, p<0.001). Best predictors of oral supplementation included a more severe illness profile at baseline, lower olanzapine-LAI dose prior to oral supplementation, supervised living arrangements, and being African-American. CONCLUSION: Supplementation of olanzapine-LAI appears to be infrequent, of relatively short duration, and reserved for more severely ill patients who may require a targeted rescue medication due to signs of impending relapse.

AMPA potentiator treatment of cognitive deficits in Alzheimer disease.

OBJECTIVE: To investigate the efficacy and safety of the positive alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid modulator LY451395 in patients with mild to moderate Alzheimer disease (AD) (Mini-Mental State Examination scores 14 to 26). METHODS: One hundred eighty-one patients were randomized to treatment in an 11-week, double-blind, placebo-controlled trial. Patients received either LY451395 0.2 mg BID for 28 days and 1.0 mg BID thereafter (n = 90) or placebo (n = 91). The primary outcome measurement was the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) with several secondary outcome measurements: Clinician's Interview-Based Impression of Change, Trail Making Part A, Stylus Tapping Test, Single Digit Modality Test, and Neuropsychiatric Inventory (NPI). RESULTS: Baseline demographics were similar between the two groups. Patients did not show any mean change from baseline in the ADAS-Cog after treatment with LY451395 for 4 weeks (p = 0.60) or 8 weeks (p = 0.83). The only secondary outcome measurement that showed changes from baseline compared with placebo was the NPI Total Score: p = 0.06 (marginal significance) after 4 weeks of treatment and p = 0.03 after 8 weeks of treatment. Ninety-two percent of LY451395-treated patients and 95% of placebo-treated patients completed the trial. Adverse events were experienced by 83% of LY451395-treated patients and 86% of placebo-treated patients, the majority of which were rated mild in severity. CONCLUSION: Patients treated with LY451395 did not show a statistically significant separation from patients taking placebo on the Alzheimer's Disease Assessment Scale-Cognitive Subscale, the primary outcome measure.

The proliferation potential protein-related (P2P-R) gene with domains encoding heterogeneous nuclear ribonucleoprotein association and Rb1 binding shows repressed expression during terminal differentiation.

Terminal differentiation is associated with repression in the expression of the proliferation potential proteins (P2P) subset of heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. We report here the cloning and characterization of a 5173-bp P2P-related (P2P-R) cDNA that contains a 4214-bp open reading frame. Probes to this cDNA detect a single 8-kb mRNA in multiple murine tissues and in proliferating 3T3T cells, but not in terminally differentiated 3T3T adipocytes. Evidence that this cDNA can encode peptides with domains for hnRNP association was established by showing that such peptides are recognized by two monoclonal antibodies known to detect core hnRNP proteins, and by showing that the C130 monoclonal antibody, produced against a cDNA-derived fusion protein, also selectively detects native P2P hnRNP proteins. In addition, P2P-R cDNA-derived fusion proteins bind single-stranded nucleic acids, and a P2P-R cDNA-derived antisense oligonucleotide selectively represses P2P expression. Because terminal differentiation is associated with modulation in Rb1 function, we assayed if products of this cDNA might interact with Rb1. Evidence that the P2P-R cDNA encodes a protein domain that binds Rb1 was established using a glutathione S-transferase fusion protein to selectively precipitate Rb1 from cellular extracts. Data also show that this binding is reduced by competition with the adenovirus E1a protein, indicating that binding occurs through the "pocket" domain of Rb1. These results establish that the P2P-R cDNA encodes protein domains involved in both hnRNP association and Rb1 binding and complement recent reports that localize Rb1 to sites of RNA processing in the nucleus.

Repression of SV40 T oncoprotein expression by DMSO.

SV40 large T oncoprotein-transformed murine mesenchymal 3T3 T stem cells (CSV3 cells) can be induced to growth arrest and then differentiate into adipocytes. When differentiation occurs, SV40 T oncoprotein expression is repressed (Estervig et al., J Virol 63:2718, 1989). To determine if repression of T oncoprotein expression can also be induced pharmacologically, the effect of a variety of agents that have been reported to effect differentiation in various cell types but not in 3T3 T or CSV3 cells was tested. This rationale suggests that if any of these agents repress T oncoprotein expression in CSV3 cells, then the results would establish that repression of T oncoprotein expression can be mediated by mechanisms independent of overt differentiation. The results show that dimethylsulfoxide (DMSO) is the only agent tested that represses T oncoprotein expression in CSV3 cells. Repression occurs in a dosage-dependent manner within 24-96 hours after exposure to DMSO. The effect of DMSO on T oncoprotein expression is mediated by posttranslational mechanisms that decrease the stability of the T oncoprotein. DMSO-induced repression of T oncoprotein expression is also associated with reversion of the transformed phenotype in CSV3 cells as demonstrated by the loss of responsiveness to a specific transformation-associated mitogen. These data support the conclusion that the pharmacological repression of T oncoprotein expression represents a form of cancer suppressor activity that can be mediated by a distinct molecular mechanism.