Understanding how p53 activity is regulated is crucial in elucidating mechanisms of cellular defense against cancer. Genetic data indicate that Mdmx as well as Mdm2 plays a major role in maintaining p53 activity at low levels in nonstressed cells. However, biochemical mechanisms of how Mdmx regulates p53 activity are not well understood. Through identification of Mdmx-binding proteins, we found that 14-3-3 proteins are associated with Mdmx. Mdmx harbors a consensus sequence for binding of 14-3-3. Serine 367 (S367) is located within the putative binding sequence for 14-3-3, and its substitution with alanine (S367A) abolishes binding of Mdmx to 14-3-3. Transfection assays indicated that the S367A mutation, in cooperation with Mdm2, enhances the ability of Mdmx to repress the transcriptional activity of p53. The S367A mutant is more resistant to Mdm2-dependent ubiquitination and degradation than wild-type Mdmx, and Mdmx phosphorylated at S367 is preferentially degraded by Mdm2. Several types of DNA damage markedly enhance S367 phosphorylation, coinciding with increased binding of Mdmx to 14-3-3 and accelerated Mdmx degradation. Furthermore, promotion of growth of normal human fibroblasts after introduction of Mdmx is enhanced by the S367 mutation. We propose that Mdmx phosphorylation at S367 plays an important role in p53 activation after DNA damage by triggering Mdm2-dependent degradation of Mdmx.

Neuropharmacological actions of a novel metabotropic glutamate receptor agonist, (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV), were examined in the isolated spinal cord of the newborn rat, and compared with those of the established agonists of (2S,1′S,2′S)‐2‐(carboxycyclopropyl)glycine (l‐CCG‐I) or (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid ((1S,3R)‐ACPD). At concentrations higher than 10 μm, DCG‐IV caused a depolarization which was completely blocked by selective N‐methyl‐d‐aspartate (NMDA) antagonists. The depolarization was pharmacologically quite different from that caused by l‐CCG‐I and (1S,3R)‐ACPD. DCG‐IV reduced the monosynaptic excitation of motoneurones rather than polysynaptic discharges in the nanomolar range without causing postsynaptic depolarization of motoneurones. DCG‐IV was more effective than l‐CCG‐I, (1S,3R)‐ACPD or l‐2‐amino‐4‐phosphonobutanoic acid (l‐AP4) in reducing the monosynaptic excitation of motoneurones. DCG‐IV (30 nm–1 μm) did not depress the depolarization induced by known excitatory amino acids in the newborn rat motoneurones, but depressed the baseline fluctuation of the potential derived from ventral roots. Therefore, DCG‐IV seems to reduce preferentially transmitter release from primary afferent nerve terminals. Depression of monosynaptic excitation caused by DCG‐IV was not affected by any known pharmacological agents, including 2‐amino‐3‐phosphonopropanoic acid (AP3), diazepam, 2‐hydroxysaclofen, picrotoxin and strychnine. DCG‐IV has the potential of providing further useful information on the physiological function of metabotropic glutamate receptors.

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This initial report from J-MACS focuses on patients' demographics, device types, survival, competing outcomes, adverse events and successful examples of system failure detection.

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the ß-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible crosstalk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

A recent study showed that granulocyte-colony stimulating factor (G-CSF) treatment improved the infarcted cardiac function. Although mobilized stem cells may affect it, the mechanism is unclear. In this study, we investigated the origins of stem cells and phenotypic changes of the migrated cells, and evaluated the efficacy of G-CSF. Eighteen C57BL/6 mice were irradiated (900 cGy) and GFP mouse-derived bone marrow cells (GFP-BMC: 10(6) cells) were injected via a tail vein followed by splenectomy 4 weeks later. Ligation of the left descending coronary artery was performed 2 weeks later. Recombinant human G-CSF (200 microg/kg/day) was injected for 3 days before and 5 days after ligation (group 1, n = 10). Saline was injected in group 2 (n = 8). Four weeks after infarction, hearts and other organs were fixed for histology. The survival rate after postoperative day 3 in group 1 was 100%, while that in group 2 was 50% (p = 0.03). Bone marrow-derived GFP cells (BMD-GFP) in group 1 (103.3+/-71.9/mm2) were located at the infarcted border area significantly more than those in group 2 (43.6+/-23.7/mm2) (p < 0.0001). BMD-GFP cells were positive for troponin I (16.6%), myosin heavy chain-slow (16.7%), and nestin (8.8%) in group 1. Ki-67-positive BMD-GFP in group 1 (10.0+/-7.0/mm2) were significantly more than those in group 2 (4.8+/-6.1/mm2) (p = 0.01). G-CSF increased the survival rate after infarction. G-CSF promoted BMC to migrate into the infarcted border area. Bone marrow was one of the origins of regenerated cardiomyocytes.

1 Neuropharmacological actions of all possible-state isomers of a-(carboxycyclopropyl)glycine (CCG), conformationally restricted analogues of glutamate, were examined for electrophysiological effects in the isolated spinal cord of the newborn rat. 2 Eight CCG stereoisomers demonstrated a large variety of depolarizing activities. Among them, the (2R, 3S, 4S) isomers of CCG (D-CCG-II) showed the most potent depolarizing activity, followed by the (2S, 3R, 4S) isomer (L-CCG-IV). 3 The depolarization evoked by L-CCG-IV, D-CCG-II and other D-CCG isomers was effectively depressed by N-methyl-D-aspartate (NMDA) antagonists. D-CCG-II was about 5 times more potent than NMDA in causing a depolarization. 4 The (2S, 3S, 4S) isomer of CCG (L-CCG-I) was more potent than L-glutamate in causing a depolarization of -spinal motoneurones. The depolarization was slightly depressed by NMDA antagonists, but residual amplitudes of responses to L-CCG-I in the presence of NMDA antagonists were almost insensitive to 6,7-dinitro-quinoxaline-2,3-dione (DNQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that L-CCG-I might be a novel potent agonist. 5 After application of the (2S, 3S, 4R) isomer of CCG (L-CCG-III), responses to L-glutamate, Dand L-aspartate were markedly enhanced. The enhancement lasted for a period of several hours without a further application of L-CCG-III. 6 L-CCG-III also caused a depolarization, but it seemed unlikely that the potentiation of the glutamate response was directly related to the depolarization evoked by L-CCG-III. 7 The potentiation might be due to inhibition of uptake processes, but L-CCG-III was superior to L-(-)-threo-3-hydroxyaspartate, a potent uptake inhibitor of L-glutamate and L-aspartate, in enhancing the response to L-glutamate in terms of amplitude and duration of responses. 8 CCG isomers should provide useful pharmacological tools for analysis of glutamate neurotransmitter systems.

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