Voluntary movements and event-related potentials in Parkinsonians (stages 1-2)

The temporal analysis of goal-directed movements has revealed that the delay of movement initiation in Parkinsonians might be decreased or abolished by altering the preparatory set and increasing the motivation level of patients. Averaged event-related potentials (ERPs) during different psychomotor reactions to light stimuli, ignore condition and difference waves between attend (reactions) and ignore conditions were analyzed in 19 Parkinsonian patients (stages 1-2) and 12 healthy subjects. A deceleration of psychomotor reactions in the patients was found to be accompanied by a decrease in ERPs, especially in frontal recording. This decrease was affected by an altered pre-stimulus preparatory set in the patients. The decrease of endogenous ERPs (difference waves) was associated to a greater extent with the motor acts than with cognitive operations. The deceleration of the voluntary movements in Parkinsonism is assumed to be connected with 'deficit' of the frontal activation due to a dysfunction of the basal ganglia.     

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene

The Visual Evoked Potential (VEP) is an electrophysiological commonly used test in investigating various neurophysiological disorders. Through the years many methods have been developed, but there are not many objective criteria in distinguishing a normal VEP waveform from an abnormal. In this communication we use the phase characteristics of the power spectrum as a criterion of distinguishing normals and abnormals. From our analysis, it was shown that the phase spectrum of a VEP has a certain periodicity in the 0- to 40-Hz region. By studying these periodicities we were able to determine the range of the period that characterizes normal and abnormal populations and to establish an experimental method for objectively examining any kind of VEP waveforms.     

Synthesis and biological investigations of 1-(tetrahydropyran-2'-yl)- and 1-(tetrahydrofuran-2'-yl)benzimidazoles and 1/2-(tetrahydropyran-2'-yl)- and 1/2-(tetrahydrofuran-2'-yl)benzotriazoles

Sets of benzimidazole and benzotriazole derivatives bearing on position 1 or 2 a tetrahydrofuranyl or tetrahydropyranyl moieties were prepared through the addition of the suitable benzazoles on 2,3-dihydrofuran and 3,4-dihydro-2H-pyran. The reactions were carried on either without solvent or in carbon tetrachloride solution. In the last case some peculiar chlorinated side products were isolated and characterized. Twenty compounds were screened for in vitro antitumoral and anti-HIV-1 activities and found poorly active or completely inactive. On the other hand several compounds exhibited good tracheal relaxant activity in vitro; compound 8, 11, 16, 24 and 26 resulted more active than theophylline in this test, while compound 11 was comparable to amrinone till the concentration of 3 micrograms/ml. Finally, compound 5 resulted endowed with a strong diuretic and saluretic activity at the dose of 3 mg/Kg, thus representing a new lead for discovering new diuretic agents.     

Synthesis and antimyoctic properties of 1-(2-alkyl-2-phenylethyl)-1H-imidazoles

The synthesis of 1-(2-alkyl-2-phenylethyl)-1H-imidazoles was accomplished starting from the corresponding phenylacetonitriles. Via alkylation, esterification, and sodium borohydride reduction-in the presence of lithium iodide-beta-phenylalconols were obtained. Mesylation of these alcohols and refluxing with imidazole in dimethylformamide furnished title compounds, which were active in vitro against dermatophytes, yeasts, other fungi, and gram-positive bacteria and in vivo as well as in vitro against Candida albicans.     

Identification and regulation of testicular interferon-gamma (IFNgamma) receptor subunits: IFNgamma enhances interferon regulatory factor-1 and interleukin-1beta converting enzyme expression

Interferon-gamma (IFNgamma) transmits its signal through a specific cell surface receptor (IFNgammaR), which consists of a primary ligand binding alpha-chain (IFNgammaR alpha) and a signaling beta-chain (IFNgammaR beta). Recent studies identified the cytokines IFNgamma, interleukin-6 (IL-6), IL-1alpha, and tumor necrosis factor-alpha in testicular cells. Therefore, we: 1) examined the expression of IFNgammaR alpha and IFNgammaR beta subunits in freshly isolated and purified rat testicular cells; 2) examined the differential regulation of receptor components by cytokines using primary cultures of Sertoli cells; 3) identified the cell signaling pathway components of testicular IFNgammaR; and 4) characterized the functional role of testicular IFNgamma using primary Sertoli cells. We demonstrated the messenger RNAs for both chains of IFNgammaR in rat testicular cells using Northern hybridization analysis. Western blot analysis and immunocytochemistry showed that both specific IFNgammaR protein subunits were present in cultured primary Leydig and Sertoli cells prepared from the testes of immature rats. The expression of both IFNgammaR component messenger RNAs in cultured Sertoli cells was increased by its specific ligand (IFNgamma), as well as IL-1alpha and tumor necrosis factor-alpha, in both a time- and dose-dependent manner. IFNgamma-activation of the Janus (JAK) tyrosine kinases, JAK1 and JAK2 proteins, indicate that IFNgammaR, expressed in the Sertoli cell, is functional. Moreover, IFNgamma modulates the expression of interferon regulatory factor (IRF)-1 and IL-1beta converting enzyme genes in Sertoli cells. Thus, our data are suggestive of a role(s) for IFN-gamma in the regulation of distinct gene expression and cell-specific sensitivity to apoptosis in the testis.     

Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.     

Stereoselective conjugation of prostaglandin A2 and prostaglandin J2 with glutathione, catalyzed by the human glutathione S-transferases A1-1, A2-2, M1a-1a, and P1-1

Prostaglandins containing an alpha,beta-unsaturated keto group, such as prostaglandin A2 (PGA2) and prostaglandin J2 (PGJ2), inhibit cell proliferation. These cyclopentenone prostaglandins may be conjugated with GSH chemically or enzymatically via glutathione S-transferases, and this has been suggested to result in inhibition of the antiproliferative mode of action. In the present study, the role of the major human GSTs in the conjugation of PGA2 and PGJ2 with GSH was investigated with purified enzymes, i.e., the Alpha-class enzymes GST A1-1 and GST A2-2, the Mu-class enzyme GST M1a-1a, and the Pi-class enzyme GST P1-1. The GSH conjugates were separated from the parent compound by HPLC and identified by fast atom bombardment mass spectrometry and 1H-NMR. Two GSH conjugates were found for both PGA2 and PGJ2, the R- and S-GSH conjugates of both prostaglandins. Incubation experiments with PGA2 and PGJ2 (70-600 microM) clearly showed the role of individual GSTs in the conjugation of PGA2 and PGJ2. Compared to the chemical reaction, enzyme activities towards PGA2 were up to 5.4 times as high (GSTA1-1) at the lowest concentration (70 microM), while at the highest concentration (600 microM) enzyme activities were up to 3.0 times as high (GST P1-1). For PGJ2, enzyme activities were up to 4.3 (GSTM1a-1a, 70 microM) and up to 3.1 (GSTM1a-1a, 600 microM) times as high. As expected, similar amounts of the R- and S-conjugates of both prostaglandins were found in the chemical reaction. Striking stereoselectivities in conjugating activities were observed for GST A1-1 and GST P1-1. GST A1-1 favors the formation of the R-GSH conjugates of both prostaglandins. GST P1-1 showed a clear selectivity with regard to the formation of the S-GSH conjugate of PGA2. However, this selectivity was not found for the formation of the S-GSH conjugate of PGJ2. GSTM1a-1a showed no stereoselectivity with regard to the GSH conjugation of both PGA2 and PGJ2. GSTA2-2 only showed some minor formation of the R-GSH conjugate of PGJ2. The possible implications of the observed stereoselectivity on the effects of PGA2 and PGJ2 are discussed.     

Expression and biochemical characterization of iron regulatory proteins 1 and 2 in Saccharomyces cerevisiae

Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms. IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activity when cellular iron is abundant or RNA-binding activity when cellular iron is scarce. In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein. To study and compare the biochemical characteristics of IRP1 and IRP2, we expressed wild-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevisiae. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affinity, resulting in the inhibition of translation of an IRE-reporter mRNA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a mutant IRP1 containing an insertion of this domain bound RNA, but lacked detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity. Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms.     

Expression of an oncogenic mutant G alpha i2 activates Ras in Rat-1 fibroblast cells

It has been reported that expression of the active mutant of heterotrimeric GTP-binding protein alpha subunit G alpha i2 transforms Rat-1 cells. However, the G alpha i2-mediated mitogenic signaling pathways remain to be elucidated. Here, we demonstrate that inducible expression of the active mutant of G alpha i2 (G alpha i2(Q205L)) activates Ras and c-Jun N-terminal kinase (JNK) in addition to extracellular signal-regulated kinase (ERK) in Rat-1 cells. Our findings suggest that Ras may play a critical role in the G alpha i2-induced transformation and G alpha i2 can transduce signals from the Gi-coupled receptor to JNK and ERK in certain types of mammalian cells.