Dose-sensitive Manner Research Articles

Following tetraploidy in an Arabidopsis ancestor, genes were removed preferentially from one homeolog leaving clusters enriched in dose-sensitive genes

Approximately 90% of Arabidopsis' unique gene content is found in syntenic blocks that were formed during the most recent whole-genome duplication. Within these blocks, 28.6% of the genes have a retained pair; the remaining genes have been lost from one of the homeologs. We create a minimized genome by condensing local duplications to one gene, removing transposons, and including only genes within blocks defined by retained pairs. We use a moving average of retained and non-retained genes to find clusters of retention and then identify the types of genes that appear in clusters at frequencies above expectations. Significant clusters of retention exist for almost all chromosomal segments. Detailed alignments show that, for 85% of the genome, one homeolog was preferentially (1.6x) targeted for fractionation. This homeolog fractionation bias suggests an epigenetic mechanism. We find that islands of retention contain "connected genes," those genes predicted-by the gene balance hypothesis-to be resistant to removal because the products they encode interact with other products in a dose-sensitive manner, creating a web of dependency. Gene families that are overrepresented in clusters include those encoding components of the proteasome/protein modification complexes, signal transduction machinery, ribosomes, and transcription factor complexes. Gene pair fractionation following polyploidy or segmental duplication leaves a genome enriched for "connected" genes. These clusters of duplicate genes may help explain the evolutionary origin of coregulated chromosomal regions and new functional modules.

N-methyl-D-aspartate antagonists and WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a cannabinoid agonist, interact to produce synergistic hypothermia.

CB(1) cannabinoid receptors mediate profound hypothermia when cannabinoid agonists are administered to rats. Glutamate, the principal excitatory neurotransmitter in the central nervous system (CNS), is thought to tonically increase body temperature by activating N-methyl-D-aspartate (NMDA) receptors. Because NMDA antagonists block cannabinoid-induced antinociception and catalepsy, intimate glutamatergic-cannabinoid interactions may exist in the CNS. The present study investigated the effect of two NMDA antagonists on the hypothermic response to WIN 55212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], a selective cannabinoid agonist, in rats. WIN 55212-2 (1-10 mg/kg i.m.) produced dose-dependent hypothermia that peaked 60 to 180 min postinjection. Dextromethorphan (5-75 mg/kg i.m.), a noncompetitive NMDA antagonist, or LY 235959 [(-)-6-[phosphonomethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-isoquinoline-2-carboxylate]](1-4 mg/kg i.m.), a competitive and highly selective NMDA antagonist, evoked hypothermia in a dose-sensitive manner, suggesting that endogenous glutamate exerts a hyperthermic tone on body temperature. A dose of dextromethorphan (10 mg/kg) that did not affect body temperature by itself potentiated the hypothermic response to WIN 55212-2 (1, 2.5, or 5 mg/kg). The enhancement was strongly synergistic, indicated by a 2.7-fold increase in the relative potency of WIN 55212-2. Similarly, a dose of LY 235959 (1 mg/kg) that did not affect body temperature augmented the hypothermia associated with a single dose of WIN 55212-2 (2.5 mg/kg), thus confirming that NMDA receptors mediated the synergy. We have demonstrated previously that CB(1) receptors mediate WIN 55212-2-evoked hypothermia in rats. The present data are the first evidence that NMDA antagonists exert a potentiating effect on cannabinoid-induced hypothermia. Taken together, these data suggest that interactions between NMDA and CB(1) receptors produce synergistic hypothermia.

A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis.

The anti-apoptotic Akt kinase is commonly activated by survival factors following plasma membrane relocalization attributable to the interaction of its pleckstrin homology (PH) domain with phosphatidylinositol 3-kinase (PI3K)-generated PI3,4-P(2) and PI3,4,5-P(3). Once activated, Akt can prevent or delay apoptosis by phosphorylation-dependent inhibition or activation of multiple signaling molecules involved in apoptosis, such as BAD, caspase-9, GSK3, and NF-kappaB and forkhead family transcription factors. Here, we describe and characterize a novel, conditional Akt controlled by chemically induced dimerization (CID). In this approach, the Akt PH domain has been replaced with the rapamycin (and FK506)-binding domain, FKBP12, to make F3-DeltaPH.Akt. To effect membrane recruitment, a myristoylated rapamycin-binding domain from FRAP/mTOR, called M-FRB, binds to lipid permeable rapamycin (and non-bioactive synthetic 'rapalogs'), leading to reversible heterodimerization of M-FRB with FKBP-DeltaPH.Akt. Like endogenous c-Akt, we show that the kinase activity of membrane-localized F3-DeltaPH.Akt correlates strongly with phosphorylation at T308 and S473; however, unlike c-Akt, phosphorylation and activation of inducible Akt (iAkt) is largely PI3K independent. CID-mediated activation of iAkt results in phosphorylation of GSK3, and contributes to NF-kappaB activation in vivo in a dose-sensitive manner. Finally, in Jurkat T cells stably expressing iAkt, CID-induced Akt activation rescued cells from apoptosis triggered by multiple apoptotic stimuli, including staurosporine, anti-Fas antibodies, PI3K inhibitors and the DNA damaging agent, etoposide. This novel inducible Akt should be useful for identifying new Akt substrates and for reversibly protecting tissue from apoptosis due to ischemic injury or immunological attack.

The canonical Wg and JNK signaling cascades collaborate to promote both dorsal closure and ventral patterning.

Elaboration of the Drosophila body plan depends on a series of cell-identity decisions and morphogenetic movements regulated by intercellular signals. For example, Jun N-terminal kinase signaling regulates cell fate decisions and morphogenesis during dorsal closure, while Wingless signaling regulates segmental patterning of the larval cuticle via Armadillo. wingless or armadillo mutant embryos secrete a lawn of ventral denticles; armadillo mutants also exhibit dorsal closure defects. We found that mutations in puckered, a phosphatase that antagonizes Jun N-terminal kinase, suppress in a dose-sensitive manner both the dorsal and ventral armadillo cuticle defects. Furthermore, we found that activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. We demonstrate that Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades.

The guanine nucleotide exchange factor trio mediates axonal development in the Drosophila embryo.

Recent analysis of Rho subfamily GTPases in Drosophila revealed roles for Rac and Cdc42 during axonogenesis. Here, we describe the identification and characterization of the Drosophila counterpart of Trio, a guanine nucleotide exchange factor (GEF) that associates with the receptor phosphatase LAR and regulates GTPase activation in vertebrate cells. Mutants deficient in trio activity display defects in both central and peripheral axon pathways reminiscent of phenotypes observed in embryos deficient in small GTPase function. Double mutant analysis shows that trio interacts with Rac in a dose-sensitive manner but not with Rho. Moreover, reduction of trio activity potentiates the phenotype of mutations in the LAR homolog Dlar, suggesting that these proteins collaborate in orchestrating the cytoskeletal events that underlie normal axonogenesis.

RAPAMYCIN (RAPA) ENHANCES TRANSFORMING GROWTH FACTOR BETA(TGF-??1) EXPRESSION IN IMMORTALIZED RAT PROXIMAL TUBULES CELLS(IRPTC)

396 The new immunosuppressant RAPA, is a potent macrolide being used in combination immunosuppression strategies with CsA. The use of CsA has significantly improved allograft survival. Concomitantly with immunosuppressive properties, CsA has been shown to induce allograft pathology, characterized by tubulointerstitial fibrosis and tubular atrophy. The induction of the cytokine TGF-β1 and its fibrogenic effect may in part be responsible for the tubulointerstitial fibrosis seen in long-term allografts. CsA has been shown in numerous animal models and cell lines to enhance the expression of TGF-β1. We have previously shown that CsA enhances TGF-β1- expression in IRPTC in a dose-dependent manner (100 to 5000ng/ml). In the salt-depleted rat model, coadministration of CsA and RAPA appeared to augment CsA-induced nephrotoxicity. We explore in the present study, whether RAPA potentiates CsA-induced TGF-β1 expression in IRPTC. At confluence, IRPTC were placed in defined serum free media for 24hr, then treated with RAPA(10;50;100; or 200 nM) or CsA(500ng/ml), RAPA (200nM) or CsA + RAPA (500ng/ml and 200nM) for 24hr, and harvested. Northern blotting (25 ug mRNA) and hybridization with a TGF-β1 cDNA probe and a cDNA GAPDH was performed and mRNA expression was normalized to GAPDH cDNA. RAPA in a dose sensitive manner, (at all concentrations) increased TGF-β1 expression. In combination with CsA or alone, RAPA augmented TGF-β1 mRNA expression without apparent synergy with CsA (at a dose of 500 ng/ml). Table Since TGF-β1 is a key fibrogenic cytokine, immunosuppressive protocols using both CsA and RAPA or RAPA alone, may be associated with increased TGF-β1 expression and subsequently higher nephrotoxicity. These results suggest that the use of the immunosuppressant RAPA, could have negative implications for long-term allograft survival and function.

RAPAMYCIN (RAPA) ENHANCES TRANSFORMING GROWTH FACTOR BETA(TGF-β1) EXPRESSION IN IMMORTALIZED RAT PROXIMAL TUBULES CELLS(IRPTC).

396 The new immunosuppressant RAPA, is a potent macrolide being used in combination immunosuppression strategies with CsA. The use of CsA has significantly improved allograft survival. Concomitantly with immunosuppressive properties, CsA has been shown to induce allograft pathology, characterized by tubulointerstitial fibrosis and tubular atrophy. The induction of the cytokine TGF-β1 and its fibrogenic effect may in part be responsible for the tubulointerstitial fibrosis seen in long-term allografts. CsA has been shown in numerous animal models and cell lines to enhance the expression of TGF-β1. We have previously shown that CsA enhances TGF-β1- expression in IRPTC in a dose-dependent manner (100 to 5000ng/ml). In the salt-depleted rat model, coadministration of CsA and RAPA appeared to augment CsA-induced nephrotoxicity. We explore in the present study, whether RAPA potentiates CsA-induced TGF-β1 expression in IRPTC. At confluence, IRPTC were placed in defined serum free media for 24hr, then treated with RAPA (10;50;100; or 200 nM) or CsA(500ng/ml), RAPA (200nM) or CsA + RAPA (500ng/ml and 200nM) for 24hr, and harvested. Northern blotting (25 ug mRNA) and hybridization with a TGF-β1 cDNA probe and a cDNA GAPDH was performed and mRNA expression was normalized to GAPDH cDNA. RAPA in a dose sensitive manner, (at all concentrations) increased TGF-β1 expression. In combination with CsA or alone, RAPA augmented TGF-β1 mRNA expression without apparent synergy with CsA(at a dose of 500 ng/ml). TableSince TGF-β1 is a key fibrogenic cytokine, immunosuppressive protocols using both CsA and RAPA or RAPA alone, may be associated with increased TGF-β1 expression and subsequently higher nephrotoxicity. These results suggest that the use of the immunosuppressant RAPA, could have negative implications for long-term allograft survival and function.

Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control.

The autonomous selector capacity of the homeotic proboscipedia (pb) gnee of the Drosophila Antennapedia Complex was tested. We introduced into the germline a P element containing a transcriptional fusion of a mini-gene for pb (normally required for formation of the labial and maxillary palps of the mouthparts) and the Hsp70 promoter. Uninduced expression of this Hsp70:pb element (HSPB) directs a novel, fully penetrant dominant transformation of antennae toward maxillary palps. Gene dosage experiments varying the number of HSPB elements indicate that the extent of the dominant transformation depends on the level of PB protein. At the same time, expression from the transgene also rescues recessive pb mutations. Finally, HSPB function may override the dominant antennal transformations caused by Antennapedia (Antp) mutations in a dose-sensitive manner, directing a switch of the antennal disc-derived appendage from ectopic leg to ectopic maxillary palp. This switch correlated with strikingly reduced ANTP protein accumulation when PB concentrations exceeded a genetically defined threshold level. These observations support a crucial role for quantitative aspects of pb function in determining segmental identity, including cross-regulatory events involved in this determination.

An increase in NMDA-sensitive [ 3H]glutamate and [ 3H]kainate binding in hippocampus 24 hours after PCP

Considerable research has identified a variety of acute PCP-induced biochemical changes in brain; but, little study has been devoted to characterizing delayed PCP-induced actions. These could potentially be associated with the prolonged psychotomimetic effects of the drug in humans. Here we studied delayed PCP-induced alterations in glutamate receptor subtype binding across a range of PCP doses, based on our previous findings of delayed regional cerebral metabolism changes with PCP. We report that 24 h after a single dose, PCP increases N- methyl- d - aspartate (NMDA)-sensitive [ 3H]glutamate binding in hippocampus (CA 1) in an apparent dose-sensitive manner; no other dose-sensitive regional changes in NMDA binding sites were apparent in a sampling of 19 brain regions. [ 3H]kainate binding sites were increased in CA 3 and dentate gyrus, but only at the high drug dose. Moreover, PCP appeared to have a general delayed effect in upregulating NMDA receptor binding in limbic-associated brain areas at its middle dose, and in upregulating [ 3H]kainate binding in neocortical and limbic area at its high dose. No PCP effects were noted on AMPA receptor binding. These delayed actions of PCP may be informative about the mechanism of PCP psychosis.

Hippocampal CRF, NE, and NMDA system interactions in memory processing in the rat.

In the present study, we investigated the neural mechanisms of corticotropin-releasing factor (CRF) and the interactions among CRF, norepinephrine (NE), and N-methyl-D-aspartate (NMDA) systems in the dentate gyrus (DG) of hippocampus in modulating the memory process of rats. One-way passive avoidance task was adopted. Results indicated that CRF (80 ng), when directly injected into the DG, consistently and significantly enhanced memory retention in rats. The noradrenergic neurotoxin DSP-4, at a high dose (4 micrograms), impaired memory. DSP-4 at a moderate dose (2 micrograms), which did not affect retention alone, antagonized the memory-enhancing effect of CRF. Similarly, the beta-adrenergic antagonist propranolol, at a high dose (8 micrograms), reduced retention. At a low dose (80 ng), which did not markedly affect retention by itself, propranolol also prevented the memory-improving effect of CRF. Moreover, direct NE infusions to the DG significantly improved retention performance in a dose-sensitive manner. Coadministration of CRF and NE did not further enhance retention. These results together suggest that CRF and NE facilitated memory probably through the same instead of independent mechanisms. In contrast, the selective NMDA receptor antagonists 2-amino-5-phosphonopentanoate (AP5) and MK801, at high doses markedly impaired memory retention (0.8 and 3.2 micrograms for AP5, 2 and 10 micrograms for MK801). At a dose of MK801 that did not significantly alter retention alone (80 ng), it completely blocked the memory-facilitating effect of CRF. These results indicate that CRF enhanced memory indirectly through NMDA receptor mediation also. Finally, MK801 at 80 ng also successfully antagonized the memory-facilitating effect of NE in the DG. We have demonstrated that MK801, at the dose chosen for interaction studies, did not markedly alter locomotor activity. These results together suggest that CRF, through a presynaptic facilitation mechanism, possibly facilitates NE release in the DG; increased NE release and stimulation of beta-adrenergic receptors in the DG result in NMDA receptor activations in the same area. This sequence of events enhance the memory consolidation process in the hippocampus and explained the neural mechanism of CRF in facilitating retention performance in rats. The same neuropeptide/neurotransmitter interactions may have other physiological and neuropathological implications.