Extended treatment with typical and atypical antipsychotic drugs differential effects on the densities of dopamine D2-like and GABAA receptors in rat striatum

In situ radioligand binding and quantitative autoradiography have been used to measure the density of striatal D1-like, D2-like, and GABAA receptors in rats treated with haloperidol at 0.01 or 0.1 mg/kg/ day or chlorpromazine, olanzapine or clozapine at 0.1 or 1.0 mg/kg/day for 1, 3 or 7 months. [3H]SCH23390 binding to D1-like receptors was not changed by any drug treatments. There were significant increases in [3H]nemonapride binding to D2-like receptors at different time points due to treatment with haloperidol, chlorpromazine and olanzapine. By contrast, treatment with clozapine and olanzapine caused a time-dependent decrease in [3H]muscimol binding to the GABAA receptor. These data suggest that treatment with atypical antipsychotic drugs, but not typical antipsychotic drugs, affect striatal GABAergic neurons. In addition, it would appear that clozapine might be unique in that it does not increase dopamine-D2 like receptor density at doses which would be predicted to have antipsychotic effects in humans. The extent to which such changes are involved in the therapeutic effects of drugs such as olanzapine and clozapine remains to be determined.     

SH3-domain-containing proteins function at distinct steps in clathrin-coated vesicle formation

Several SH3-domain-containing proteins have been implicated in endocytosis by virtue of their interactions with dynamin; however, their functions remain undefined. Here we report the efficient reconstitution of ATP-, GTP-, cytosol- and dynamin-dependent formation of clathrin-coated vesicles in permeabilized 3T3-L1 cells. The SH3 domains of intersectin, endophilin I, syndapin I and amphiphysin II inhibit coated-vesicle formation in vitro through interactions with membrane-associated proteins. Most of the SH3 domains tested selectively inhibit late events involving membrane fission, but the SH3A domain of intersectin uniquely inhibits intermediate events leading to the formation of constricted coated pits. These results suggest that interactions between SH3 domains and their partners function sequentially in endocytic coated-vesicle formation.     

Physiological effects and biochemical properties of a serum protein that produces positive inotropic and chronotropic effects on isolated guinea pig atria

Isolated left and right guinea pig atria were used as a bioassay for the detection of an endogenous cardioactive substance in bovine serum. Serum, buffer exchanged to Krebs-Henseleit solution, produced positive inotropic and chronotropic effects on the isolated guinea pig atria. The cardiotonic effects were unaffected by the combined presence of propranolol and methysergide (both 10(-6)M) and were also dissimilar in time course from other known cardiotons such as catecholamines and cardiac glycosides. Following ultrafiltration (using XM100A Amicon membranes), activity was found solely in the retentate fractions and was therefore probably due to a large molecular weight (> 100 kDa) substance or a small molecule bound to a large protein. The cardioactive factor (CF) in the whole serum was heat labile, sensitive to acidification, exposure to potassium bromide and equilibration to physiological buffers of a low ionic strength. Isolation by conventional protein purification techniques was unsuccessful due to the labile nature of the active molecule(s) when exposed to non-physiological experimental conditions. Physical and biochemical properties of the CF which may help avoid inactivation are discussed for future experiments aimed at elucidating the nature and identity of the cardiotonic principle.     

Effect of reactive oxygen species on the metabolism of tryptophan in rat brain: Influence of age

In an earlier study, oxidation of tryptophan hydroxylase was implicated as its affinity was decreased with aging in rat brain. To establish any potential link between its oxidative damage and aging, we have determined the activities of antioxidant enzymes in midbrain, pons and medulla of 2, 12 and 24 month old Fisher 344 BNF1 rats. The results obtained suggest that the activities of antioxidant enzymes varied considerably with age and brain regions studied. Activities of Cu/Zn superoxide dismutase and glutathione peroxidase were found to increase from 2 to 12 months and then decrease in 24 month old rats. However catalase activity decreased consistently with the age. A parallel increase in the carbonyl content was observed in these brain regions indicating the oxidation of proteins. Reactive oxygen species when included in the incubation mixture decreased the activity of tryptophan hydroxylase in a concentration dependent manner. The loss of tryptophan hydroxylase activity induced by hydrogen peroxide and superoxide anion was prevented by catalase. However superoxide dismutase did not provide such protection. Sulfhydryl agents, cysteine, glutathione and dithiothreitol partially prevented the loss of activity. These studies suggest an involvement of reactive oxygen species for sulfhydryl oxidation of tryptophan hydroxylase in aging.     

Origins of chromosomal rearrangement hotspots in the human genome: evidence from the <Emphasis Type="Italic">AZFa</Emphasis>deletion hotspots

Background  

The origins of the recombination hotspots that are a common feature of both allelic and non-allelic homologous recombination in the human genome are poorly understood. We have investigated, by comparative sequencing, the evolution of two hotspots of non-allelic homologous recombination on the Y chromosome that lie within paralogous sequences known to sponsor deletions resulting in male infertility.     

P-type ATPase heavy metal transporters with roles in essential zinc homeostasis in Arabidopsis

Arabidopsis thaliana has eight genes encoding members of the type 1_B heavy metal–transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.     

Microsomal triglyceride transfer protein and its role in apoB-lipoprotein assembly

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are necessary for lipoprotein assembly. ApoB consists of five structural domains, betaalpha(1)-beta(1)-alpha(2)-beta(2)-alpha(3). We propose that MTP contains three structural motifs (N-terminal beta-barrel, central alpha-helix, and C-terminal lipid cavity) and three functional domains (lipid transfer, membrane associating, and apoB binding). MTP's lipid transfer activity is required for the assembly of lipoproteins. This activity renders nascent apoB secretion-competent and may be involved in the import of triglycerides into the lumen of endoplasmic reticulum. In addition, MTP binds to apoB with high affinity involving ionic interactions. MTP interacts at multiple sites in the N-terminal betaalpha(1) structural domain of apoB. A novel antagonist that inhibits apoB-MTP binding decreases apoB secretion. Furthermore, site-directed mutagenesis and deletion analyses that inhibit apoB-MTP binding decrease apoB secretion. Lipids modulate protein-protein interactions between apoB and MTP. Lipids associated with MTP increase apoB-MTP binding whereas lipids associated with apoB decrease this binding. Thus, specific antagonist, site-directed mutagenesis, deletion analyses, and modulation studies support the notion that apoB-MTP binding plays a role in lipoprotein biogenesis. However, specific steps in lipoprotein assembly that require apoB-MTP binding have not been identified. ApoB-MTP binding may be important for the prevention of degradation and lipidation of nascent apoB.     

IL-1 receptor-associated kinase 1 regulates susceptibility to organ-specific autoimmunity

Infections often precede the development of autoimmunity. Correlation between infection with a specific pathogen and a particular autoimmune disease ranges from moderately strong to quite weak. This lack of correspondence suggests that autoimmunity may result from microbial activation of a generic, as opposed to pathogen-specific host-defense response. The Toll-like receptors, essential to host recognition of microbial invasion, signal through a common, highly conserved pathway, activate innate immunity, and control adaptive immune responses. To determine the influence of Toll/IL-1 signaling on the development of autoimmunity, the responses of wild-type (WT) mice and IL-1R-associated kinase 1 (IRAK1)-deficient mice to induction of experimental autoimmune encephalomyelitis were compared. C57BL/6 and B6.IRAK1-deficient mice were immunized with MOG 35-55/CFA or MOG 35-55/CpG DNA/IFA. WT animals developed severe disease, whereas IRAK1-deficient mice were resistant to experimental autoimmune encephalomyelitis, exhibiting little or no CNS inflammation. IRAK1-deficient T cells also displayed impaired Th1 development, particularly during disease induction, despite normal TCR signaling. These results suggest that IRAK1 and the Toll/IL-1 pathway play an essential role in T cell priming, and demonstrate one means through which innate immunity can control subsequent development of autoimmunity. These findings may also help explain the association between antecedent infection and the development or exacerbations of some autoimmune diseases.