Activation of the Jnk signaling pathway by a dual-specificity phosphatase, JSP-1.

The mitogen-activated protein kinases (MAPKs) are integral to the mechanisms by which cells respond to physiological stimuli, such as growth factors, hormones, and cytokines, and to a wide variety of environmental stresses. The MAPKs, which are stimulated by phosphorylation of a TXY motif in their activation loop, are components of signal transduction cascades in which sequential activation of protein kinases culminates in their activation and their subsequent phosphorylation of various effector proteins that mediate the physiological response. MAPKs are also subject to dephosphorylation and inactivation, both by enzymes that recognize the residues of the TXY motif independently and by dual specificity phosphatases, which dephosphroylate both Tyr and Ser/Thr residues. We report the identification and characterization of a novel dual specificity phosphatase. Contrary to expectation, this broadly expressed enzyme did not inactivate MAPKs in transient cotransfection assays but instead displayed the capacity to function as a selective activator of the MAPK Jnk, hence the name, Jnk Stimulatory Phosphatase-1 (JSP-1). This study illustrates a new aspect of the regulation of MAPK-dependent signal transduction and raises the possibility that JSP-1 may offer a different perspective to the study of various inflammatory and proliferative disorders associated with dysfunctional Jnk signaling.

Targeted expression of human CuZn superoxide dismutase gene in mouse central nervous system.

Copper zinc superoxide dismutase (CuZnSOD) is an important enzyme for the detoxification of reactive oxygen species. Particularly in the central nervous system (CNS), reactive oxygen species are often associated with acute brain injuries and chronic neurodegeneration. It has been demonstrated in vivo that there is an inverse correlation between CuZnSOD activity and neuronal death after acute brain injury. To further understand the protective role of CuZnSOD upon neurons, we have generated transgenic mouse lines with targeted expression of the human CuZnSOD gene (SOD1) that is driven by a rat neuron-specific enolase gene promoter in neurons of the CNS. The transgenic SOD1 expression was restricted to the CNS identified by reverse transcriptase polymerase chain reaction and SOD gel electrophoresis assays. The CuZnSOD activity was significantly increased in the brain stem of the transgenic mice. Immunostaining of human CuZnSOD activity showed that Purkinje cells in the cerebellar cortex were the most intensely stained neurons in the CNS of the transgenic mice.

Novel mutations in an otherwise strictly conserved domain of CuZn superoxide dismutase.

All mutations in the human gene for CuZn superoxide dismutase (CuZnSOD) reported to date are associated with the disease amyotrophic lateral sclerosis (ALS). These mutations, mostly of a familial nature (ALS 1, MIM 105400), span all of the coding region of this enzyme except for a highly conserved centrally located domain that includes all of exon III. We describe the identification and characterization of two mutations in this region, both found in mice. One mutation, a glutamate to lysine amino acid substitution was found in position 77 (E77K) of the strain SOD1/Ei distributed by the Jackson Laboratory. The other mutation, a lysine to glutamate substitution at position 70 (K70E) of a human transgene, was discovered in mouse line TgHS/SF-155. Enzyme activity measurements and heterodimer analysis of the CuZn SOD variant in SOD1/Ei suggest a mild loss of activity, which differs from the enzyme activity losses detected in patients with autosomal dominant ALS 1. Similarly, the presence of the mutant transgene in TgHS/SF 155 does not produce any phenotypic manifestations.

Transgenic copper/zinc superoxide dismutase modulates susceptibility to type I diabetes.

A growing body of evidence suggests that active oxygen is an important participant in the destruction of the pancreatic beta cell, which, in turn, leads to type I or insulin-dependent diabetes mellitus. Consequently, genetic factors predisposing susceptibility to insulin-dependent diabetes mellitus may include those that determine active oxygen metabolism. A direct test of this hypothesis is provided by a transgenic model for increased activity of Cu/Zn superoxide dismutase (EC 1.15.1.1), a principal radical scavenging enzyme. Here we demonstrate that elevated levels of this enzyme provided by a Cu/Zn superoxide dismutase transgene enhance the tolerance of pancreatic beta cells to oxidative stress-induced diabetogenesis. These results show that this transgenic approach holds promise for revealing the role of reactive oxygen in autoimmune models of diabetogenesis as well as in other models of disease pathology in which active oxygen has been implicated.

Saccharomyces cerevisiae BUF protein binds to sequences participating in DNA replication in addition to those mediating transcriptional repression (URS1) and activation.

The heteromeric BUF protein was originally shown to bind to URS1 elements which are situated upstream of many genes in Saccharomyces cerevisiae and mediate negative control of their transcription. Among the genes regulated through the URS1 site and the proteins interacting with it are those participating in carbon, nitrogen, and inositol metabolism; electron transport; meiosis; sporulation; and mating-type switching. We show here that pure BUF protein, in addition to binding to the negatively acting URS1 site, also binds to CAR1 sequences supporting transcriptional activation (upstream activation sequences). To determine the BUF protein structure, we cloned and sequenced the BUF1 and BUF2 genes and found them to be identical to the RF-A (RP-A) gene whose products participate in yeast DNA replication as single-stranded DNA binding proteins. These data argue that BUF protein-binding sites serve multiple roles in transcription and replication.

Purification of the heteromeric protein binding to the URS1 transcriptional repression site in Saccharomyces cerevisiae.

The protein that binds to the URS1 site situated upstream of many genes in Saccharomyces cerevisiae is a central element responsible for global negative control of transcription in this organism. Among the genes whose expression is regulated by this protein are those that participate in nitrogen metabolism, carbon metabolism, electron transport, inositol metabolism, heat shock response, meiosis, and sporulation. This factor, binding URS1 factor (BUF), has been purified and shown to be a heteromeric protein composed of 37.5- and 73.5-kDa monomers. The heteromeric form of BUF is stably maintained both in solution and bound to its DNA target site.

The yeast UME6 gene product is required for transcriptional repression mediated by the CAR1 URS1 repressor binding site.

URS1 is known to be a repressor binding site in Saccharomyces cerevisiae that negatively regulates expression of many genes including CAR1 (arginase), several required for sporulation, mating type switching, inositol metabolism, and oxidative carbon metabolism. In addition to the proteins previously shown to directly bind to the URS1 site, we show here that the UME6 gene product is required for URS1 to mediate repression of gene expression in the absence of inducer. We also show that mutations in the CAR80 (CARGRI) gene are allelic to those in UME6.

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion.

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

[The prevalence of smoking in psychiatric patients. The effect of "institutionalization"]. / La prevalenza del fumo di tabacco nei pazienti psichiatrici. L'influenza dell'istituzionalizzazione.

The prevalence and other parameters concerning tobacco smoking were assessed in 100 outpatients with psychiatric disorders of axis I and II, according to DSM-III R criteria, and compared with those of 85 male psychiatric inpatients with forensic problems, recruited in a psychiatric hospital. The use of tobacco smoking was increased in psychiatric outpatients vs. the general pupulation and was very heightened in schizophrenic, as reported in earlier investigations; in the forensic sample both schizophrenic and non schizophrenic patients showed a very high prevalence. Therefore, the factor "institutionalization" does not modify the prevalence of tobacco smoking in schizophrenic--whose "smoking disposition" appears independent of the different social environments--while increasing the use in non schizophrenic patients. The positive correlation between tobacco smoking and schizophrenia is largely supported by these data and demands further investigation of its pathophysiologic mechanisms.

A cis-acting element present in multiple genes serves as a repressor protein binding site for the yeast CAR1 gene.

Induction of the arginase (CAR1) gene expression in Saccharomyces cerevisiae has previously been shown to require participation of a cis-dominantly regulated upstream repression sequence (URS). Deletion of this element results in high-level expression of the CAR1 gene without inducer. To determine the structure of the CAR1 URS element, we performed a saturation mutagenesis. Results of the mutagenic analysis indicated that the CAR1 URS was a 9-base-pair palindromic sequence, 5'-AGCCGCCGA-3'. A DNA fragment containing this sequence was shown to bind one or more proteins by a gel shift assay. DNA fragments containing point mutations that completely eliminated URS function were not effective competitors in this assay, whereas those which supported URS function were effective competitors. Sequences in the 5'-flanking regions of 14 other genes were found to be homologous to the CAR1 URS. These sequences were shown to support varying degrees of URS function in the expression vector assay, to bind protein as demonstrated by the gel shift assay, and to compete with a DNA fragment containing the CAR1 URS for protein binding. These results indicate that the CAR1 URS element possesses the characteristics of a repressor binding site. Further, they are consistent with the suggestion that sites homologous to the CAR1 URS may be situated in the 5'-flanking regions of multiple unrelated yeast genes. The widespread occurrence of this element raises the possibility that it is the target site for one or more negatively acting general transcription factors.