Foot shock facilitates reward seeking in an experience-dependent manner.

Animals organize reward seeking around aversive events. An abundance of research shows that foot shock, as well as a shock-associated cue, can elicit freezing and suppress reward seeking. Yet, there is evidence that experience can flip the effect of foot shock to facilitate reward seeking. Here we examined cue suppression, foot shock suppression and foot shock facilitation of reward seeking in a single behavioural setting. Male Long Evans rats received fear discrimination consisting of danger, uncertainty, and safety cues. Discrimination took place over a baseline of rewarded nose poking. With limited experience (1-2 sessions), all cues and foot shock suppressed reward seeking. With continued experience (10-16 sessions), suppression became specific to shock-associated cues, foot shock briefly suppressed, then facilitated reward seeking. Our results provide a means of assessing positive properties of foot shock, and may provide insight into maladaptive behaviour around aversive events.

Variability of TSNA in U.S. Tobacco and Moist Smokeless Tobacco Products.

Tobacco-specific nitrosamines (TSNAs) have been of concern to the public health community for decades and their reduction through agricultural practices, plant breeding, and tobacco processing has also been a decades-long industry effort. Despite those efforts, TSNAs, though lower, continue to be constituents of concern in tobacco products. This paper examines the TSNA levels of dark air-cured, dark fire-cured, and burley tobaccos purchased in the United States by U.S. Smokeless Tobacco Company LLC (USSTC) and of nine finished USSTC moist smokeless tobacco products. TSNA values of the incoming purchased tobaccos and the finished products showed considerable variability. For the incoming tobaccos, the coefficient of variation was generally more than 100 % for each tobacco type and for each of the measured TSNAs. The relative TSNA variability of the finished tobacco products was also considerable, averaging approximately 25 %. It was also found that the measured values for the finished products averaged well above the proposed FDA NNN proposed product standard of 1.0 µg/g dry weight. Because of the large variability in NNN values, products would have to average well below FDA's proposed product standard to be consistently compliant.

Photorhabdus luminescens W-14 insecticidal activity consists of at least two similar but distinct proteins. Purification and characterization of toxin A and toxin B.

Both the bacterium Photorhabdus luminescens alone and its symbiotic Photorhabdus-nematode complex are known to be highly pathogenic to insects. The nature of the insecticidal activity of Photorhabdus bacteria was investigated for its potential application as an insect control agent. It was found that in the fermentation broth of P. luminescens strain W-14, at least two proteins, toxin A and toxin B, independently contributed to the oral insecticidal activity against Southern corn rootworm. Purified toxin A and toxin B exhibited single bands on native polyacrylamide gel electrophoresis and two peptides of 208 and 63 kDa on SDS-polyacrylamide gel electrophoresis. The native molecular weight of both the toxin A and toxin B was determined to be approximately 860 kDa, suggesting that they are tetrameric. NH2-terminal amino acid sequencing and Western analysis using monospecific antibodies to each toxin demonstrated that the two toxins were distinct but homologous. The oral potency (LD50) of toxin A and toxin B against Southern corn rootworm larvae was determined to be similar to that observed with highly potent Bt toxins against lepidopteran pests. In addition, it was found that the two peptides present in toxin B could be processed in vitro from a 281-kDa protoxin by endogenous P. luminescens proteases. Proteolytic processing was shown to enhance insecticidal activity.

Inhibition of Diabrotica Larval Growth by Patatin, the Lipid Acyl Hydrolase from Potato Tubers.

Patatin, the nonspecific lipid acyl hydrolase from potato (Solanum tuberosum L.) tubers, dose-dependently inhibits the growth of southern corn rootworm (SCR) and western corn rootworm when fed to them on artificial diet. The 50% growth reduction levels are somewhat cultivar dependent, ranging from 60 to 150 [mu]g/g diet for neonate SCR larvae. A single patatin isoform also inhibits larval growth. Neonate SCR continuously exposed to patatin are halted in larval development. Treatment with di-isopropylfluorophosphate essentially eliminates patatin's phospholipase, galactolipase, and acyl hydrolase activities. SCR growth inhibition is eliminated also, indicating that patatin's serine hydrolase activity is responsible for the observed activities. Patatin-mediated phospholipolysis is highly pH and cultivar dependent, with specific activities up to 300-fold less at pH 5.5 than at pH 8.5. Esterase or phospholipase activities do not correlate with insect growth inhibition. Galactolipase activity, being cultivar and pH independent, correlates significantly with SCR growth inhibition. Insect-growth inhibition of patatin is significantly reduced with increased dietary cholesterol levels. In conclusion, patatin represents a new class of insect-control proteins with a novel mode of action possibly involving lipid metabolism.

Proteolysis of the 85-kilodalton crystalline cysteine proteinase inhibitor from potato releases functional cystatin domains.

The protein crystals found in potato (Solanum tuberosum L.) tuber cells consist of a single 85-kD polypeptide. This polypeptide is an inhibitor of papain and other cysteine proteinases and is capable of binding several proteinase molecules simultaneously (P. Rodis, J.E. Hoff [1984] Plant Physiol 74: 907-911). We have characterized this unusual inhibitor in more detail. Titrations of papain activity with the potato papain inhibitor showed that there are eight papain binding sites per inhibitor molecule. The inhibition constant (Ki) value for papain inhibition was 0.1 nM. Treatment of the inhibitor with trypsin resulted in fragmentation of the 85-kD polypeptide into a 32-kD polypeptide and five 10-kD polypeptides. The 32-kD and 10-kD fragments all retained the ability to potently inhibit papain (Ki values against papain were 0.5 and 0.7 nM, respectively) and the molar stoichiometries of papain binding were 2 to 3:1 and 1:1, respectively. Other nonspecific proteinases such as chymotrypsin, subtilisin Carlsberg, thermolysin, and proteinase K also cleaved the 85-kD inhibitor polypeptide into functional 22-kD and several 10-kD fragments. The fragments obtained by digestion of the potato papain inhibitor with trypsin were purified by reverse-phase high-performance liquid chromatography, and the N-terminal amino acid sequence was obtained for each fragment. Comparison of these sequences showed that the fragments shared a high degree of homology but were not identical. The sequences were homologous to the N termini of members of the cystatin superfamily of cysteine proteinase inhibitors. Therefore, the inhibitor appears to comprise eight tandem cystatin domains linked by preteolytically sensitive junctions. We have called the inhibitor potato multicystatin (PMC). By immunoblot analysis and measurement of papain inhibitory activity, PMC was found at high levels in potato leaves (up to 0.6 microgram/g fresh weight tissue), where it accumulated under conditions that induce the accumulation of other proteinase inhibitors linked to plant defense. PMC may have a similar defensive role, for example in protecting the plant from phytophagous insects that utilize cysteine proteinases for dietary protein digestion.

Microdialysis of noradrenaline in rostral ventrolateral medulla after intravenous methionine enkephalin administration in anesthetized rats.

The objective of this research was to define the role of central and peripheral opioid receptors for the regulation of cardiovascular action. Cardiovascular effects of methionine-enkephalin (met-enkephalin) after intracisternal, intravenous or direct administration into the rostral ventrolateral medulla (C1 area) were compared in inactin-anesthetized Sprague-Dawley rats. A microdialysis probe was stereotaxically implanted in the C1 area to dialyze monoamines during intravenous administration of met-enkephalin. An intravenous injection of met-enkephalin decreased both arterial pressure and heart rate in a dose-dependent manner. There were no cardiovascular responses to intracisternal dosages of up to 10 micrograms/kg, but as little as 0.1 micrograms/kg met-enkephalin decreased arterial pressure and heart rate after a direct injection into the C1 area. Onset of the met-enkephalin effect was similar regardless of drug doses after intravenous administration; however, duration and magnitude of the peptide's action and time to peak effect were directly related to the dose. Intravenous infusion of 100 micrograms/kg/min met-enkephalin increased the extracellular concentration of noradrenaline in the C1 area. There was a differential blockade by naloxone of the hypotensive action of met-enkephalin after intravenous or C1 administration. This study suggests the importance of both central and peripheral sites(s) of met-enkephalin for its cardiovascular action. Additionally, the data suggest that the C1 area is a communication site between catecholamines and opioid peptides for cardiovascular regulation.

Purification and properties of nuclease SP.

Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.

Binding of meso-tetrakis(N-methylpyridiniumyl)porphyrin isomers to DNA: quantitative comparison of the influence of charge distribution and copper(II) derivatization.

Factors influencing the binding of tetracationic porphyrin derivatives to DNA have been comprehensively evaluated by equilibrium dialysis, stopped-flow kinetics, etc., for mesotetrakis (4-N-methylpyridiniumyl)porphyrin [TMpyP (4)]. Technical difficulties have previously precluded a comprehensive study of metalloporphyrins. Since electrostatic interactions with the DNA and metal derivatization of the porphyrins have important consequences, we have investigated in greater detail two isomers of TMpyP (4) (meso-tetrakis(3-N-methylpyridiniumyl)porphyrin, [TMpyP (3)] and meso-tetrakis(2-N-methylpyridiniumyl)porphyrin [TMpyP (2)]) in which the position of the charged centers has been varied. A comprehensive study of the Cu(II) derivatives, e.g., CuTMpyP (4), was possible since the difficulties encountered previously with Ni(II) compounds were not a problem with Cu(II) porphyrins [J. A. Strickland, L. G. Marzilli, M. K. Gay, and W. D. Wilson (1988) Biochemistry 27, 8870-8878]. At 25 degrees C, the apparent equilibrium constants [Kobs] decreased with increasing [Na+] for all porphyrins. The Kobs values were comparable for TMpyP (4) and TMpyP (3) binding to either polyd(G-C).polyd(G-C) [poly[d(G-C)2]] or poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2]]. For the copper(II) porphyrins, the Kobs values were about fivefold greater. The Kobs value for CuTMpyP (2) binding to poly[d(G-C)2] was too small to measure under typical salt conditions; however, Kobs for binding to poly[d(A-T)2] was about two orders of magnitude smaller than those found for CuTMpyP (4) or CuTMpyP (3). Application of the condensation theory for polyelectrolytes suggests about three charge interactions when CuTMpyP (4), CuTMpyP (3), and TMpyP (3) bind to poly[d(G-C)2] or poly[d(A-T)2], a result comparable to that reported for TMpyP (4). At 20 degrees C and 0.115 M [Na+], incorporation of copper decreased the rates of dissociation from poly[d(A-T)2] by a 100-fold compared to those reported for TMpyP (4) but had little effect on the rates of dissociation from poly[d(G-C)2]. Also, movement of the H3CN+ group from the fourth to the third position of the pyridinium ring enhanced the rates of dissociation from poly[d(A-T)2] but decreased the rates of dissociation from poly[d(G-C)2]. From polyelectrolyte theory, the [Na+] dependence of the dissociation rates from poly[d(G-C)2] is consistent with intercalative binding, while that for poly[d(A-T)2] is consistent with an outside binding model. For calf thymus [CT] DNA at 20 degrees C, a greater decrease in the AT than in the GC imino 1H-nmr signal was observed upon addition of CuTMpyP (2), suggesting selective outside binding to the AT regions.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood ethanol level and physiologic measurements during ethanol-induced hypertension.

Plasma norepinephrine levels and alpha 2-adrenoceptor binding in central nervous system areas involved in blood pressure regulation were determined during the development of ethanol-induced hypertension in rats. Blood pressure was increased after the 4th week of ethanol feeding. Plasma norepinephrine levels of the ethanol-fed group were lower than those of the control group at most time periods studied even though blood pressure was elevated. 3H-Clonidine binding to alpha 2-adrenoceptors was decreased in the anterior and posterior hypothalamus of ethanol-fed rats throughout most of the study. Blood pressure in the ethanol-fed group was elevated when blood ethanol concentration was high, whereas blood pressure in this group was similar to that of the control group when blood ethanol concentration was low. Brain and plasma samples were taken during low blood ethanol which may explain the lack of positive findings. These results suggest that ethanol is a pressor agent and must be present to exert its cardiovascular effects. These findings suggest the importance of blood ethanol concentration at the time of study and also suggest that the blood pressure elevating effects of ethanol may be central rather than peripheral in origin.

Porphyrin and metalloporphyrin binding to DNA polymers: rate and equilibrium binding studies.

Interactions of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin [TMpyP(4)] with poly[d(G-C)].poly[d(G-C)] [poly[d(G-C)2] and poly[d(A-T)].poly[d(A-T)] [poly[d(A-T)2] were studied by equilibrium dialysis and stopped-flow dissociation kinetics as a function of [Na+]. Metalloderivatives of TMpyP(4), NiTMpyP(4), and ZnTMpyP(4) were also investigated. The apparent equilibrium binding constants (Kobs) were approximately the same for TMpyP(4) binding to either poly[d(G-C)2] or poly[d(A-T)2] and decreased with increasing [Na+]. The slopes of the plots of log Kobs vs log [Na+] were similar, with values close to -2.7. Contrary to implications in previously reported studies, these data do not indicate that TMpyP(4) prefers to bind to GC sites at low ionic strength and to AT sites at high ionic strength. In contrast, binding of ZnTMpyP(4) to these two polymers is very different. Comparisons of Kobs values at 0.065 M [Na+] indicate that ZnTMpyP(4) binding to AT sites is approximately 200 times more favorable than binding to GC sites, a finding in agreement with previous qualitative observations. Although the binding of the Zn species to the GC polymer was too weak for us to assess the salt effect, the plot of log Kobs vs log [Na+] gave a slope of -2.0 for ZnTMpyP(4) binding to poly[d(A-T)2]. Application of condensation theory for polyelectrolytes suggests similar charge interactions for ZnTMpyP(4) and for TMpyP(4) binding to poly[d(A-T)2]. Likewise, the rates of dissociation from poly[d(A-T)2] were similar for TMpyP(4) and ZnTMpyP(4) [and also NiTMpyP(4)]. However, whereas TMpyP(4) [and NiTMpyP(4)] dissociation from poly[d(G-C)2] was measurable, that for ZnTMpyP(4) was too fast to measure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of acute and chronic ethanol on the agonist responses of vascular smooth muscle.

We studied the effect of ethanol in vitro on the response of isolated rat aortas to phenylephrine and angiotensin II. We also examined the effect of chronic ethanol consumption on the phenylephrine response and the effect of ethanol in vitro on that response during the development of ethanol-induced hypertension. In acute experiments the depression of the phenylephrine dose-response produced by ethanol in vitro was greater than that for angiotensin II. Comparing the depression of these agonist dose-responses by ethanol to the depression by the receptor blockers, verapamil, prazosin and saralasin, suggests that ethanol may act like an alpha 1-adrenoceptor blocker. During chronic ethanol consumption two opposing changes occurred: (1) desensitization to phenylephrine during weeks 6-18 and, (2) tolerance to depression by ethanol in vitro during weeks 4-10. These opposing changes may cancel each other which suggests that the hypertension due to chronic ethanol consumption is probably not due to an action of ethanol on the vasculature.

Elective aortocoronary bypass grafting in the elderly.

Operative mortality, postoperative morbidity and follow up data were analyzed retrospectively from 122 consecutive patients, over 65 years old undergoing elective aortocoronary bypass grafting for symptomatic relief of angina pectoris at the Plains Health Centre, Regina, Saskatchewan, from January 1980 to December 1985. There were two in-hospital deaths (operative mortality 1.6%). Definite perioperative myocardial infarction occurred in 12 patients (9.8%). The 120 survivors were followed for a mean of 32 months. There were three deaths during follow-up, giving a five-year probability of survival of 93%. Twenty-three clinical events (including deaths) occurred during follow-up, giving a five-year probability of event free survival of 72%. Ninety percent of patients say they are pleased with their operation. Seventy-eight percent are currently angina free. These data illustrate the effectiveness of aortocoronary bypass grafting in low risk elderly patients with symptomatic coronary artery disease.

Acute effects of ethanol on baroreceptor reflex control of heart rate and on pressor and depressor responsiveness in rats.

In rats anesthetized with alpha-chloralose, doses of 0.1, 0.5, and 1 g/kg of ethanol produced an upward shift of baroreflex curves constructed by plotting the heart rate response against mean arterial pressure following evoked rises in mean arterial pressures by phenylephrine or angiotensin II. Whereas the upward shift of baroreceptor curves may be related, at least in part, to a higher base-line heart rate after ethanol, the data showed that the 1 g/kg dose of ethanol significantly depressed baroreflex sensitivity, suggesting that higher doses of ethanol impair baroreflex-mediated bradycardia. The phenylephrine, but not the angiotensin II or the nitroprusside, dose-response curves were shifted to the right after ethanol, indicating a decreased pressor responsiveness and suggesting that ethanol may have alpha-adrenergic blocking activity. This effect was also obtained in conscious rats. That this effect was not influenced by changes in baroreflex sensitivity was supported by the finding that a similar shift of the phenylephrine pressor-response curve was obtained in bilaterally vagotomized and hexamethonium-treated rats. Whether this effect of ethanol on baroreflex control of heart rate was influenced by anesthesia was investigated in conscious rats; the 1 g/kg dose of ethanol that produced the most significant decrease in baroreflex sensitivity was used in these experiments. Ethanol was still able to significantly inhibit baroreflex sensitivity in conscious rats, but the upward shift of the baroreflex curve and the elevated base-line heart rate no longer occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Must antidepressants be anticholinergic to inhibit muricide?

Two classical tricyclic antidepressants possessing anticholinergic side effects, amitriptyline and desipramine, were compared to newer antidepressants lacking such activity, mianserin, trazodone, and bupropion, for their ability to inhibit muricidal behavior. As has been previously shown for the tricyclic antidepressants, the newer antidepressants without anticholinergic activity also depressed mouse-killing behavior. Scopolamine HBr, at a dose which lacked antimuricidal activity, was tested for its ability to potentiate the antimuricidal effect of these antidepressants. Although potentiation was not demonstrated, there was a trend for scopolamine to enhance the antimuricidal effect of all drugs tested, regardless of whether or not they had anticholinergic activity.

Primary inoculation tuberculosis.

A case of primary inoculation tuberculosis of the finger is described, in which Mantoux test conversion reverted after prompt surgical and medical treatment. The pathology of primary inoculation tuberculosis is reviewed briefly, and the rationale for excision of the early tuberculous lesion is discussed.