ZNF313 is a novel cell cycle activator with an E3 ligase activity inhibiting cellular senescence by destabilizing p21(WAF1.).

ZNF313 encoding a zinc-binding protein is located at chromosome 20q13.13, which exhibits a frequent genomic amplification in multiple human cancers. However, the biological function of ZNF313 remains largely undefined. Here we report that ZNF313 is an ubiquitin E3 ligase that has a critical role in the regulation of cell cycle progression, differentiation and senescence. In this study, ZNF313 is initially identified as a XIAP-associated factor 1 (XAF1)-interacting protein, which upregulates the stability and proapoptotic effect of XAF1. Intriguingly, we found that ZNF313 activates cell cycle progression and suppresses cellular senescence through the RING domain-mediated degradation of p21(WAF1). ZNF313 ubiquitinates p21(WAF1) and also destabilizes p27(KIP1) and p57(KIP2), three members of the CDK-interacting protein (CIP)/kinase inhibitor protein (KIP) family of cyclin-dependent kinase inhibitors, whereas it does not affect the stability of the inhibitor of CDK (INK4) family members, such as p16(INK4A) and p15(INK4B). ZNF313 expression is tightly controlled during the cell cycle and its elevation at the late G1 phase is crucial for the G1-to-S phase transition. ZNF313 is induced by mitogenic growth factors and its blockade profoundly delays cell cycle progression and accelerates p21(WAF1)-mediated senescence. Both replicative and stress-induced senescence are accompanied with ZNF313 reduction. ZNF313 is downregulated during cellular differentiation process in vitro and in vivo, while it is commonly upregulated in many types of cancer cells. ZNF313 shows both the nuclear and cytoplasmic localization in epithelial cells of normal tissues, but exhibits an intense cytoplasmic distribution in carcinoma cells of tumor tissues. Collectively, ZNF313 is a novel E3 ligase for p21(WAF1), whose alteration might be implicated in the pathogenesis of several human diseases, including cancers.

Emergency rescue primary stenting for atherosclerotic basilar artery occlusion with acute thrombosis. A case report.

SUMMARY: We demonstrate endovascular stent deployment for the treatment of atherosclerotic basilar artery occlusion with acute thrombosis. Application of a microstent without previous balloon dilatation resulted in vessel reopening and good clinical improvement. Emergency primary stent application can be technically feasible and improve the outcome in acute basilar artery occlusion and clinical status.

Caspase-cleaved TRAF1 negatively regulates the antiapoptotic signals of TRAF2 during TNF-induced cell death.

Tumor necrosis factor (TNF) signaling leads to pleiotropic responses in a wide range of cell types, in part by activating antiapoptotic and proapoptotic pathways. Previous studies have suggested that TNF receptor-associated factor (TRAF) 2 can mediate crucial antiapoptotic signals during TNF stimulation. However, it is unclear how the antiapoptotic signals via TRAF2 in TNF-R1 signaling is regulated. Here we show that TRAF1 is cleaved by caspase-8 into two fragments during apoptosis induced by TNF. Overexpression of the C-terminal cleavage product, TRAF1-c, increased TNF-induced cell death of hybridoma T cells. Importantly, we demonstrate that the cleavage product of TRAF1 coimmunoprecipitates with TRAF2 that is released from the TNF-R1 complex in response to prolonged TNF treatment. These results indicate that caspase-dependent cleavage of TRAF1 generates TRAF1-c fragments that are able to bind TRAF2, and then sequester TRAF2 from the TNF-R1 complex, rendering cells, at least in part, sensitive to TNF.

Differential regulation of cAMP-mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors.

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of alpha-melanocyte-stimulating hormone (alpha-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, alpha-MSH-ND was the most efficient alpha-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of alpha-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor-ligand complex by NMR, [Gln6]alpha-MSH-ND and [Lys6]alpha-MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30-50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor-ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.

Distinct functions of the two isoforms of dopamine D2 receptors.

Signalling through dopamine D2 receptors governs physiological functions related to locomotion, hormone production and drug abuse. D2 receptors are also known targets of antipsychotic drugs that are used to treat neuropsychiatric disorders such as schizophrenia. By a mechanism of alternative splicing, the D2 receptor gene encodes two molecularly distinct isoforms, D2S and D2L, previously thought to have the same function. Here we show that these receptors have distinct functions in vivo; D2L acts mainly at postsynaptic sites and D2S serves presynaptic autoreceptor functions. The cataleptic effects of the widely used antipsychotic haloperidol are absent in D2L-deficient mice. This suggests that D2L is targeted by haloperidol, with implications for treatment of neuropsychiatric disorders. The absence of D2L reveals that D2S inhibits D1 receptor-mediated functions, uncovering a circuit of signalling interference between dopamine receptors.

Type I beta-turn conformation is important for biological activity of the melanocyte-stimulating hormone analogues.

In order to define which structure of alpha-melanocyte-stimulating hormone (MSH) analogues plays a critical role for ligand-receptor interaction and selectivity, we analysed receptor-binding and cAMP-generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized alpha-MSH analogues. Compared with [Ahx4]alpha-MSH, the linear MTII designated as alpha-MSH-ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of alpha-MSH-ND remarkably decreased the receptor-binding and cAMP-generating activity. Meanwhile, maximum cAMP-generating activity was observed at a higher concentration (10(-5) M) of alpha-MSH-ND(6-10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]alpha-MSH-ND(6-10) lost its cAMP-generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of alpha-MSH-ND revealed a stable type I beta-turn structure, [Gln6]alpha-MSH-ND(6-10) revealed a tight gamma-turn composed of Gln6-D-Phe7-Arg8. Replacement of the His6 residue of alpha-MSH-ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP-generating activity in both the MC3R and the MC4R. The structure of [Gln6]alpha-MSH-ND exhibited a stable type I' beta-turn comprising Asp5, Gln6, D-Phe7 and Arg8. [Lys6]alpha-MSH-ND showed a greatly reduced binding affinity and cAMP-generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]alpha-MSH-ND also demonstrated a gamma-turn conformation around Lys6-DPhe7-Arg8. From the above results, we conclude that a type I beta-turn conformation comprising the residues Asp5-His6-(D-Phe7)-Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.

G protein-mediated mitogen-activated protein kinase activation by two dopamine D2 receptors.

Two isoforms of dopamine D2 receptor, D2L (long) and D2S (short), differ by the insertion of 29 amino acids specific to D2L within the putative third intracellular loop of the receptor, which appears to be important in selectivity for G-protein coupling. We have generated D2L- and D2S-expressing Chinese hamster ovary (CHO) cells, and regulation of the mitogen-activated protein kinase (MAPK) pathway was examined in these cells. Both D2L and D2S mediated a rapid and transient activation of MAPK with dominant activation of p42-kDa MAPK. Pertussis toxin treatment completely abrogated stimulation of MAPK mediated by D2L and D2S, demonstrating that both receptors couple to pertussis toxin-sensitive G proteins in this signaling. Stimulation of MAPK mediated by both D2L and D2S receptor was markedly attenuated by coexpression of the C-terminus of beta-adrenergic receptor kinase (betaARKct), which selectively inhibits Gbetagamma-mediated signal transduction. Further analysis of D2L- and D2S-mediated MAPK activation demonstrated that D2L-mediated MAPK activation was not significantly affected by PKC depletion or partially affected by genistein. In contrast, D2S-mediated MAPK activation was potentially inhibited by PKC depletion and genistein was capable of completely inhibiting D2S-mediated MAPK activation. Together, these results suggest that D2L- and D2S-mediated MAPK activation is predominantly Gbetagamma subunit-mediated signaling and that protein kinase C and tyrosine phosphorylations are involved in these signaling pathways.

Antiproliferative role of dopamine: loss of D2 receptors causes hormonal dysfunction and pituitary hyperplasia.

The function of dopamine (DA) in the nervous system is paralleled by its neuroendocrine control of pituitary gland functions. Here, we document the neuroendocrine function of dopamine by studying the pituitary gland of mice lacking DA D2 receptors (D2R). These mice present a striking, progressive increase in lactotroph number, which ultimately leads to tumors in aged animals. Females develop tumors much earlier than males. An estrogen-mediated lactotroph proliferation cannot account for this sexual dimorphism, since D2R-null females are hypoestrogenic and, thus, have estrogen levels similar to males. In contrast, prolactin levels are six times higher in females than in males. We show that active prolactin receptors are present in the pituitary and their expression increases in concomitance with tumor expansion. These results point to prolactin as an autocrine proliferative factor in the pituitary gland. Additionally, they demonstrate an antiproliferative function for DA regulated through D2 receptor activation.

Abnormal synaptic plasticity in the striatum of mice lacking dopamine D2 receptors.

Dopamine D2 receptors (D2Rs) are of crucial importance in the striatal processing of motor information received from the cortex. Disruption of the D2R gene function in mice results in a severe locomotor impairment. This phenotype has analogies with Parkinson's disease symptoms. D2R-null mice were used to investigate the role of this receptor in the generation of striatal synaptic plasticity. Tetanic stimulation of corticostriatal fibers produced long-term depression (LTD) of EPSPs in slices from wild-type (WT) mice. Strikingly, recordings from D2R-null mice showed the converse: long-term potentiation (LTP). This LTP, unlike LTD, was blocked by an NMDA receptor antagonist. In magnesium-free medium, LTP was also revealed in WT mice and found to be enhanced by L-sulpiride, a D2R antagonist, whereas it was reversed into LTD by LY 17555, a D2R agonist. In D2R-null mice this modulation was lost. Thus, our study indicates that D2Rs play a key role in mechanisms underlying the direction of long-term changes in synaptic efficacy in the striatum. It also shows that an imbalance between D2R and NMDA receptor activity induces altered synaptic plasticity at corticostriatal synapses. This abnormal synaptic plasticity might cause the movement disorders observed in Parkinson's disease.

Dopamine D2 receptors in signal transduction and behavior.

The dopamine D2 receptor belongs to the family of seven transmembrane domain G-protein-coupled receptors and is highly expressed in the central nervous system and the pituitary gland. The binding of dopamine to the D2 receptor is crucial for the regulation of diverse physiological functions, such as the control of locomotor activity and the synthesis of peptide hormones. Two alternatively spliced transcripts are generated from the D2 receptor gene and code for the D2L and D2S isoforms, which are 444 and 415 amino acids in length, respectively. These isoforms exhibit similar pharmacological characteristics and are expressed in the same cell types, with a ratio that normally favors expression of the longer isoform. The D2L isoform differs from D2S by the insertion of 29 amino acids in the putative third intracellular loop of the receptor. This loop is involved in the coupling of the receptor to different G proteins. Experiments have shown that the D2 isoforms have different G-protein-coupling affinities, suggesting that these receptors might serve different functions in vivo. Additionally, this difference in coupling affinity could be a mechanism to amplify the signal transduced by the binding of dopamine to D2 receptors. Important insights into D2 receptor function in vivo have been obtained by knocking out the D2 gene in mice. The Parkinsonian-like phenotype of D2-null mice demonstrates the importance of the D2 receptor for locomotor function.

High-level expression of functional human gamma-glutamyl transpeptidase using the baculovirus system.

The understanding of the structure and function of gamma-glutamyl transpeptidase (GGT) has been hindered by the difficulty of obtaining large quantities of functional enzyme. A recombinant baculovirus, encoding the human hepatoma cell (Hep G2) GGT, was easily purified using a histochemical procedure to reveal GGT activity. Infected insect cells synthesized a large amount of enzymatically active GGT representing up to 10% of the total cell extract protein. The GGT specific activity of the infected cells was 13 units per mg of protein which is the highest GGT expression level reported to date, 260-times more than in Hep G2 cells. The recombinant protein displayed an apparent molecular mass (M(r), 58,000 for the heavy subunit), immunoreactivity and catalytic features similar to those of the native protein. The high-level expression of functional GGT should provide an excellent tool to further study the structure-function relationships of the protein.

Parkinsonian-like locomotor impairment in mice lacking dopamine D2 receptors.

Dopaminergic neuronal pathways arise from mesencephalic nuclei and project axons to the striatum, cortex, limbic system and hypothalamus. Through these pathways dopamine affects many physiological functions, such as the control of coordinated movement and hormone secretion. Here we have studied the physiological involvement of the dopamine D2 receptors in dopaminergic transmission, using homologous recombination to generate D2-receptor-deficient mice. Absence of D2 receptors leads to animals that are akinetic and bradykinetic in behavioural tests, and which show significantly reduced spontaneous movements. This phenotype presents analogies with symptoms characteristic of Parkinson's disease. Our study shows that D2 receptors have a key role in the dopaminergic control of nervous function. These mice have therapeutic potential as a model for investigating and correcting dysfunctions of the dopaminergic system.

Tissue- and developmental-stage-specific methylation in the two kidney promoters of the rat gamma-glutamyl transpeptidase gene.

We have investigated, using DNA methylation patterning, the site-specific methylation of promoters I and II of the rat gamma-glutamyl transpeptidase gene. This analysis was done in fetal, newborn and adult rat kidney, in which promoters I and II are progressively active during development, as well as in rat liver, which never expresses mRNAs from these two promoters. During kidney development, a progressive demethylation occurs in the promoter I and II region, specially at the level of the most proximal MspI site of promoter II. A progressive reorganization of the methylated sites within the 5' end of the gene also occurs during liver development.

Repetitive 5-azacytidine treatments of Fao cells induce a stable and strong expression of gamma-glutamyl transpeptidase.

The role of DNA methylation in the expression of the rat gamma-glutamyl transpeptidase (GGT) gene was assessed in the Fao cell line using a hypomethylating agent, 5-azacytidine. Ten repetitive treatments of the cells, with 8 microM 5-azacytidine for 24 h, led to 13- and 80-fold increases, respectively, in GGT activity and in GGT mRNA level. The DNA methylation patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I indicated that the GGT gene, highly methylated in Fao cells, became strongly demethylated after 5-azacytidine treatments. Thus, DNA demethylation increases the expression of the GGT gene. 5-Azacytidine treatments also increased, but to a lesser extent, mRNAs level for actin, albumin, mitochondrial aspartate aminotransferase, aldolase B mRNAs (12- to 16-fold) as well as for tubulin, gluthathione transferase, and tyrosine aminotransferase mRNAs (2- to 5-fold). The GGT gene expression was further studied in B4 cells, cloned from the demethylated Fao cell population. This clone B4 exhibited a stable and strong GGT activity and a highly demethylated GGT gene. Among the three GGT mRNA I, II, or III, transcribed from three different promoters of the single rat GGT gene, only mRNA III was detected in Fao cells and was increased in clone B4, indicating that the demethylation acts on the promoter for mRNA III. The analysis of the differentiation state of B4 cells, as compared to Fao cells, showed a loss of the regulation of GGT and aspartate aminotransferase genes by dexamethasone, as well as a loss of the gluconeogenic pathway. Interestingly, B4 cells have retained many other specific functions of hepatic differentiation and have acquired alpha-fetoprotein expression; thus this clone exhibits the characteristics of a hepatic fetal phenotype.

DNA methylation patterns of the rat gamma-glutamyl transpeptidase gene in embryonic, adult and neoplastic liver.

The methylation status of the rat gamma-glutamyl transpeptidase (GGT) gene was investigated during liver development and hepatocarcinogenesis. The analysis with the restriction enzymes MspI/HpaII revealed that, during ontogeny, there is a progressive methylation of the GGT gene that coincides with a progressive decrease in GGT activity. Thus, there is an inverse correlation between methylation and expression of the GGT gene, suggesting a role for DNA methylation in the regulation of the gene during normal differentiation. The methylation patterns of the GGT gene in liver tumours induced by aflatoxin B1 exhibit heterogeneity. Nevertheless, a band of 5.7 kb was observed in all the DNA samples from aflatoxin B1-induced tumours which was not present in control liver DNA. The specificity of the DNA methylation changes was assessed using nafenopin, which induces hepatic tumours without elevation of GGT activity. We conclude that, during hepatocarcinogenesis, there is a modification of the DNA methylation pattern of the GGT gene, but there is no simple correlation with GGT activity. In no case was the GGT gene methylation in hepatocarcinogenesis found to be equivalent to the pattern observed in fetal liver. Thus if methylation is involved in the regulation of GGT gene transcription, the mechanisms must be different in fetal liver and hepatocarcinoma.

Significant changes of bronchial responsiveness to methacholine after early asthmatic reaction to toluene diisocyanate (TDI) in a TDI-sensitive asthmatic worker.

Current asthma is often diagnostically excluded by the presence of normal bronchial responsiveness. We report on a TDI-induced occupational asthma patient with normal bronchial responsiveness. He had suffered from shortness of breath during and after TDI exposure for several months. His initial methacholine bronchial challenge test showed a negative response. The bronchoprovacation test with TDI showed an isolated immediate bronchoconstriction. The following methacholine bronchial challenge tests revealed that the bronchial hyperresponsiveness developed seven hours after the TDI challenge (methacholine PC20:5.1 mg/ml), progressed up until 24 hours, and returned to normal on the seventh day. This case provides evidence that the response of the airway to TDI may not always be accompanied by bronchial hyperresponsiveness to methacholine. Screening programs utilizing methacholine challenges may not always identify TDI-sensitized asthmatic workers.

An alternatively processed mRNA specific for gamma-glutamyl transpeptidase in human tissues.

Human gamma-glutamyl transpeptidase (GGT)1 is composed of two subunits derived from a single precursor (Nash, B., and Tate, S.S. (1984) J. Biol. Chem. 259, 678-685; Finidori, J., Laperche, Y., Tsapis, R., Barouki, R., Guellaën, G., and Hanoune, J. (1984) J. Biol. Chem. 259, 4687-4690) consisting of 569 amino acids (Laperche, Y., Bulle, F., Aissani, T., Chobert, M.N., Aggerbeck, M., Hanoune, J., and Guellaën, G. (1986) Proc Natl. Acad. Sci. U.S.A. 83, 937-941). In the present study we report the cloning of an altered form of this precursor from human liver. We have isolated two clones, one 2,632 base pairs (bp) long from a fetal liver cDNA library and one 926 bp long from an adult liver cDNA library, each containing a 22-bp insertion that introduces a premature stop codon and shortens the open reading frame to 1,098 bp when compared with known human cDNA sequences specific for GGT. Sequence analysis of a human genomic GGT clone shows that this insertion of 22 bp is generated by a splicing event involving an alternative 3'-acceptor site. By polymerase chain reaction experiments we demonstrate that the alternatively spliced mRNA is present in polysomes from the microsomal fraction of a human hepatoma cell line (Hep G2) and thus could encode an altered GGT molecule of 39,300 Da (366 amino acids) encompassing most of the heavy subunit which is normally 41,500 Da (380 amino acids). The altered mRNA is detected in various human tissues including liver, kidney, brain, intestine, stomach, placenta, and mammary gland. This report is the first demonstration of an alternative primary sequence in the mRNA coding for GGT, a finding that could be related to the presence of some inactive forms of GGT detected in human tissues.

Different gamma-glutamyl transpeptidase mRNAs are expressed in human liver and kidney.

In human, the two subunits of gamma-glutamyl transpeptidase (GGT) arise from a common precursor encoded by a multigene family. Until now, a single specific coding sequence for this precursor (type I) has been identified in human placenta and liver. In the present study, we have isolated from a human kidney cDNA library, a GGT specific clone (0.8 Kb). The sequence of which (type II) i) covers the carboxy terminal part of the GGT precursor, ii) exhibits 22 point mutations and a 30 bp deletion as compared to the type I GGT sequence. The sequencing of a human genomic clone reveals that this type II GGT mRNA is encoded by a different gene than the type I GGT mRNA. Both type I and type II GGT mRNAs are expressed in human liver, while almost exclusively type II GGT mRNA is detected in human kidney.