Altered expression and coregulation of dopamine signalling genes in schizophrenia and bipolar disorder.

INTRODUCTION: signalling through dopamine receptors is of critical importance in the brain and is implicated in schizophrenia and bipolar disorder, but its underlying molecular mechanisms remain poorly understood. MATERIALS AND METHODS: using a yeast two-hybrid approach, we previously identified 11 novel dopamine receptor-interacting proteins. Here we compare gene expression levels for 17 genes [including all 11 dopamine receptor interacting proteins, all 5 dopamine receptors (DRD1-DRD5) and DARPP-32] by real-time polymerase chain reaction, using prefrontal cortex post mortem brain samples from 33 schizophrenic, 32 bipolar disorder and 34 control subjects. RESULTS: the expression of C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 genes was altered in schizophrenia and/or bipolar disorder samples relative to controls (P < 0.05). Hierarchical clustering analysis revealed the expression of these five genes (C14ORF28, GNB2L1, MLLT3, DARPP-32, DRD2) is closely correlated in patients. However, in controls, DRD2 expression in relation to the other genes appears to be very different, suggesting abnormal DRD2 activity is an important trigger in the pathophysiology of schizophrenia and bipolar disorder. CONCLUSIONS: our data suggest: (i) C14ORF28, GNB2L1, MLLT3, DRD2 and DARPP-32 are important in the pathogenesis of schizophrenia and bipolar disorder; (ii) these two disorders share common disease-related mechanisms linked to dopamine signalling; (iii) the expression of these genes is closely correlated; and (iv) DRD2 provides the initial trigger in the pathogenesis of these disorders.

Paraptosis: mediation by MAP kinases and inhibition by AIP-1/Alix.

Programmed cell death (pcd) may take the form of apoptotic or nonapoptotic pcd. Whereas cysteine aspartyl-specific proteases (caspases) mediate apoptosis, the mediators of nonapoptotic cell death programs are much less well characterized. Here, we report that paraptosis, an alternative, nonapoptotic cell death program that may be induced by the insulin-like growth factor I receptor (among other inducers), is mediated by mitogen-activated protein kinases (MAPKs) and inhibited by AIP-1/Alix. The inhibition by AIP-1/Alix is specific for paraptosis since apoptosis was not inhibited. Caspases were not activated in this paradigm, nor were caspase inhibitors effective in blocking cell death. However, insulin-like growth factor I receptor (IGFIR)-induced paraptosis was inhibited by MEK-2-specific inhibitors and by antisense oligonucleotides directed against c-jun N-terminal kinase-1 (JNK-1). These results suggest that IGFIR-induced paraptosis is mediated by MAPKs, and inhibited by AIP-1/Alix.

Concentration of hydroxyproline in blood: a biological marker in occupational exposure to asbestos and its relationship with Pi*Z and Pi*S polymorphism in the alpha-1 antitrypsin gene.

BACKGROUND: Hydroxyproline (OHP) is one of the most abundant amino acids in collagen and, in general, it provides a good measure of overall collagen catabolism. METHODS: Asbestos workers suffering from asbestosis (cases n = 85); asbestos exposed workers without asbestosis (exposed controls, EC, n = 86), and non-exposed population (non-exposed controls, NEC, n = 122) were studied. The concentration of free OHP in whole blood was measured following the Pico-Tag procedure. RESULTS: Concentration of OHP in blood was significantly different in the three groups studied (P < 0.001), being higher in cases (19.8 +/- 14.7 micromol/L) than in EC (16 +/- 12.4) and NEC (13.5 +/- 6.7). When all individuals were grouped and stratified by the Pi*S and Pi*Z polymorphisms in the alpha-1-antitrypsin gene, the highest OHP levels were detected in the Pi*S homozygotes, one of the asbestosis-at risk-genotypes (Pi*S homozygotes, x = 24.5 +/- 11.7; Pi*S heterozygotes, x = 16.6 +/- 10.0; wild type, wt, x = 15.9 +/- 11.8). CONCLUSIONS: Blood OHP concentration could be used for monitoring human exposure to asbestos, either as a marker for occupational monitoring or as an additional clinical parameter in diagnostic exploration of asbestosis.

Pi*S and Pi*Z alpha 1 antitrypsin polymorphism and the risk for asbestosis in occupational exposure to asbestos.

Alpha 1 antitrypsin is a highly polymorphic anti-elastase enzyme, especially active in the protection of alveoli and liver. Here we studied the distribution of two deficient alleles Pi*Z and Pi* S, in 194 asbestos workers, of whom 100 were asbestosis cases, and 94 were controls without disease (exposed controls, EC). A second group of controls without asbestos exposure (non-exposed controls, NEC; n=122) was also included. Multivariate analysis adjusted by age and smoking habit showed ninefold risk for asbestosis in Pi*Z heterozygous individuals and 5.9-fold risk for Pi*S homozygous although differences were only significant in the first case (cases vs. EC: OR 8.9; p=0.04). Considering both genotypes (Pi*Z heterozygous, Pi*S homozygous) we obtained an OR of 8 (p=0.01). Our results suggest that the alpha 1 antitrypsin polymorphisms, especially Pi*Z, could help to predict asbestosis risk and confirm the high prevalence of the Pi*S allele in Spain.

Glutathione S-transferase (GSTM1 and GSTT1)-dependent risk for colorectal cancer.

BACKGROUND: The deleted GSTT1 and GSTM1 genotypes (null genotypes) resulting in loss of transferase activity are found in 10-20% and 50-60% of the population, respectively. PATIENTS AND METHODS: The GSTT1- and GSTM1-dependent risk for sporadic colorectal cancer (CRC) was studied in 247 incident CRC cases and 296 hospital-based controls. RESULTS: The GSTT1-null genotype was found to be 1.5 times more prevalent in CRC patients (17.4%) compared with controls (11.1%) (crude OR 1.6; p = 0.03). The GSTM1-null genotype was found to be equally prevalent in cases and controls (53%). Multivariate analysis showed a significant 1.7-fold risk for CRC associated with the GSTT1-null genotypes (p = 0.04) and this increased to 2.9 for smokers (p = 0.02). CONCLUSION: This study provides evidence of gene-environment interaction and illustrates the importance of further research into the role of genetic susceptibility for CRC.

Correlations of partial and extensive methylation at the p14(ARF) locus with reduced mRNA expression in colorectal cancer cell lines and clinicopathological features in primary tumors.

p14(ARF) is a putative tumor suppressor gene thought to modify the levels of p53. CpG sites within the 5'-flanking region and exon 1beta of p14(ARF) are targets of aberrant methylation and transcriptional silencing in human colorectal cancer (CRC). Here we have developed methylation-specific polymerase chain reaction (MSPCR) methods to detect methylation of CpG sites in p14(ARF) in CRC cell lines and primary CRC tumors, and correlated p14(ARF) mRNA expression with methylation in CRC cell lines using competitive quantitative reverse transcription-polymerase chain reaction methods. Ten CRC cell lines were studied; three (DLD-1, HCT15 and SW48) showed extensive methylation and six (Colo320, SW480, HT29, Caco2, SW837 and WiDr) were unmethylated; the other cell line, LoVo, showed partial methylation that affected exon 1beta but not the immediate upstream CpG sites. p14(ARF) mRNA was expressed at extremely low levels in fully methylated cell lines and at 10(4)- to 10(5)-fold higher levels in unmethylated cell lines. p14(ARF) expression in the partially methylated LoVo cell line was intermediate. Treatment of LoVo cells with 2 microM 5-aza-2'-deoxycytidine for 72 h was associated with marked (100-fold) induction of mRNA levels. Of 119 primary CRCs, 18% contained p14(ARF) methylation, although partial methylation was the most common pattern observed (in 67% of methylated tumors). Methylation of p14(ARF) was often accompanied by p16(INK4a) methylation; however, 50% of p14(ARF) methylated tumors contained unmethylated p16(INK4a). Methylation at p14(ARF) was associated with female gender, greater age, proximal anatomic location and poor differentiation, but not stage at diagnosis. A two-step MSPCR method for assaying p14(ARF) methylation in human tumors is described.

NAD(P)H:quinone oxidoreductase-dependent risk for colorectal cancer and its association with the presence of K-ras mutations in tumors.

NAD(P)H:quinone oxidoreductase (NQO1) is a polymorphic enzyme involved in the detoxification of potentially mutagenic and carcinogenic quinones. The homozygous C609T NQO1 genotype resulting in loss of reductase activity is found in 2-20% of individuals. In the present study, the NQO1-dependent risk for sporadic colorectal cancer (CRC) was studied in 247 incident CRC cases and 296 hospital-based controls recruited during 1996-1997. Four subgroups of cases were studied: (i) all CRCs; (ii) a molecular CRC subgroup (n = 117, cases with molecular tumor analyses); (iii) within the molecular subgroup those tumors with K-ras mutations in codon 12 (CRC K12); (iv) within the molecular subgroup those tumors with K-ras mutations in codon 13 (CRC K13). The C609T NQO1 genotype was found to be twice as prevalent in all CRC patients (6.8%) compared with controls (3%) and six times more common in the subset CRC K12 (20%). Multivariant analyses in the overall population of 247 cases and 296 controls showed a significant age and gender adjusted risk for CRC associated with the C609T NQO1 genotype (OR 2.9, 95% CI 1.19-6.97; P = 0.01) or with any variant genotype (the low activity allele frequency, i.e. heterozygotes plus homozygotes) (OR 1.41, 95% CI 1.02-1.92; P = 0.03). Within cases of the molecular subgroup (n = 117) the C609T NQO1 genotype was associated with the presence of K-ras codon 12 mutation (OR 6.5 95%, CI 1.39-34.9; P = 0.003). Logistic regression showed an age and gender adjusted risk for K-ras codon 12 mutant CRC associated with the C609T NQO1 genotype (OR 10.5, 95% CI 2.99-36.7; P: = 0.0002) or with any variant NQO1 genotype (OR 2.23, 95% CI 1.23-4.00; P = 0.007) compared with the control group. Genetically determined variations in NQO1 may modify the risk for CRC and these risks may be greatest for tumors containing K-ras codon 12 mutations. CRC with K-ras codon 12 mutations may represent a distinct and etiologically more homogeneous subtype of the disease, which may be associated with toxicants that are metabolized via a NQO1-dependent pathway.

Human huntingtin-associated protein (HAP-1) gene: genomic organisation and an intragenic polymorphism.

The huntingtin-associated protein (HAP-1) interacts with the Huntington disease gene product, huntingtin. It is predominantly expressed in the brain and shows an increased affinity for mutant huntingtin. We have sequenced an 18,656bp genomic region encompassing the entire human HAP-1 gene and determined its genomic organisation, with 11 exons spanning 12.1kb. We have also found an intragenic polymorphism within intron 6 of HAP-1. We have recently shown that HAP-1 maps to a region of the genome which has been implicated in a variety of neurological conditions, including progressive supranuclear palsy (PSP), a late-onset atypical parkinsonian disorder. The detailed characterisation of the genomic organisation of HAP-1 and the presence of an intragenic polymorphism will be helpful in evaluating its role in different disorders, using candidate gene approaches.

Mth1 receives the signal given by the glucose sensors Snf3 and Rgt2 in Saccharomyces cerevisiae.

We have determined that the mutant genes DGT1-1 and BPC1-1, which impair glucose transport and catabolite repression in Saccharomyces cerevisiae, are allelic forms of MTH1. Deletion of MTH1 had only slight effects on the expression of HXT1 or SNF3, but increased expression of HXT2 in the absence of glucose. A two-hybrid screen revealed that the Mth1 protein interacts with the cytoplasmic tails of the glucose sensors Snf3 and Rgt2. This interaction was affected by mutations in Mth1 and by the concentration of glucose in the medium. A double mutant, snf3 rgt2, recovered sensitivity to glucose when MTH1 was deleted, thus showing that glucose signalling may occur independently of Snf3 and Rgt2. A model for the possible mode of action of Snf3 and Rgt2 is presented.

The use erythrocyte glutathione as a predictive marker for malignant melanoma.

BACKGROUND: Glutathione (GSH) may provide defense against reactive oxygen species (ROS) generated by ultraviolet radiation. Furthermore, some authors have demonstrated a relationship between the GSH of peripheral blood erythrocytes (GSHe) and resistance to chemotherapy. PATIENTS & METHODS: To observe the influence of GSH on the genesis and evolution of Malignant Melanoma (MM), we assessed the concentration of GSH in erythrocytes (GSHe) in MM patients (n = 566) and controls (n = 164) by the method of Beutler (1963). RESULTS: No differences were found between the two groups (5.94 +/- 1.61 cases vs 6.08 +/- 1.49 mmol/gr Hb, controls; p > 0.05). Fifty seven patients with poor evolution (disease-free survival < 2 years) had higher GSH levels than the remaining patients (6.35 +/- 1.83 vs 5.83 +/- 1.62 mmol/g Hb; p < 0.01). GSHe increased significantly after antineoplastic therapy (4.75 +/- 1.26 vs 7.73 +/- 1.39 mmoVg Hb; p < 0.001), thus indicating a possible role in chemoresistance. 2 CONCLUSIONS: GSHe is not related to the risk of developing MM. GSHe may be related to the evolution of MM, being higher in patients who suffer relapse or metastasis. GSHe increases significantly during cytostatic treatment.

Disruption and basic functional analysis of six novel ORFs of chromosome XV from Saccharomyces cerevisiae.

We report the disruption and functional analysis of six open reading frames (ORFs) from chromosome XV, namely YOL155c, YOL154w, YOL119c, YOL118c, YOR301w and YOR306c, in FY1679 and CEN.PK2 backgrounds. We constructed replacement cassettes and cloned each ORF into the pRS416 centromeric plasmid. No obvious phenotype was observed for the corresponding deleted strains with respect to growth, mating or sporulation. YOL155c encodes a protein with a secretion signal and putative GPI-anchor recognition site and is possibly a cell wall protein, although its deletion did not present morphogenetic defects under any of the conditions tested. Although YOL119c and YOR306c are members of the monocarboxylate permease family, the growth of the double disruptant in acetate, lactate and pyruvate was similar to that of the parental strains.

Aberrant methylation of p16INK4a in anatomic and gender-specific subtypes of sporadic colorectal cancer.

Colorectal cancer (CRC) occurring in the proximal colon and among women may represent a distinct subtype of the disease. In the present study of 120 sporadic CRCs, we used methylation-specific PCR to test whether methylation of the CpG island in the 5' region of the p16INK4a tumor suppressor gene was associated with anatomical location, gender, or other clinicopathological characteristics. Overall, 18.3% of the tumors had detectable p16INK4a methylation. A marked preponderance of methylated tumors occurred within the proximal colon; cancers occurring proximal to the sigmoid colon were 13.1 times more likely to contain methylated p16INK4a compared with distal tumors. In addition, female patients were 8.8 times more likely than males to have methylation-positive cancers, and p16INK4a methylation was also associated with poorly differentiated tumors. The localization of tumors with p16INK4a methylation within the proximal colon and among female patients specifically adds to a growing database of molecular alterations that define important subtypes of sporadic CRC. The potentially reversible nature of CpG methylation may provide novel therapeutic opportunities for this increasing subtype of the disease, which, due to anatomical location, presents a great challenge for early detection.

Limitations in the use of glutathione S-transferase P1 in urine as a marker for bladder cancer.

BACKGROUND: GST pi (GSTPl) is overexpressed in bladder cancer and desquamation of the tumour may produce detectable levels of urinary GSTPl which could be used as a marker for the early diagnosis of bladder cancer. MATERIALS AND METHODS: A preliminary study in 27 transitional cell carcinoma (TCC) patients and 20 controls, using an ELISA methodl is presented here. RESULTS: 55.5% of TCC patients were positive for GSTP1, while all control samples were negative. Some of the GSTP1 positive cases also gave positive results for haematuria, which indicates that a limitation of this marker involves the contamination of the urine with erythocyte GSTP1. In 5 cases (18.5%) without haematuria detectable levels of GST pi were found. CONCLUSIONS: Further studies would be required to assess the advantages of this technique over clas sical cytology or as a complement to it, especially in patients in a phase of temporary negative haematuria.

Glutathione and glutathione S-transferases in human squamous cell carcinomas of the larynx and GSTM1 dependent risk.

BACKGROUND: The aim of the present study was to establish the risk of squamous cell carcinoma (SCC) of the larynx associated with the congenital absence of glutathione S-transferase M1 (GSTM1), and to describe the expression of the isoenzymes GSTA1/2, GSTP1-1, and GSTM1 and glutathione (GSH) content in healthy and tumoral larynx tissue. MATERIAL AND METHODS: Blood samples from 160 SCC male patients and 158 controls were phenotyped for GSTM1 by ELISA. Using 37 paired samples (normal and tumour specimens) from cancer patients we carried out a descriptive study of enzyme activity by ELISA (GSTs) and Ellman's as say (GSH) RESULTS: GSTM1 null phenotype was more common in the SCC group than in controls (OR 1.9, CIs 1.18-3.05, p = 0.004). Total GST activity was higher in tumour samples than in matched healthy tissue (2.2-fold, p-0.00001), being largely determined by GSTP1-1 (1.9-fold increased in malignant tissue; p = 0.0003). The GSH content was also significantly higher in SCC than in normal mucosa (1.9-fold, p = 0.0007). CONCLUSIONS: We confirmed the GSTM1-dependent risk for larynx cancer among smokers. The overexpression of the GST/GSH system in tumours reported here indicates their possible role in chemoresistance to pharmacological therapy.

A series of vectors to construct lacZ fusions for the study of gene expression in Schizosaccharomyces pombe.

We have constructed a series of plasmids to facilitate the fusion of promoters with or without coding regions of genes of Schizosaccharomyces pombe to the lacZ gene of Escherichia coli. These vectors carry a multiple cloning region in which fission yeast DNA may be inserted in three different reading frames with respect to the coding region of lacZ. The plasmids were constructed with the ura4+ or the his3+ marker of S. pombe. Functionality of the plasmids was tested measuring in parallel the expression of fructose 1,6-bisphosphatase and beta-galactosidase under the control of the fbp1+ promoter in different conditions.

Influence of smoking in the glutathione-S-transferase M1 deficiency--associated risk for squamous cell carcinoma of the bladder in schistosomiasis patients in Egypt.

In this study we show an effect of the glutathione-S-transferase M1 (GSTM1) null phenotype on the risk for squamous cell carcinoma (SCC) of the bladder among male smokers in Egypt, with an adjusted odds ratio of 4.8 (95% confidence interval: 1.06-21.77). However, no overall effect of the GSTM1 null phenotype on the risk for bladder SCC was observed.

DNA sequence analysis of a 10 624 bp fragment of the left arm of chromosome XV from Saccharomyces cerevisiae reveals a RNA binding protein, a mitochondrial protein, two ribosomal proteins and two new open reading frames.

We have determined the sequence of a 10624 bp DNA segment located in the left arm of chromosome XV of Saccharomyces cerevisiae. The sequence contains eight open reading frames (ORFs) longer than 100 amino acids. Two of them do not present significant homology with sequences found in the databases. The product of ORF o0553 is identical to the protein encoded by the gene SMF1. Internal to it there is another ORF, o0555 that is apparently expressed. The proteins encoded by ORFs o0559 and o0565 are identical to ribosomal proteins S19.e and L18 respectively. ORF o0550 encodes a protein with an RNA binding signature including RNP motifs and stretches rich in asparagine, glutamine and arginine.

Analysis of the DNA sequence of a 15,500 bp fragment near the left telomere of chromosome XV from Saccharomyces cerevisiae reveals a putative sugar transporter, a carboxypeptidase homologue and two new open reading frames.

We report the sequence of a 15.5 kb DNA segment located near the left telomere of chromosome XV of Saccharomyces cerevisiae. The sequence contains nine open reading frames (ORFs) longer than 300 bp. Three of them are internal to other ones. One corresponds to the gene LGT3 that encodes a putative sugar transporter. Three adjacent ORFs were separated by two stop codons in frame. These ORFs presented homology with the gene CPS1 that encodes carboxypeptidase S. The stop codons were not found in the same sequence derived from another yeast strain. Two other ORFs without significant homology in databases were also found. One of them, O0420, is very rich in serine and threonine and presents a series of repeated or similar amino acid stretches along the sequence.

DNA sequence analysis of a 13 kbp fragment of the left arm of yeast chromosome XV containing seven new open reading frames.

The sequence of a 13 kbp fragment located in the vicinity of the left telomere of chromosome XV (cosmid pEOA179) has been determined. Seven new open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB629, AOA342, AOC231, AOE555, AOE236, AOA236 and AOE1045). Three of them show no identity with proteins deposited in the data banks. ORF AOB629 (629 amino acids) has some similarity with previously described ferric reductases from Saccharomyces cerevisiae and Schizosaccharomyces pombe. ORF AOA342 encodes a polypeptide reminiscent of dihydroflavonol-4-reductases from a number of plant species. AOE236 displays a high level of identity when compared with peroxisomal membrane proteins previously cloned from the methylotrophic yeast Candida boidinii. Finally, AOE1045 encodes a large protein (1045 residues) with some identity with a hypothetical 147 kDa protein identified during the sequencing of Caenorhabditis elegans chromosome 3.

Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.