Alterations in serotonergic responsiveness during cocaine withdrawal in rats: similarities to major depression in humans

BACKGROUND: Withdrawal from long-term cocaine use is accompanied by symptoms resembling major depression. Because acute cocaine affects serotonin (5-HT) neurons, and 5-HT dysfunction is implicated in the pathophysiology of depression, we evaluated the effects to 5-HT agonists in rats withdrawn from repeated injections of cocaine (15 mg/kg i.p., b.i.d., 7 days) or saline. METHODS: In the first study, prolactin (PRL) responses elicited by the 5-HT-releasing agent fenfluramine, the 5-HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), and the 5-HT2A/2C agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) were examined as indices of postsynaptic 5-HT receptor function. In a second study, specific responses induced by 8-OH-DPAT, namely inhibition of brain 5-HT synthesis and stimulation of feeding, were examined as correlates of 5-HT1A autoreceptor function. RESULTS: Prior treatment with cocaine did not modify fenfluramine-evoked PRL release; however, the PRL secretory response to 8-OH-DPAT was blunted and the PRL response to DOI was potentiated after chronic cocaine treatment. Cocaine exposure did not alter the inhibitory effect of 8-OH-DPAT on 5-HT synthesis. 8-OH-DPAT-induced feeding was influenced by prior cocaine, but this effect was secondary to pronounced baseline hyperphagia in the cocaine-treated group. CONCLUSIONS: These data indicate that withdrawal from chronic cocaine renders specific subpopulations of postsynaptic 5-HT1A receptors subsensitive and 5-HT2A/2C receptors supersensitive. No evidence for cocaine-induced changes in 5-HT1A autoreceptor responsiveness was found. A survey of the literature reveals similarities in the profile of 5-HT dysfunction between rats withdrawn from cocaine and humans diagnosed with depression. We propose that withdrawal from chronic cocaine in rats may serve as a useful animal model of depressive disorders.     

Preservation of myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest with 2, 3-butanedione monoxime

One proposed contributory mechanism for depressed ventricular performance after hypothermic, hyperkalemic cardioplegic arrest is a reduction in myocyte contractile function caused by alterations in intracellular calcium homeostasis. Because 2,3-butanedione monoxime decreases intracellular calcium transients, this study tested the hypothesis that 2,3-butanedione monoxime supplementation of the hyperkalemic cardioplegic solution could preserve isolated myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Myocytes were isolated from the left ventricles of six pigs. Magnitude and velocity of myocyte shortening were measured after 2 hours of incubation under normothermic conditions (37 degrees C, standard medium), hypothermic, hyperkalemic cardioplegic arrest (4 degrees C in Ringer's solution with 20 mEq potassium chloride and 20 mmol/L 2,3-butanedione monoxime). Because beta-adrenergic agonists are commonly employed after cardioplegic arrest, myocyte contractile function was examined in the presence of the beta-agonist isoproterenol (25 nmol/L). Hypothermic, hyperkalemic cardioplegic arrest and rewarming reduced the velocity (32%) and percentage of myocyte shortening (27%, p < 0.05). Supplementation with 2,3 butanedione monoxime normalized myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Although beta-adrenergic stimulation significantly increased myocyte contractile function under normothermic conditions and after hypothermic, hyperkalemic cardioplegic arrest, contractile function of myocytes exposed to beta-agonist after hypothermic, hyperkalemic cardioplegic arrest remained significantly reduced relative to the normothermic control group. Supplementation with 2,3-butanedione monoxime restored beta-adrenergic responsiveness of myocytes after hypothermic, hyperkalemic cardioplegic arrest. Thus, supplementation of a hyperkalemic cardioplegic solution with 2,3-butanedione monoxime had direct and beneficial effects on myocyte contractile function and beta-adrenergic responsiveness after cardioplegic arrest. A potential mechanism for the effects of 2,3-butanedione monoxime includes modulation of intracellular calcium transients or alterations in sensitivity to calcium. Supplementation with 2,3-butanedione monoxime may have clinical utility in improving myocardial contractile function after hypothermic, hyperkalemic cardioplegic arrest.     

Influence of fumonisin B1, present in Fusarium moniliforme culture material, and T-2 toxin on turkey poults

Diets containing 300 mg fumonisin B1 (FB1)/kg of feed and 5 mg T-2 toxin/kg of feed singly or in combination were fed to female turkey poults (Nicholas Large White) from day of hatch to 21 d of age. When compared with controls, 21-d body weight gains were reduced 21% by FB1, 26% by T-2, and 47% by the combination. the efficiency of feed utilization was adversely affected by FB1 and the combination of FB1 and T-2. Relative weights (grams/100 g BW) of the liver and gizzard were increased in poults fed the FB1 and the combination diets; whereas, the relative weight of the pancreas was increased in all treated groups. All poults were scored for oral lesions using a scale of 1 to 4 (1 = no visible lesions, 4 = severe lesions). Oral lesions were present in all poults fed the T-2 diet (average score of 3.29) or the combination diet (average score of 3.54). Serum concentration of cholesterol was decreased and lactate dehydrogenase activity was increased in poults fed the FB1 and combination diets. The activity of aspartate aminotransferase and the values for red blood cells, hemoglobin, and hematocrit were increased only in poults fed the combination diet. Inorganic phosphorus concentration was decreased only in poults fed the combination diet. The increased toxicity in poults fed the combination diet for most variables can best be described as additive, although some variables not altered by FB1 or T-2 singly were significantly affected by the combination, indicating that the combination may pose a potentially greater problem to the turkey industry than either of the mycotoxins individually.     

Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY

The bacterial chemotaxis protein CheY is activated in vivo by the covalentphosphorylation of a single aspartate residue at position 57. However, thisphosphate linkage is unstable (t1/2 approximately 20 s at roomtemperature), thereby precluding many biochemical analyses. Here we presenta synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) withchemical stability of the phosphate linkage enhanced by several orders ofmagnitude relative to the native protein. Starting with CheY D57C, asite-specific mutant of CheY with a unique cysteine residue in place of theaspartate at position 57, two sequential disulfide exchange reactions wereperformed to form the final product 'CheY D57C-SPO3' with a thiophosphatemoiety covalently bonded to the protein in a disulfide linkage. Massspectral analysis showed that the desired analog was present at 70-80% ofthe total protein. The disulfide linkage had a t1/2 of 8 days at 4 degreesC. Biochemical characterization of CheY D57C-SPO3 included assessment ofconformational properties using tryptophan fluorescence, evaluation ofmetal binding properties and measurement of binding interactions with thechemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group ata nearly identical location as native CheY-phosphate, the analog was unableto emulate CheY-phosphate function, thereby supporting the idea that thereare very precise geometric requirements for successful CheY activation.     

Full-arch implant framework casting accuracy: preliminary in vitro observation for in vivo testing

PURPOSE: Conventional techniques for implant metal framework fabrication produce error of a magnitude that is inconsistent with the passive-fit requirement for osseointegrated implants. To understand the correlation between prosthesis fit and the implant-tissue response, evaluation of the interface tissue reactions to customary levels of fit is required. The purpose of this study is to determine the accuracy of torch casting full arch frameworks using a high palladium alloy and a ringless phosphate-bonded investment technique. MATERIALS AND METHODS: Three different variables were considered relative to casting accuracy effect. The first variable, completeness of mold-fill, compared cast specimens where the entire sprue system was filled as part of the casting and cast specimens without the sprue system filled. The second variable, phosphate-bonded investment special liquid concentrations, compared groups of castings produced from 0%, 12%, 25%, and 50% special liquid. The third variable, investment mold shape, compared casting produced from a conventional ringless mold shape with a modified ringless mold shape where the investment in the same horizontal plane as the pattern was equal in thickness at the internal and external surfaces. Horizontal and vertical distances on the wax pattern and resulting framework were measured using a machinists microscope to determine casting error. Combined vertical and horizontal error was used for comparison between groups (one-way analysis of variance). RESULTS: No significant differences existed among the three groups compared (P > 0.05). The mean error comparison between the complete and incomplete mold-fill groups showed no statistical difference, while the incomplete fill group was found to be more porous. The mean error of all groups (0.130 mm) exceeded the recommended level of fit needed to satisfy the passive fit requirement by more than 10-fold. CONCLUSIONS: These results verify clinical observation and suggest that the use of conventional lost wax casting technique to cast one-piece full arch implant frameworks is both imprecise and inaccurate as judged against the passive fit requirement. The consequences of screw-fastening misfitting prostheses to osseointegrated implants is currently under investigation.     

Protein analysis of human von Ebner saliva and a method for its collection from the foliate papillae

The lingual serous glands of von Ebner are located close to the foliate and circumvallate papillae. Saliva secreted by these glands provides the immediate environment of the taste buds, and it has been hypothesized that it modulates taste perception. The purpose of this study was to develop a technique for collection of unstimulated and stimulated saliva from human von Ebner glands. Saliva was collected under resting conditions and after application of various gustatory stimuli (sweet, sour, salt, and bitter) by insertion of periostrips into the folds of the foliate papillae of healthy human volunteers. Stimulated saliva was also collected in glass microcapillaries or micropipettes. The flow-rates of unstimulated von Ebner saliva were 2.3 +/- 0.6 (S.E.) microL/min and 4.5 +/- 1.2 (S.E.) microL/min with 1% citric acid stimulation. The protein content was 2.5 +/- 0.5 (S.E.) mg/mL. The SDS gel electrophoretic profile of von Ebner saliva revealed two protein bands of Mr 18,000 that were identified on Western blots as von Ebner gland (VEG) proteins. Although lingual lipase activity was detected at very low levels by enzyme assay, this protein was not detected on Western blots. This collection technique should prove useful for analysis of specific functions associated with secretion from von Ebner glands.     

Cloning, expression and sequence analysis of the SphI restriction-modification system

SphI, a type II restriction-modification (R-M) system from the bacterium Streptomyces phaeochromogenes, recognizes the sequence 5'-GCATGC. The SphI methyltransferase (MTase)-encoding gene, sphIM, was cloned into Escherichia coli using MTase selection to isolate the clone. However, none of these clones contained the restriction endonuclease (ENase) gene. Repeated attempts to clone the complete ENase gene along with sphIM in one step failed, presumably due to expression of SphI ENase gene, sphIR, in the presence of inadequate expression of sphIM. The complete sphIR was finally cloned using a two-step process. PCR was used to isolate the 3' end of sphIR from a library. The intact sphIR, reconstructed under control of an inducible promoter, was introduced into an E. coli strain containing a plasmid with the NlaIII MTase-encoding gene (nlaIIIM). The nucleotide sequence of the SphI system was determined, analyzed and compared to previously sequenced R-M systems. The sequence was also examined for features which would help explain why sphIR unlike other actinomycete ENase genes seemed to be expressed in E. coli.     

Effects of phentermine on responding maintained under multiple fixed-ratio schedules of food and cocaine presentation in the rhesus monkey

Drugs that decrease drug-maintained responding at doses that do not decrease other behaviors in animals may be suitable candidates for development as medications to treat drug abuse in humans. The present study examined whether this effect could be obtained with phentermine, a drug that has been reported to decrease cocaine intake in humans. Rhesus monkeys were trained under multiple fixed-ratio 30-response schedules of food and i.v. cocaine delivery. Phentermine was always given as a slow, i.v. infusion. Acute treatment with phentermine (0.3-10 mg/kg) decreased cocaine-maintained responding at doses that did not decrease, or decreased less, food-maintained responding for each of three unit doses of cocaine (10-100 microg/kg/injection). Subacute treatment with phentermine (3 or 5.6 mg/kg, daily) also decreased cocaine-maintained responding more than food-maintained responding. After subacute treatment was terminated, rates of cocaine-maintained responding generally recovered to levels comparable to those seen during untreated control sessions. Phentermine (0.3-3 mg/kg) did not generally increase responding associated with a very low (1 microg/kg/injection) unit dose of cocaine, suggesting that the decrease in cocaine-maintained responding at higher unit doses was not the result of a leftward shift in the cocaine unit dose-effect function. Phentermine (0.1-3 mg/kg) decreased responding maintained by 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909) (30 microg/kg/injection) at doses similar to those that decreased food-maintained responding. These results show that phentermine is effective in decreasing cocaine self-administration and suggest that it may be an effective medication for cocaine abuse.