Investigation of Turner syndrome in schizophrenia.

Both Turner syndrome and schizophrenia are relatively infrequent conditions. Consequently, individuals having both illnesses are rare. Previous reviews of sex chromosome abnormalities in schizophrenia have focused primarily on the presence of supernumerary X-chromosomes. After identifying two female patients with schizophrenia and Turner syndrome, we reevaluated the available literature that survey female schizophrenics for the presence of chromosomal abnormalities. Eleven patients with Turner syndrome were identified among 6,483 females with schizophrenia in non-case-report studies. These survey results indicate that Turner syndrome occurs approximately three-fold more frequently in schizophrenic females than in the general female population (P < 0.02). Including 6 other case reports and our 2 cases, a total of 19 females with both schizophrenia and Turner syndrome were reported. Interestingly, whereas most Turner syndrome patients have the 45,X karyotype, the majority (18/19) of women with both illnesses have a mosaic karyotype (P < 0.0002). Given the potential role of genes on the X-chromosome in the pathogenesis of schizophrenia, the study of unique populations with abnormalities in this chromosome, such as women with Turner syndrome, may offer clues into this illness.

Drug metabolism and atypical antipsychotics.

The introduction of the atypical antipsychotics clozapine, risperidone, olanzapine, quetiapine and sertindole for the treatment of schizophrenia has coincided with an increased awareness of the potential of drug-drug interactions, particularly involving the cytochrome P450 (CYP) enzymes. The current literature describing the pharmacokinetics of the metabolism of these agents, including their potential to influence the metabolism of other medications, is reviewed. Clozapine appears to be metabolized primarily by CYP1A2 and CYP3A4, with additional contributions by CYP2C19 and CYP2D6. In addition, clozapine may inhibit the activity of CYP2C9 and CYP2C19, and induce CYP1A, CYP2B and CYP3A. Risperidone is metabolized by CYP2D6, and possibly CYP3A4. In vitro data indicate that olanzapine is metabolized by CYP1A2 and CYP2D6. Quetiapine is metabolised by CYP3A4 and sertindole by CYP2D6. There is, however, a general paucity of in vivo data regarding the metabolism of the atypical antipsychotics, indicating a need for further research in this area.

Studies on the activity of barnase toxins in vitro and in vivo.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains which are responsible for cell binding (domain Ia, amino acids 1-252), translocation into the cytosol (domain II, amino acids 253-364) and ADP-ribosylation activity (domain III, amino acids 405-613). We have previously described (Prior, T. I., FitzGerald, D. J., and Pastan, I. (1992) Biochem. 31, 3555-3559) a molecule composed of amino acids 1-412 of PE and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase (Bar), and shown that this protein (PE1-412-Bar) is toxic to cells due to its ribonuclease activity, which had been delivered to the cytosol. We have now used this model to evaluate the role played by the carboxyl end of domain II (amino acids 347-364), domain Ib, and the amino end of domain III (amino acids 405-412) in the translocation event. Toxins completely lacking domain III, termed PE1-380-Bar, or both domains Ib and III, termed PE1-364-Bar, were equally cytotoxic to a murine fibroblast cell line (L929) as was PE1-412-Bar. Extending the deletion to include part of the E-helix and all of the F-helix of domain II (amino acids 347-364) resulted in a toxin (PE1-346-Bar) that was 10-fold less toxic. Previously tested on only murine cell lines, we demonstrate that barnase toxins are cytotoxic also to a variety of human cell lines. Cytotoxicity was assessed by measuring inhibition of DNA synthesis. Surprisingly, PE1-380-Bar is not lethal when injected into mice, either intraperitoneally or intravenously, at 9 nmol, which is 2200-fold more than the amount required for killing by PE (4 pmol). In cell culture these barnase-containing toxins are 100-fold less toxic to murine fibroblast cells than PE. Barnase toxin has a greater survival time in the blood of mice than PE, with a half-life of 102 min. We conclude that domain II is sufficient to transport proteins into the cytosol. Further, since domain Ia can be replaced with other cell targeting moieties, we propose that barnase-toxins should be evaluated for utility in targeted cancer therapy.

Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol.

Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains. Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation. Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic. However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process. To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens. This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE. A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic. Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.

Barnase toxin: a new chimeric toxin composed of pseudomonas exotoxin A and barnase.

We have constructed a chimeric toxin composed of Pseudomonas exotoxin A (PE) and the extracellular ribonuclease of Bacillus amyloliquefaciens, barnase. The chimeric protein, termed PE-Bar, reacted with both anti-PE and anti-barnase antisera and had both ADP ribosylation and ribonuclease activities. The chimeric toxin was cytotoxic to the murine fibroblast cell line L929 and to a murine hybridoma resistant to PE. A mutant form of PE-Bar lacking ADP-ribosylating activity was still cytotoxic to L929 cells. Because treatment of cells prelabeld with [3H]uridine resulted in a decrease in their RNA content, we conclude that this cytotoxic effect was due to the ribonuclease activity of barnase molecules that had been translocated to the cytosol. It is now possible to construct chimeric toxins with two or more enzymatic activities that can be delivered to the cytosol of the target cells.

Cytotoxic activity of a recombinant fusion protein between insulin-like growth factor I and Pseudomonas exotoxin.

A chimeric toxin in which the cell binding domain of Pseudomonas exotoxin was replaced with mature human insulin-like growth factor I (IGF-I) was produced in Escherichia coli. This protein, IGF-I-PE40, was cytotoxic to human cell lines derived from a variety of tumor types, with a breast carcinoma line (MCF-7) and two hepatoma lines (HEP3B and HEPG2) showing the highest sensitivity to the toxin. The specificity of IGF-I-PE40 cytotoxicity was confirmed through competition with excess IGF-I and through blockage of toxin binding using an antibody specific to the type I IGF receptor. A potential interaction between the toxin and soluble IGF-binding proteins was also demonstrated. IGF-I-PE40 may be useful in the selective elimination of cells bearing the type I IGF receptor.

Nucleotide sequence of fruA, the gene specifying enzyme IIfru of the phosphoenolpyruvate-dependent sugar phosphotransferase system in Escherichia coli K12.

The Enzyme IIfru of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate by Escherichia coli, is specified by a gene designated fruA. The nucleotide sequence of a 2.5 kb PvuII restriction fragment spanning fruA+, cloned on a plasmid, was determined. This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator. The amino acid sequence deduced from this DNA corresponds to that of a protein of 563 amino acids (57.5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions. There is relatively little overall homology between this protein and those of other Enzymes II of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues (His381) of Enzyme IIfru and those surrounding the particular histidines of other Enzymes II, and of HPr, known to be involved in phosphorylation. A plasmid carrying the complete fruA+ nucleotide sequence, but not that of any other functional protein, fully restored the ability of fruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined species Enzyme IIfru.

Inactivation of the beta-adrenergic receptor in cardiac muscle by dithiols.

The effect of sulfhydryl reagents on binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol hydrochloride [-)-[3H]DHA) to a microsomal fraction of rabbit ventricular muscle was studied. Incubation with the disulfide reducing agents dithiothreitol (DTT), 2-mercaptoethanol, and reduced glutathione resulted in loss of (-)-[3H]DHA binding. At 500 microM DTT, less than 50% of specific binding activity remained; at 100 mM, binding was completely eliminated. 2-Mercaptoethanol and reduced glutathione were less effective than DTT at inhibiting binding activity. The total binding capacity (Bmax) decreased from 155.4 fmol mg-1 of protein, in the absence of DTT, to 92.4 and 77.5 fmol mg-1 at 0.25 and 0.7 mM DTT, respectively. The equilibrium dissociation constant (KD) increased from 7.6 nM, in the absence of DTT, to 10.3 nM at 0.25 mM DTT and to 20.8 nM at 0.7 mM DTT. Thus, DTT-induced decline in (-)-[3H]DHA binding results from a decrease in both the number and affinity of membrane binding sites for the tracer. Receptors could be protected from DTT inactivation by preincubation with beta-adrenergic ligands. Oxidants could not reverse inactivation, with the exception of o-iodosobenzoate which was only partially effective. Thus, the beta-adrenergic receptor of rabbit ventricular muscle contains essential disulfide moietie(s) which can be inactivated by reducing thiols.