Analysis of gene expression in two large schizophrenia cohorts identifies multiple changes associated with nerve terminal function.

Schizophrenia is a severe psychiatric disorder with a world-wide prevalence of 1%. The pathophysiology of the illness is not understood, but is thought to have a strong genetic component with some environmental influences on aetiology. To gain further insight into disease mechanism, we used microarray technology to determine the expression of over 30 000 mRNA transcripts in post-mortem tissue from a brain region associated with the pathophysiology of the disease (Brodmann area 10: anterior prefrontal cortex) in 28 schizophrenic and 23 control patients. We then compared our study (Charing Cross Hospital prospective collection) with that of an independent prefrontal cortex dataset from the Harvard Brain Bank. We report the first direct comparison between two independent studies. A total of 51 gene expression changes have been identified that are common between the schizophrenia cohorts, and 49 show the same direction of disease-associated regulation. In particular, changes were observed in gene sets associated with synaptic vesicle recycling, transmitter release and cytoskeletal dynamics. This strongly suggests multiple, small but synergistic changes in gene expression that affect nerve terminal function.

Expression of GRK2 is increased in the left ventricles of cardiomyopathic hamsters.

Reduced beta-adrenergic responsiveness in the heart is a characteristic feature of heart failure. G protein-coupled receptor kinase 2 (GRK2) phosphorylates beta-adrenoceptors in an agonist-dependent manner, causing receptor uncoupling and desensitisation. Elevated levels of both GRK2 mRNA and activity have been shown to occur in the failing human heart (Ungerer et al. (1992) Circulation 87: 454-463). We have analysed levels of GRK2 protein in heart tissue from the cardiomyopathic Syrian hamster CHF 147 and compared these to GRK2 levels in age-matched, non-cardiomyopathic control hamsters (CHF 148). GRK2 protein levels were found to be significantly increased in the left ventricles of the cardiomyopathic hamsters compared to the controls. The relative amounts of GRK2 in the cardiomyopathic hamsters, as compared to normal controls, increased with age from 2-fold at 100 days to 5-fold at 350 days. These animals should provide a useful model for testing the effect of GRK2 inhibitors on the development of heart failure.

Chronic systemic administration of salmeterol to rats promotes pulmonary beta(2)-adrenoceptor desensitization and down-regulation of G(s alpha).

1. The aim of the present study was to examine the effects of chronic infusion of the long-acting agonist salmeterol on pulmonary beta(2)-adrenoceptor function in Sprague-Dawley rats in vivo and to elucidate the molecular basis of any altered state. 2. Systemic administration of rats with salmeterol for 7 days compromised the ability of salmeterol and prostaglandin E(2) (PGE(2)), given acutely by the intravenous route, to protect against ACh-induced bronchoconstriction when compared to rats treated identically with vehicle. 3. beta(1)- and beta(2)-adrenoceptor density was significantly reduced in lung membranes harvested from salmeterol-treated animals, which was associated with impaired salmeterol- and PGE(2)-induced cyclic AMP accumulation ex vivo. 4. Three variants of G(s alpha) that migrated as 42, 44 and 52 kDa peptides on SDS polyacrylamide gels were detected in lung membranes prepared from both groups of rats but the intensity of each isoform was markedly reduced in rats that received salmeterol. 5. The activity of cytosolic, but not membrane-associated, G-protein receptor-coupled kinase was elevated in the lung of salmeterol-treated rats when compared to vehicle-treated animals. 6. The ability of salmeterol, administered systemically, to protect the airways of untreated rats against ACh-induced bronchoconstriction was short-acting (t(off) approximately 45 min), which contrasts with its long-acting nature when given to asthmatic subjects by inhalation. 7. These results indicate that chronic treatment of rats with salmeterol results in heterologous desensitization of pulmonary G(s)-coupled receptors. In light of previous data obtained in rats treated chronically with salbutamol, we propose that a primary mechanism responsible for this effect is a reduction in membrane-associated G(s alpha). The short-acting nature of salmeterol, when administered systemically, and the reduction in beta-adrenoceptor number may be due to metabolism to a biologically-active, short-acting and non-selective beta-adrenoceptor agonist.

Albuterol-induced downregulation of Gsalpha accounts for pulmonary beta(2)-adrenoceptor desensitization in vivo.

The aim of the present study was to develop a chronic in vivo model of pulmonary beta(2)-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E(2), but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. beta(2)-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsalpha expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin-induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein-coupled receptor kinase-2, but the extent to which these events contributed to beta(2)-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsalpha representing a primary molecular etiology.

Expression of EGF and HIV envelope glycoprotein.

1. The S. cerevisiae alpha-factor prepro leader is functional and is correctly processed in P. pastoris. 2. P. pastoris has a high secretory capacity, but yields can be severely reduced by extracellular proteases. This problem can be reduced by altering the medium composition, e.g., adjusting the pH or by adding casamino acids. 3. A rapid DNA dot-blot technique can be used for mass screening of transformants to obtain high-copy-number, high-expressing strains. 4. For mEGF, which is an efficiently secreted protein, there was a good correlation between gene dosage and yield, and maximum levels were obtained at high copy number. 5. Vectors conferring resistance to G418 have been developed for the selection of high-copy-number transformants. These vectors can also be used to isolate a series of transformants with increasing copy number of optimizing the expression of genes where high copy number may be detrimental. 6. The HIV-1 ENV gene was not expressed in P. pastoris owing to fortuitous termination of transcription within AT-rich regions. This is a species-specific phenomenon, since full-length HIV-1 ENV transcripts are produced in S. cerevisiae. The problem was overcome by synthesizing the relevent portion of the gene with increased GC content. 7. ENV was hyperglycosylated and immunologically inactive when secreted by P. pastoris. The yield was reduced by extracellular proteases, but like mEGF, this could be significantly improved by altering the pH of the culture medium and by adding casamino acids. 8. In single-copy integrants, transcripts from the semisynthetic HIV-1 ENV gene were almost as abundant as endogenous AOX1. Transcript levels increased progressively with increasing copy number, showing that the AOX1 promoter is not greatly limited by the level of trans-activating factors.

Studies on the localization and expression of nitric oxide synthase using histochemical techniques.

This review provides an update on the variety of histochemical techniques available for the cellular localization and expression of nitric oxide synthase in formalin-fixed tissue sections. The techniques of immunohistochemistry and NADPH-diaphorase histochemistry are discussed and the suitability of various types of probes and reporters which are useful for in situ detection of nitric oxide synthase mRNA expression are assessed. Figures are also included which illustrate the techniques described and protocols for in situ hybridization and NADPH-diaphorase histochemistry.

Production of a phosphorylated GST::HPV-6 E7 fusion protein using a yeast expression vector and glutathione S-transferase fusions.

A Saccharomyces cerevisiae GAL7 expression vector for the production of protein fusions to glutathione S-transferase (GST) has been constructed. Using this vector, a GST fusion to human papillomavirus type 6 (HPV-6) E7 protein was produced and purified by affinity chromatography in a single step, at a yield of 2 micrograms/ml of culture. The E7 portion of the fusion protein was phosphorylated, in contrast to the same product made in Escherichia coli. Therefore, yeast GST vectors may be of specific use in producing phosphoproteins, or proteins with other eukaryotic post-translational modifications, in preparative amounts for in vitro analysis.

Rapid selection using G418 of high copy number transformants of Pichia pastoris for high-level foreign gene expression.

Pichia pastoris is a methylotrophic yeast increasingly important in the production of therapeutic proteins. Expression vectors are based on the methanol-inducible AOX1 promoter and are integrated into the host chromosome. In most cases high copy number integration has been shown to be important for high-level expression. Since this occurs at low frequency during transformation, we previously used DNA dot blot screens to identify suitable clones. In this paper we report the use of vectors containing the Tn903 kanr gene conferring G418-resistance. Initial experiments demonstrated that copy number showed a tight correlation with drug-resistance. Using a G418 growth inhibition screen, we readily isolated a series of transformants, containing progressively increasing numbers (1 to 12) of a vector expressing HIV-1 ENV, which we used to examine the relationship between copy number and foreign mRNA levels. Northern blot analysis indicated that ENV mRNA levels from a single-copy clone were nearly as high as AOX1 mRNA, and increased progressively with increasing copy number so as to greatly exceed AOX1 mRNA. We have also developed protocols for the selection, using G418, of high copy number transformants following spheroplast transformation or electroporation. We anticipate that these protocols will simplify the use of Pichia as a biotechnological tool.

The intracellular production and secretion of HIV-1 envelope protein in the methylotrophic yeast Pichia pastoris.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120 (ENV), is required in large quantities for immunological studies and as a potential vaccine component. We have expressed the DNA encoding gp120 in a highly efficient expression system based on the methylotrophic yeast, Pichia pastoris. The native gene was found to contain a sequence which resembled a Saccharomyces cerevisiae polyadenylation consensus and acted as a premature polyadenylation site in P. pastoris, resulting in the production of truncated mRNA. As full-length mRNA was produced in S. cerevisiae, this indicates differences in mRNA 3'-end formation between the two yeasts. Inactivation of this site by site-directed mutagenesis revealed several additional fortuitous polyadenylation sites within the gene. We have designed and constructed a 69%-synthetic gene with increased G + C content which overcomes this transcriptional problem, giving rise to full-length mRNA. High levels of intracellular, insoluble, unglycosylated ENV were produced [1.25 mg/ml in high-density (2 x 10(10) cells per ml) fermentations]. ENV also was secreted from P. pastoris using the S. cerevisiae alpha-factor prepro secretion leader and the S. cerevisiae invertase signal sequence. However, a high proportion of the secreted product was found to be hyperglycosylated, in contrast to other foreign proteins secreted from P. pastoris. There also was substantial proteolytic degradation, but this was minimized by maintaining a low pH on induction. Insoluble, yeast-derived ENV proteins are being considered as vaccine antigens and the P. pastoris system offers an efficient method of production.

Amino acid misincorporation during high-level expression of mouse epidermal growth factor in Escherichia coli.

To determine whether the high-level expression of foreign proteins in Escherichia coli can lead to frequent translational errors, we analyzed amino acid misincorporation in mouse epidermal growth factor (mEGF) produced as a TrpE fusion protein. The mEGF DNA does not encode phenylalanine and determining the phenylalanine content of the purified protein will measure missense errors. Using this approach, we found an error frequency of about 1 in 40 for codons differing by a single base from those for phenylalanine. This is at least ten times higher than the error rate found for normal E. coli protein synthesis and may be due to limiting supply of charged tRNAs and GTP, brought about by the high-level production of the heterologous protein. The unexpectedly high error rate has implications for the clinical use of E. coli-derived therapeutic proteins.