Alcohol preference in mice lacking the Avpr1a vasopressin receptor

[Arg(8)]-vasopressin (Avp), a nonapeptide hormone, is known to regulate blood pressure, water balance, and a variety of behaviors such as anxiety, aggression, and bonding. Although some evidence that Avp modifies ethanol consumption and some of the effects of ethanol on behavior have been reported, the role of Avp in alcohol consumption and preference is poorly understood. The Avp1a receptor (Avpr1a) is ubiquitously expressed in the central nervous system. To determine the role of Avp signaling on the behavioral effects of alcohol, we examined voluntary ethanol consumption in mice with targeted disruptions of the Avpr1a knockout (Avpr1a KO) gene. Avpr1a KO mice displayed both increased ethanol consumption and preference compared with wild-type (WT) mice. Enhanced ethanol consumption was dramatically and reversibly reduced by treatment with N-methyl-D-aspartic acid antagonists. Basal glutamate release was elevated around the striatum in Avpr1a KO mice. Elevation of extracellular glutamate was also produced in WT mice by local application of an Avpr1a antagonist though a dialysis probe, and this elevation was quickly reversed by stopping the perfusion. These results suggest that Avp can inhibit the release of glutamate from the presynaptic terminal via the Avp1a receptor and that elevation of glutamate levels owing to loss of the inhibitory effect via Avp-Avpr1a signaling may play an important role in the preference for ethanol.     

Development and validation of genomic simple sequence repeat markers in <Emphasis Type="Italic">Erianthus arundinaceus</Emphasis>

Erianthus arundinaceus, a member of the Saccharum complex, is of interest as a potential resource for sugarcane improvement and as a bioenergy crop. Genetic analyses of germplasm collections of E. arundinaceus are being used increasingly. To expand the genomic resources in E. arundinaceus, we aimed at developing simple sequence repeat markers. Using pyrosequencing on the 454 GS FLX system, we sequenced genomic DNA from “JW630” collected in Japan. A total of 1682 candidate loci were used to design the primers, and 1234 primer pairs amplified fragments of the expected size in the primer screening with three wild E. arundinaceus accessions (JW630, “JW4,” and “IJ76-349”). The efficiency of genotyping was validated with a subset of 174 primer pairs and 8 E. arundinaceus accessions. Of these primer pairs, 171 amplified fragments in all accessions tested and 162 detected polymorphic loci. The average values of genetic parameters were estimated as 0.30 (range, 0.09–0.49) for polymorphic information content, 1.65 (0.00–5.87) for marker index, and 2.78 (0.00–8.75) for resolving power. Using these parameters, we selected 61 primer pairs with large discriminatory power for the analyzed loci. Of the 174 primer pairs, 45 (25.9%) were also applicable to Saccharum and 33 (19.0%) to Miscanthus species. These markers would provide a valuable tool for estimating genetic diversity and constructing linkage maps in E. arundinaceus, which would be useful for genetic study and breeding.     

Increase in hippocampal cell death after treatment with kainate in zinc deficiency

Susceptibility to kainate-induced seizures is enhanced by zinc deficiency. To evaluate kainate-induced excitotoxicity in zinc deficiency, the relationship between kainate-induced seizures and hippocampal cell death was examined in control and zinc-deficient mice. Mice were fed a control and zinc-deficient diet for 4 weeks, and then intraperitoneally injected with 12 mg/kg kainate every 60 min three times. The rate of dead mice to the total mice was higher in zinc-deficient group than in control group 3 days after the last injection of kainate. In the survivals, which exhibited tonic convulsions in both control and zinc-deficient groups, kainate-induced hippocampal cell death was also analyzed by cresyl violet staining. Neuronal loss was more observed in the CA1, CA2 and CA3 pyramidal cell layers of zinc-deficient group than those of the control group. TUNEL-positive cells were significantly more detected in the CA1 and CA3 pyramidal cell layers of zinc-deficient group. These results demonstrate that kainate-induced hippocampal cell death occurs more easily in zinc deficiency. Extracellular zinc concentration detected with ZnAF-2 was significantly decreased in the hippocampal CA3 of zinc-deficient mice, in agreement with the previous data measured by in vivo microdialsysis. Synaptically released zinc may be less involved in kainate-induced hippocampal cell death in zinc deficiency.     

JNK antagonizes Akt-mediated survival signals by phosphorylating 14-3-3

Life and death decisions are made by integrating a variety of apoptotic and survival signals in mammalian cells. Therefore, there is likely to be a common mechanism that integrates multiple signals adjudicating between the alternatives. In this study, we propose that 14-3-3 represents such an integration point. Several proapoptotic proteins commonly become associated with 14-3-3 upon phosphorylation by survival-mediating kinases such as Akt. We reported previously that cellular stresses induce c-Jun NH2-terminal kinase (JNK)-mediated 14-3-3zeta phosphorylation at Ser184 (Tsuruta, F., J. Sunayama, Y. Mori, S. Hattori, S. Shimizu, Y. Tsujimoto, K. Yoshioka, N. Masuyama, and Y. Gotoh. 2004. EMBO J. 23:1889-1899). Here, we show that phosphorylation of 14-3-3 by JNK releases the proapoptotic proteins Bad and FOXO3a from 14-3-3 and antagonizes the effects of Akt signaling. As a result of dissociation, Bad is dephosphorylated and translocates to the mitochondria, where it associates with Bcl-2/Bcl-x(L). Because Bad and FOXO3a share the 14-3-3-binding motif with other proapoptotic proteins, we propose that this JNK-mediated phosphorylation of 14-3-3 regulates these proapoptotic proteins in concert and makes cells more susceptible to apoptotic signals.     

Conformational changes of p97 during nucleotide hydrolysis determined by small-angle X-Ray scattering

Valosin-containing protein (VCP)/p97 is an AAA family ATPase that has been implicated in the removal of misfolded proteins from the endoplasmic reticulum and in membrane fusion. p97 forms a homohexamer whose protomers consist of an N-terminal (N) domain responsible for binding to effector proteins, followed by two AAA ATPase domains, D1 and D2. Small-angle X-ray scattering (SAXS) measurements of p97 in the presence of AMP-PNP (ATP state), ADP-AlF(x) (hydrolysis transition state), ADP, or no nucleotide reveal major changes in the positions of the N domains with respect to the hexameric ring during the ATP hydrolysis cycle. Nucleotide binding and hydrolysis experiments indicate that D2 inhibits nucleotide exchange by D1. The data suggest that the conversion of the chemical energy of ATP hydrolysis into mechanical work on substrates involves transmission of conformational changes generated by D2 through D1 to move N.     

A single amino acid substitution in the active site of Escherichia coli aspartate transcarbamoylase prevents the allosteric transition

Modeling of the tetrahedral intermediate within the active site of Escherichia coli aspartate transcarbamoylase revealed a specific interaction with the side-chain of Gln137, an interaction not previously observed in the structure of the X-ray enzyme in the presence of N-phosphonacetyl-L-aspartate (PALA). Previous site-specific mutagenesis experiments showed that when Gln137 was replaced by alanine, the resulting mutant enzyme (Q137A) exhibited approximately 50-fold less activity than the wild-type enzyme, exhibited no homotropic cooperativity, and the binding of both carbamoyl phosphate and aspartate were extremely compromised. To elucidate the structural alterations in the mutant enzyme that might lead to such pronounced changes in kinetic and binding properties, the Q137A enzyme was studied by time-resolved, small-angle X-ray scattering and its structure was determined in the presence of PALA to 2.7 angstroms resolution. Time-resolved, small-angle X-ray scattering established that the natural substrates, carbamoyl phosphate and L-aspartate, do not induce in the Q137A enzyme the same conformational changes as observed for the wild-type enzyme, although the scattering pattern of the Q137A and wild-type enzymes in the presence of PALA were identical. The overall structure of the Q137A enzyme is similar to that of the R-state structure of wild-type enzyme with PALA bound. However, there are differences in the manner by which the Q137A enzyme coordinates PALA, especially in the side-chain positions of Arg105 and His134. The replacement of Gln137 by Ala also has a dramatic effect on the electrostatics of the active site. These data taken together suggest that the side-chain of Gln137 in the wild-type enzyme is required for the binding of carbamoyl phosphate in the proper orientation so as to induce conformational changes required for the creation of the high-affinity aspartate-binding site. The inability of carbamoyl phosphate to create the high-affinity binding site in the Q137A enzyme results in an enzyme locked in the low-activity low-affinity T state. These results emphasize the absolute requirement of the binding of carbamoyl phosphate for the creation of the high-affinity aspartate-binding site and for inducing the homotropic cooperativity in aspartate transcarbamoylase.     

Influence of nucleotide effectors on the kinetics of the quaternary structure transition of allosteric aspartate transcarbamylase

We report the effects of allosteric effectors, ATP, CTP and UTP on the kinetics of the quaternary structure change of Escherichia coli ATCase during the enzyme reaction with physiological substrates. Time-resolved, small-angle, X-ray scattering of solutions allows direct observation of structural transitions over the entire time-course of the enzyme reaction initiated by fast mixing of the enzyme and substrates. In the absence of effectors, all scattering patterns recorded during the reaction are consistent with a two-state, concerted transition model, involving no detectable intermediate conformation that differs from the less active, unliganded T-state and the more active, substrate-bound R-state. The latter predominates during the steady-state phase of enzyme catalysis, while the initial T-state is recovered after substrate consumption. The concerted character of the structural transition is preserved in the presence of all effectors. CTP slightly shifts the dynamical equilibrium during a shortened steady state toward T while the additional presence of UTP makes the steady state vanishingly short. The return transition to the T conformation is slowed significantly in the presence of inhibitors, the effect being most severe in the presence of UTP. While ATP increases the apparent T to R rate, it also increases the duration of the steady-state phase, an apparently paradoxical observation. This observation can be accounted for by the greater increase in the association rate constant of aspartate, promoted by ATP, while the nucleotide produces a lesser degree of increase in the dissociation rate constant. Under our experimental conditions, using high concentrations of both enzyme and substrate, it appears that this very mechanism of activation turns the activator into an efficient inhibitor. The scattering patterns recorded in the presence of ATP support the view that ATP alters the quaternary structure of the substrate-bound enzyme, an effect reminiscent of the reported modification of PALA-bound R-state by Mg-ATP.     

Production of unnatural glucosides of curcumin with drastically enhanced water solubility by cell suspension cultures of Catharanthus roseus

Catharanthus roseus cell suspension cultures converted exogenously supplied curcumin to a series of glucosides, none of which has been found in nature so far. The efficiency of glucosylation was dependent on culture stage of the cells and medium sucrose concentration. Methyl jasmonate and salicylic acid enhanced the glucoside formation only when they were added to the cultures prior to the addition of curcumin. The glucoside yield was 2.5 micromol/g fresh weight of the cells at an optimal culture condition. The water solubility of curcumin-4',4"-O-beta-D-digentiobioside was 0.65 mmol/ml, which was 20 million-fold higher than that of curcumin.     

alpha 1-Adrenergic receptor subtypes differentially control the cell cycle of transfected CHO cells through a cAMP-dependent mechanism involving p27Kip1

Three distinct subtypes of alpha(1)-adrenergic receptors (alpha(1)A-, alpha(1)B-, and alpha(1)D-AR) play a prominent role in cell growth. However, little is known about subtype-specific effects on cell proliferation. The activation of alpha(1)A- or alpha(1)B-AR inhibits serum-promoted cell proliferation, whereas alpha(1)D-AR activation does not show such an inhibitory effect. Notably, cell-cycle progression was blocked at G(1)/S transition after activation of alpha(1)A/alpha(1)B-AR but not of alpha(1)D-AR. In agreement with the differential cell proliferation effect, cAMP production was increased after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR, whereas all alpha(1)-AR subtypes are associated with inositol 1,4,5-trisphosphate production and mitogen-activated protein kinase activation in a similar fashion. Furthermore, the serum-induced reduction in the levels of the cyclin-dependent kinase inhibitor, p27(Kip1), was blocked after activation of alpha(1)A/alpha(1)B-AR but not alpha(1)D-AR. These results show that alpha(1)-AR subtypes differentially activate the cAMP/p27(Kip1) pathway and thereby have differential inhibitory effects on cell proliferation. Subtype-dependent effects should be taken into consideration when assessing the physiological response of native cells where alpha(1)-AR subtypes are generally co-expressed.     

Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus

Platelet-activating factor acetylhydrolase (PAF-AH) is a phospholipase A2 associated with lipoproteins that hydrolyzes platelet-activating factor (PAF) and oxidized phospholipids. We have developed an ELISA for PAF-AH that is more sensitive than previous methods, and have quantified HDL-associated and non-HDL-associated PAF-AH in healthy, hyperlipidemic, and diabetic subjects. In healthy subjects, plasma total PAF-AH concentration was positively correlated with PAF-AH activity and with plasma total cholesterol, triacylglycerol, LDL cholesterol and apolipoprotein B (apoB) concentrations (all P < 0.01). HDL-associated PAF-AH concentration was correlated positively with plasma apoA-I and HDL cholesterol. Subjects with hyperlipidemia (n = 73) and diabetes mellitus (n = 87) had higher HDL-associated PAF-AH concentrations than did controls (P < 0.01). Non-HDL-associated PAF-AH concentration was lower in diabetic subjects than in controls (P < 0.01). Both hyperlipidemic and diabetic subjects had lower ratios of PAF-AH to apoB (P < 0.01) and higher ratios of PAF-AH to apoA-I (P < 0.01) than did controls. Our results show that the distribution of PAF-AH mass between HDLs and LDLs is determined partly by the concentrations of the lipoproteins and partly by the mass of enzyme per lipoprotein particle, which is disturbed in hyperlipidemia and diabetes mellitus.