Cocaine-predictive stimulus induces drug-seeking behavior and neural activation in limbic brain regions after multiple months of abstinence: Reversal by D1 antagonists

The conditioning of cocaine's subjective actions with environmental stimuli may be a critical factor in long-lasting relapse risk associated with cocaine addiction. To study the significance of learning factors in persistent addictive behavior as well as the neurobiological basis of this phenomenon, rats were trained to associate discriminative stimuli (SD) with the availability of i.v. cocaine vs. nonrewarding saline solution, and then placed on extinction conditions during which the i.v. solutions and SDs were withheld. The effects of reexposure to the SD on the recovery of responding at the previously cocaine-paired lever and on Fos protein expression then were determined in two groups. One group was tested immediately after extinction, whereas rats in the second group were confined to their home cages for an additional 4 months before testing. In both groups, the cocaine SD, but not the non-reward SD, elicited strong recovery of responding and increased Fos immunoreactivity in the basolateral amygdala and medial prefrontal cortex (areas Cg1/Cg3). The response reinstatement and Fos expression induced by the cocaine SD were both reversed by selective dopamine D1 receptor antagonists. The undiminished efficacy of the cocaine SD to elicit drug-seeking behavior after 4 months of abstinence parallels the long-lasting nature of conditioned cue reactivity and cue-induced cocaine craving in humans, and confirms a significant role of learning factors in the long-lasting addictive potential of cocaine. Moreover, the results implicate D1-dependent neural mechanisms within the medial prefrontal cortex and basolateral amygdala as substrates for cocaine-seeking behavior elicited by cocaine-predictive environmental stimuli.

An improved helper-dependent adenoviral vector allows persistent gene expression after intramuscular delivery and overcomes preexisting immunity to adenovirus

Helper-dependent adenoviral vectors deleted of all viral coding sequences have shown an excellent gene expression profile in a variety of animal models, as well as a reduced toxicity after systemic delivery. What is still unclear is whether long-term expression and therapeutic dosages of these vectors can be obtained also in the presence of a preexisting immunity to adenovirus, a condition found in a high proportion of the adult human population. In this study we performed intramuscular delivery of helper-dependent vectors carrying mouse erythropoietin as a marker transgene. We found that low doses of helper-dependent adenoviral vectors can direct long-lasting gene expression in the muscles of fully immunocompetent mice. The best performance—i.e., 100% of treated animals showing sustained expression after 4 months—was achieved with the latest generation helper-dependent backbones, which replicate and package at high efficiency during vector propagation. Moreover, efficient and prolonged transgene expression after intramuscular injection was observed with limited vector load also in animals previously immunized against the same adenovirus serotype. These data suggest that human gene therapy by intramuscular delivery of helper-dependent adenoviral vectors is feasible.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

A Photoperiod-Insensitive Barley Line Contains a Light-Labile Phytochrome B1

Barley (Hordeum vulgare L.) is a long-day plant whose flowering is enhanced when the photoperiod is supplemented with far-red light, and this promotion is mediated by phytochrome. A chemically mutagenized dwarf cultivar of barley was selected for early flowering time (barley maturity daylength response [BMDR]-1) and was made isogenic with the cultivar Shabet (BMDR-8) by backcrossing. BMDR-1 was found to contain higher levels of both phytochrome A and phytochrome B in the dark on immunoblots with monoclonal antibodies from oat (Avena sativa L.) that are specific to different members of the phytochrome gene family. Phytochrome A was light labile in both BMDR-1 and BMDR-8, decreasing to very low levels after 4 d of growth in the light. Phytochrome B was light stable in BMDR-8, being equal in both light and darkness. However, phytochrome B became light labile in BMDR-1 and this destabilization of phytochrome B appeared to make BMDR-1 insensitive to photoperiod. In addition, both the mutant and the wild type lacked any significant promotion of flowering in response to a pulse of far-red light given at the end of day, and the end-of-day, far-red inhibition of tillering is normal in both, suggesting that phytochrome B is not involved with these responses in barley.

Change in Apoplastic Aluminum during the Initial Growth Response to Aluminum by Roots of a Tolerant Maize Variety1

Root elongation, hematoxylin staining, and changes in the ultrastructure of root-tip cells of an Al-tolerant maize variety (Zea mays L. C 525 M) exposed to nutrient solutions with 20 μm Al (2.1 μm Al3+ activity) for 0, 4, and 24 h were investigated in relation to the subcellular distribution of Al using scanning transmission electron microscopy and energy-dispersive x-ray microanalysis on samples fixed by different methods. Inhibition of root-elongation rates, hematoxylin staining, cell wall thickening, and disturbance of the distribution of pyroantimoniate-stainable cations, mainly Ca, was observed only after 4 and not after 24 h of exposure to Al. The occurrence of these transient, toxic Al effects on root elongation and in cell walls was accompanied by the presence of solid Al-P deposits in the walls. Whereas no Al was detectable in cell walls after 24 h, an increase of vacuolar Al was observed after 4 h of exposure. After 24 h, a higher amount of electron-dense deposits containing Al and P or Si was observed in the vacuoles. These results indicate that in this tropical maize variety, tolerance mechanisms that cause a change in apoplastic Al must be active. Our data support the hypothesis that in Al-tolerant plants, Al can rapidly cross the plasma membrane; these data clearly contradict the former conclusions that Al mainly accumulates in the apoplast and enters the symplast only after severe cell damage has occurred.

Temporal and Spatial Patterns of Accumulation of the Transcript of Myo-Inositol-1-Phosphate Synthase and Phytin-Containing Particles during Seed Development in Rice1

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

The Regulation of Photosynthetic Electron Transport during Nutrient Deprivation in Chlamydomonas reinhardtii1

The light-saturated rate of photosynthetic O2 evolution in Chlamydomonas reinhardtii declined by approximately 75% on a per-cell basis after 4 d of P starvation or 1 d of S starvation. Quantitation of the partial reactions of photosynthetic electron transport demonstrated that the light-saturated rate of photosystem (PS) I activity was unaffected by P or S limitation, whereas light-saturated PSII activity was reduced by more than 50%. This decline in PSII activity correlated with a decline in both the maximal quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers (PSII centers capable of performing a charge separation but unable to reduce the plastoquinone pool). In addition to a decline in the light-saturated rate of O2 evolution, there was reduced efficiency of excitation energy transfer to the reaction centers of PSII (because of dissipation of absorbed light energy as heat and because of a transition to state 2). These findings establish a common suite of alterations in photosynthetic electron transport that results in decreased linear electron flow when C. reinhardtii is limited for either P or S. It was interesting that the decline in the maximum quantum efficiency of PSII and the accumulation of the secondary quinone electron acceptor of PSII nonreducing centers were regulated specifically during S-limited growth by the SacI gene product, which was previously shown to be critical for the acclimation of C. reinhardtii to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150–2159).

Overexpression of Nitrate Reductase in Tobacco Delays Drought-Induced Decreases in Nitrate Reductase Activity and mRNA1

Transformed (cauliflower mosaic virus 35S promoter [35S]) tobacco (Nicotiana plumbaginifolia L.) plants constitutively expressing nitrate reductase (NR) and untransformed controls were subjected to drought for 5 d. Drought-induced changes in biomass accumulation and photosynthesis were comparable in both lines of plants. After 4 d of water deprivation, a large increase in the ratio of shoot dry weight to fresh weight was observed, together with a decrease in the rate of photosynthetic CO2 assimilation. Foliar sucrose increased in both lines during water stress, but hexoses increased only in leaves from untransformed controls. Foliar NO3− decreased rapidly in both lines and was halved within 2 d of the onset of water deprivation. Total foliar amino acids decreased in leaves of both lines following water deprivation. After 4 d of water deprivation no NR activity could be detected in leaves of untransformed plants, whereas about 50% of the original activity remained in the leaves of the 35S-NR transformants. NR mRNA was much more stable than NR activity. NR mRNA abundance increased in the leaves of the 35S-NR plants and remained constant in controls for the first 3 d of drought. On the 4th d, however, NR mRNA suddenly decreased in both lines. Rehydration at d 3 caused rapid recovery (within 24 h) of 35S-NR transcripts, but no recovery was observed in the controls. The phosphorylation state of the protein was unchanged by long-term drought. There was a strong correlation between maximal extractable NR activity and ambient photosynthesis in both lines. We conclude that drought first causes increased NR protein turnover and then accelerates NR mRNA turnover. Constitutive NR expression temporarily delayed drought-induced losses in NR activity. 35S-NR expression may therefore allow more rapid recovery of N assimilation following short-term water deficit.

Antisense RNA to the type I insulin-like growth factor receptor suppresses tumor growth and prevents invasion by rat prostate cancer cells in vivo.

Prostate carcinoma is the second leading cause of death from malignancy in men in the United States. Prostate cancer cells express type I insulin-like growth factor receptor (IGF-IR) and prostate cancer selectively metastazises to bone, which is an environment rich in insulin-like growth factors (IGFs), thereby supporting a paracrine action for cancer cell proliferation. We asked whether the IGF-IR is coupled to tumorigenicity and invasion of prostate cancer. When rat prostate adenocarcinoma cells (PA-III) were stably transfected with an antisense IGF-IR expression construct containing the ZnSO4-inducible metallothionein-1 transcriptional promoter, the transfectants expressed high levels of IGF-IR antisense RNA after induction with ZnSO4, which resulted in dramatically reduced levels of endogenous IGF-IR mRNA. A significant reduction in expression both of tissue-type plasminogen activator and of urokinase-type plasminogen activator occurred in PA-III cells accompanying inhibition of IGF-IR. Subcutaneous injection of either nontransfected PA-III or PA-III cells transfected with vector minus the IGF-IR insert into nude mice resulted in large tumors after 4 weeks. However, mice injected with IGF-IR antisense-transfected PA-III cells either developed tumors 90% smaller than controls or remained tumor-free after 60 days of observation. When control-transfected PA-III cells were inoculated over the abraded calvaria of nude mice, large tumors formed with invasion of tumor cells into the brain parenchyma. In contrast, IGF-IR antisense transfectants formed significantly smaller tumors with no infiltration into brain. These results indicate an important role for the IGF/IGF-IR pathway in metastasis and provide a basis for targeting IGF-IR as a potential treatment for prostate cancer.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

31P magnetic resonance spectroscopy of the Sherpa heart: a phosphocreatine/adenosine triphosphate signature of metabolic defense against hypobaric hypoxia.

Of all humans thus far studied, Sherpas are considered by many high-altitude biomedical scientists as most exquisitely adapted for life under continuous hypobaric hypoxia. However, little is known about how the heart is protected in hypoxia. Hypoxia defense mechanisms in the Sherpa heart were explored by in vivo, noninvasive 31P magnetic resonance spectroscopy. Six Sherpas were examined under two experimental conditions [normoxic (21% FiO2) and hypoxic (11% FiO2) and in two adaptational states--the acclimated state (on arrival at low-altitude study sites) and the deacclimating state (4 weeks of ongoing exposure to low altitude). Four lowland subjects were used for comparison. We found that the concentration ratios of phosphocreatine (PCr)/adenosine triphosphate (ATP) were maintained at steady-state normoxic values (0.96, SEM = 0.22) that were about half those found in normoxic lowlanders (1.76, SEM = 0.03) monitored the same way at the same time. These differences in heart energetic status between Sherpas and lowlanders compared under normoxic conditions remained highly significant (P < 0.02) even after 4 weeks of deacclimation at low altitudes. In Sherpas under acute hypoxia, the heart rate increased by 20 beats per min from resting values of about 70 beats per min, and the percent saturation of hemoglobin decreased to about 75%. However, these perturbations did not alter the PCr/ATP concentration ratios, which remained at about 50% of the values expected in healthy lowlanders. Because the creatine phosphokinase reaction functions close to equilibrium, these steady-state PCr/ATP ratios presumably coincided with about 3-fold higher free adenosine diphosphate (ADP) concentrations. Higher ADP concentrations (i.e., lower [PCr]/[ATP] ratios) were interpreted to correlate with the Km values for ADP-requiring kinases of glycolysis and to reflect elevated carbohydrate contributions to heart energy needs. This metabolic organization is postulated as advantageous in hypobaria because the ATP yield per O2 molecule is 25-60% higher with glucose than with free fatty acids (the usual fuels utilized in the human heart in postfasting conditions).

Staphylococcal enterotoxins modulate interleukin 2 receptor expression and ligand-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription (Stat proteins).

Staphylococcal enterotoxins (SE) stimulate T cells expressing the appropriate variable region beta chain of (V beta) T-cell receptors and have been implicated in the pathogenesis of several autoimmune diseases. Depending on costimulatory signals, SE induce either proliferation or anergy in T cells. In addition, SE can induce an interleukin-2 (IL-2) nonresponsive state and apoptosis. Here, we show that SE induce dynamic changes in the expression of and signal transduction through the IL-2 receptor (IL-2R) beta and gamma chains (IL-2R beta and IL-2R gamma) in human antigen-specific CD4+ T-cell lines. Thus, after 4 hr of exposure to SEA and SEB, the expression of IL-2R beta was down-regulated, IL-2R gamma was slightly up-regulated, while IL-2R alpha remained largely unaffected. The changes in the composition of IL-2Rs were accompanied by inhibition of IL-2-induced tyrosine phosphorylation of the Janus protein-tyrosine kinase 3 (Jak3) and signal transducers and activators of transcription called Stat3 and Stat5. In parallel experiments, IL-2-driven proliferation was inhibited significantly. After 16 hr of exposure to SE, the expression of IL-2R beta remained low, while that of IL2R alpha and IL2R gamma was further up-regulated, and ligand-induced tyrosine phosphorylation of Jak3 and Stat proteins was partly normalized. Yet, IL-2-driven proliferation remained profoundly inhibited, suggesting that signaling events other than Jak3/Stat activation had also been changed following SE stimulation. In conclusion, our data suggest that SE can modulate IL-2R expression and signal transduction involving the Jak/Stat pathway in CD4+ T-cell lines.

Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats.

We investigated the cellular and molecular events associated with the increase in sodium transport across the alveolar epithelium of rats exposed to hyperoxia (85% O2 for 7 days followed by 100% O2 for 4 days). Alveolar type II (ATII) cell RNA was isolated and probed with a cDNA for one of the rat colonic epithelial sodium channel subunits (alpha rENaC). The alpha rENaC mRNA (3.7-kb transcript) increased 3-fold in ATII cell RNA isolated from rats exposed to 85% O2 for 7 days and 6-fold after 4 days of subsequent exposure to 100% O2. In situ hybridization revealed increased expression of alpha rENaC mRNA transcripts in both airway and alveolar epithelial cells of hyperoxic rats. When immunostained with a polyclonal antibody to kidney sodium channel protein, ATII cells from hyperoxic rats exhibited a significant increase in the amount of immunogenic protein present in both the plasma membrane and the cytoplasm. When patched in the whole-cell mode, ATII cells from hyperoxic rats exhibited amiloride and 5-(N-ethyl-N-isopropyl)-2',4'-amiloride (EIPA)-sensitive currents that were 100% higher compared with those obtained from air-breathing rats. Single-channel sodium currents (mean conductance of 25 pS) were seen in ATII cells patched in both the inside-out and cell-attached modes. The number and open probability of these channels increased significantly during exposure to hyperoxia. Exposure to sublethal hyperoxia up-regulated both alpha rENaC mRNA and the functional expression of sodium channels in ATII cells.

N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia.

Recent results have demonstrated that the spin trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) reduces infarct size due to middle cerebral artery occlusion (MCAO), even when given after ischemia. The objective of the present study was to explore whether PBN influences recovery of energy metabolism. MCAO of 2-hr duration was induced in rats by an intraluminal filament technique. Brains were frozen in situ at the end of ischemia and after 1, 2, and 4 hr of recirculation. PBN was given 1 hr after recirculation. Neocortical focal and perifocal ("penumbra") areas were sampled for analyses of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glycogen, glucose, and lactate. The penumbra showed a moderate-to-marked decrease and the focus showed a marked decrease in PCr and ATP concentrations, a decline in the sum of adenine nucleotides, near-depletion of glycogen, and an increase in lactate concentration after 2 hr of ischemia. Recirculation for 1 hr led to only a partial recovery of energy state, with little further improvement after 2 hr and signs of secondary deterioration after 4 hr, particularly in the focus. After 4 hr of recirculation, PBN-treated animals showed pronounced recovery of energy state, with ATP and lactate contents in both focus and penumbra approaching normal values. Although an effect of PBN on mitochondria cannot be excluded, the results suggest that PBN acts by preventing a gradual compromise of microcirculation. The results justify a reevaluation of current views on the pathophysiology of focal ischemic damage and suggest that a therapeutic window of many hours exists in stroke.

Elimination of residual metastatic prostate cancer after surgery and adjunctive cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy

Cancer relapse after surgery is a common occurrence, most frequently resulting from the outgrowth of minimal residual disease in the form of metastases. We examined the effectiveness of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade as an adjunctive immunotherapy to reduce metastatic relapse after primary prostate tumor resection. For these studies, we developed a murine model in which overt metastatic outgrowth of TRAMP-C2 (C2) prostate cancer ensues after complete primary tumor resection. Metastatic relapse in this model occurs reliably and principally within the draining lymph nodes in close proximity to the primary tumor, arising from established metastases present at the time of surgery. Using this model, we demonstrate that adjunctive CTLA-4 blockade administered immediately after primary tumor resection reduces metastatic relapse from 97.4 to 44%. Consistent with this, lymph nodes obtained 2 weeks after treatment reveal marked destruction or complete elimination of C2 metastases in 60% of mice receiving adjunctive anti-CTLA-4 whereas 100% of control antibody-treated mice demonstrate progressive C2 lymph node replacement. Our study demonstrates the potential of adjunctive CTLA-4 blockade immunotherapy to reduce cancer relapse emanating from minimal residual metastatic disease and may have broader implications for improving the capability of immunotherapy by combining such forms of therapy with other cytoreductive measures including surgery.