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Overall Abstract: The dopamine (DA) hypothesis of schizophrenia is among the most enduring biological theories in psychiatry, and it has received some substantial modifications. For example, a major revision of the theory was made over 2 decades ago proposing that “schizophrenia can be characterized by hypodopaminergia in mesocortical and hyperdopaminergia in mesolimbic dopamine neurons”. It was also recently proposed that DA striatal hyperfunction as a “final common pathway” for the schizophrenia psychosis. Although DA dysfunction is clearly linked to psychotic symptoms, evidence for dopamine’s involvement in the negative and cognitive symptoms is much less clear-cut. Thus, other preceding abnormalities such as glutamatergic, GABAergic and neurotrophic systems, may play a crucial role in the etiopathophysiology, which could result in the dopaminergic dysfunction as a final common pathway for psychosis. It is critical to delineate the molecular and cellular mechanisms of such crosstalk that may lead to schizophrenia.

Possible phosphorylation sites on the Purkinje cell α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunits were identified using in vitro kinase assays of 17 synthetic peptides derived from the transmembrane-3 (TM3) domain to the end of C -terminal of a rat glutamate receptor 2 (GluR2). Only two peptides containing Ser-662 and Ser-696 were found to be efficiently phosphorylated by protein kinase C (PKC). The peptide including Ser-696 was also phosphorylated by protein kinase G (PKG). Another peptide containing Thr-692 of a rat GluRA, clone almost identical to GluR1, was phosphorylated by PKC but not by PKG. Antisera recognizing phosphorylated AMPA receptor subunits at GluR2 Ser-696 or the homologous sites of GluR1/3/4 were produced, and the specificity of one of them, named 12P3, was established by enzyme-linked immunosorbent assay (ELISA), immunoblot and immunoprecipitation analyses. 12P3-immunocytochemistry on cerebellar slices demonstrated an AMPA-induced transient AMPA receptor phosphorylation, which appeared in Purkinje cell dendrites as well as somata immediately after AMPA treatment and disappeared after 20 min. This antibody may be a useful tool to study the role of AMPA receptor phosphorylation in producing synaptic plasticity.

Recombinant beta-1,4-galactosyltransferase which synthesizes the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains was obtained as a soluble form from Escherichia coli by transfection of the human cDNA lacking the transmembrane segment. Kinetic study revealed that the soluble transferase has the same apparent Km values toward sugar nucleotide and sugar acceptors as those of mouse membrane-bound beta-1,4-galactosyltransferase previously characterized [Nakazawa et al. (1991) Eur. J. Biochem. 196, 363-368]. However, the Vmax value of this transferase was low when compared to that of the mammalian transferase, probably due to the instability of the transferase caused by the lack of protein glycosylation. The soluble transferase was purified from the E. coli lysates almost to homogeneity by chromatography on DEAE-Sepharose and alpha-lactalbumin-Sepharose columns. Using this purified transferase, the acceptor specificity of the transferase has been studied. The results showed that the transferase has apparent Km values of 170, 190, and 830 microM for agalacto-poly-N-acetyllactosamine, lacto-N-triose II, and lacto-N-triaosylceramide, respectively, but has apparently no activity toward glucosylceramide. These results suggest that the beta-1,4-galactosyltransferase may be involved in the synthesis of poly-N-acetyllactosamine, lacto-N-neotetraose, and probably lacto-N-neotetraosylceramide in addition to the formation of the Gal beta 1-->4GlcNAc group of glycoprotein sugar chains and lactose.

We inserted a full-length murine cDNA, which had been isolated from F9 embryonal carcinoma cells by using a bovine lactose synthetase A protein cDNA as a probe, in a mammalian expression vector (pCMGT1) and expressed it in COS-1 cells to characterize the pCMGT1-directed enzyme. The galactosyltransferase activity toward asialo-agalacto-transferrin (AsAg-Tf) in the pCMGT1-transfected cells was approximately eightfold higher than that in mock- or non-transfected cells. In contrast, no difference was observed in the specific activity of galactose transfer between pCMGT1-transfected cells and mock- or non-transfected cells when asialo-ovine submaxillary mucin were used as an acceptor. Since almost all [3H]galactose incorporated into the AsAg-Tf was released by digestion with streptococcal beta-galactosidase, most of the linkage created by this enzyme was in the Gal beta 1-4GlcNAc group. The acceptor specificity of the pCMGT1-directed enzyme was changed from N-acetylglucosamine to glucose by adding alpha-lactalbumin in the reaction mixture. Alpha-Lactalbumin also partially inhibited the galactose transfer to AsAg-Tf. The kinetic study revealed that the apparent Km values of the pCMGT1-directed enzyme for N-acetylglucosamine, AsAg-Tf and UDP-Gal are 2 mM, 60 microM and 24 microM, respectively. These results indicated that the murine cDNA isolated from F9 cells encodes an active enzyme which catalyzes not only the lactose synthesis but also the transfer of galactose to N-acetylglucosamine residues of Asn-linked sugar chains of glycoproteins in a beta 1-4 linkage.

Expression of immediate early genes (IEGs) was examined following long-term desensitization of cerebellar Purkinje cells. This form of desensitization, which may underlie synaptic long-term depression (LTD), was evoked by co-administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and 8-bromo cGMP (8-Br-cGMP). Among the IEGS examined with in situ hybridization and immunohistochemistry, combined application of 8-Br-cGMP and AMPA synergistically enhanced the expression of c-Fos and Jun-B in Purkinje cells. This may suggest a role for active transcriptional complexes such as AP-1 (c-Fos/Jun-B), which could be formed following conjoint inputs to Purkinje cells and which may help to establish cerebellar long-term plasticity.

A full-length cDNA clone for mouse N-acetylglucosamine (beta 1-4)galactosyltransferase (beta 1-4GT) [EC 2.4.1.90] and several clones diverged from the beta 1-4GT cDNA were isolated from a mouse F9 cDNA library and then sequenced. The beta 1-4GT cDNA has an open reading frame consisting of 399 amino acids. The homology at the amino acid level is 80 and 91% as to the partial sequences of bovine and human milk beta 1-4GT, respectively. The general enzyme structure of the beta 1-4GT seems to be similar to that of a rat beta-galactoside (alpha 2-6) sialyltransferase. Junctions of the common and divergent regions of cDNA have dinucleotides, AG, suggesting that the variety of cDNA clones is generated through alternative splicing.