Study of ligand-based virtual screening tools in computer-aided drug design

Virtual screening is a central technique in drug discovery today. Millions of molecules can be tested in silico with the aim to only select the most promising and test them experimentally. The topic of this thesis is ligand-based virtual screening tools which take existing active molecules as starting point for finding new drug candidates. One goal of this thesis was to build a model that gives the probability that two molecules are biologically similar as function of one or more chemical similarity scores. Another important goal was to evaluate how well different ligand-based virtual screening tools are able to distinguish active molecules from inactives. One more criterion set for the virtual screening tools was their applicability in scaffold-hopping, i.e. finding new active chemotypes. In the first part of the work, a link was defined between the abstract chemical similarity score given by a screening tool and the probability that the two molecules are biologically similar. These results help to decide objectively which virtual screening hits to test experimentally. The work also resulted in a new type of data fusion method when using two or more tools. In the second part, five ligand-based virtual screening tools were evaluated and their performance was found to be generally poor. Three reasons for this were proposed: false negatives in the benchmark sets, active molecules that do not share the binding mode, and activity cliffs. In the third part of the study, a novel visualization and quantification method is presented for evaluation of the scaffold-hopping ability of virtual screening tools.

Evolution of Bioaffinity Reagents by Phage Display

Antibodies are natural binding proteins produced in vertebrates as a response to invading pathogens and foreign substances. Because of their capability for tight and specific binding, antibodies have found use as binding reagents in research and diagnostics. Properties of cloned recombinant antibodies can be further improved by means of in vitro evolution, combining mutagenesis with subsequent phage display selection. It is also possible to isolate entirely new antibodies from vast naïve or synthetic antibody libraries by phage display. In this study, library techniques and phage display selection were applied in order to optimise binding scaffolds and antigen recognition of antibodies, and to evolve new and improved bioaffinity reagents. Antibody libraries were generated by random and targeted mutagenesis. Expression and stability were mainly optimised by the random methods whereas targeted randomisation of the binding site residues was used for optimising the binding properties. Trinucleotide mutagenesis allowed design of defined randomisation patterns for a synthetic antibody library. Improved clones were selected by phage display. Capture by a specific anti- DHPS antibody was exploited in the selection of improved phage display of DHPS. Efficient selection for stability was established by combining phage display selection with denaturation under reducing conditions. Broad-specific binding of a generic anti-sulfonamide antibody was improved by selection with one of the weakest binding sulfonamides. In addition, p9 based phage display was studied in affinity selection from the synthetic library. A TIM barrel protein DHPS was engineered for efficient phage display by combining cysteinereplacement with random mutagenesis. The resulting clone allows use of phage display in further engineering of DHPS and possibly use as an alternative-binding scaffold. An anti-TSH scFv fragment, cloned from a monoclonal antibody, was engineered for improved stability to better suite an immunoassay. The improved scFv tolerates 8 – 9 °C higher temperature than the parental scFv and should have sufficient stability to be used in an immunoanalyser with incubation at 36 °C. The anti-TSH scFv fragment was compared with the corresponding Fab fragment and the parental monoclonal antibody as a capturing reagent in a rapid 5-min immunoassay for TSH. The scFv fragment provided some benefits over the conventionally used Mab in anayte-binding capacity and assay kinetics. However, the recombinant Fab fragment, which had similar kinetics to the scFv, provided a more sensitive and reliable assay than the scFv. Another cloned scFv fragment was engineered in order to improve broad-specific recognition of sulfonamides. The improved antibody detects different sulfonamides at concentrations below the maximum residue limit (100 μg/kg in EU and USA) and allows simultaneous screening of different sulfonamide drug residues. Finally, a synthetic antibody library was constructed and new antibodies were generated and affinity matured entirely in vitro. These results illuminate the possibilities of phage display and antibody engineering for generation and optimisation of binding reagents in vitro and indicate the potential of recombinant antibodies as affinity reagents in immunoassays.

Effects of Silica Based Biomaterials on Bone Marrow Derived Cells - Material Aspects of Bone Regeneration

Silica based biomaterials, such as melt-derived bioactive glasses and sol-gel glasses, have been used for a long time in bone healing applications because of their ability to form hydroxyapatite and to stimulate stem cell proliferation and differentiation. In this study, bone marrow derived cells were cultured with bioactive glass and sol-gel silica, and seeded into porous polymer composite scaffolds that were then implanted femorally and subcutaneously in rats to monitor their migration inside host tissue. Bone marrow derived cells were also injected intraperitoneally. Transplanted cells migrated to various tissues inside the host, including the lung, liver spleen, thymus and bone marrow. The method of transplantation affected the time frame of cell migration, with intraperitoneal injection being the fastest and femoral implantation the slowest, but not the target tissues of migration. Transplanted donor cells had a limited lifetime in the host and were later eliminated from all tested tissues. Bioactive glass, however, affected the implanted cells negatively. When it was present in the scaffold no donor cells were found in any of the tested host tissues. Bioactive glass S53P4 was found to support both osteoblastic and osteoclastic phenotype of bone marrow derived cells, but it was resistant to the resorbing effect of osteoclastic bone marrow derived cells, showing that bioactive glass is rather dissolved through physicochemical reactions than resorbed by cells. Fast-dissolving silica sol gel in microparticulate form was found to increase collagen formation by bone marrow derived cells, while slow dissolving silica microparticles enhanced their proliferation, suggesting that the dissolution rate of silica controls the response of bone marrow derived cells.

Wood as a model material for medical biomaterials. In vivo and in vitro studies with bone and Betula pubescens Ehrh

Novel biomaterials are needed to fill the demand of tailored bone substitutes required by an ever‐expanding array of surgical procedures and techniques. Wood, a natural fiber composite, modified with heat treatment to alter its composition, may provide a novel approach to the further development of hierarchically structured biomaterials. The suitability of wood as a model biomaterial as well as the effects of heat treatment on the osteoconductivity of wood was studied by placing untreated and heat‐treated (at 220 C , 200 degrees and 140 degrees for 2 h) birch implants (size 4 x 7mm) into drill cavities in the distal femur of rabbits. The follow‐up period was 4, 8 and 20 weeks in all in vivo experiments. The flexural properties of wood as well as dimensional changes and hydroxyl apatite formation on the surface of wood (untreated, 140 degrees C and 200 degrees C heat‐treated wood) were tested using 3‐point bending and compression tests and immersion in simulated body fluid. The effect of premeasurement grinding and the effect of heat treatment on the surface roughness and contour of wood were tested with contact stylus and non‐contact profilometry. The effects of heat treatment of wood on its interactions with biological fluids was assessed using two different test media and real human blood in liquid penetration tests. The results of the in vivo experiments showed implanted wood to be well tolerated, with no implants rejected due to foreign body reactions. Heat treatment had significant effects on the biocompatibility of wood, allowing host bone to grow into tight contact with the implant, with occasional bone ingrowth into the channels of the wood implant. The results of the liquid immersion experiments showed hydroxyl apatite formation only in the most extensively heat‐treated wood specimens, which supported the results of the in vivo experiments. Parallel conclusions could be drawn based on the results of the liquid penetration test where human blood had the most favorable interaction with the most extensively heat‐treated wood of the compared materials (untreated, 140 degrees C and 200 degrees C heat‐treated wood). The increasing biocompatibility was inferred to result mainly from changes in the chemical composition of wood induced by the heat treatment, namely the altered arrangement and concentrations of functional chemical groups. However, the influence of microscopic changes in the cell walls, surface roughness and contour cannot be totally excluded. The heat treatment was hypothesized to produce a functional change in the liquid distribution within wood, which could have biological relevance. It was concluded that the highly evolved hierarchical anatomy of wood could yield information for the future development of bulk bone substitutes according to the ideology of bioinspiration. Furthermore, the results of the biomechanical tests established that heat treatment alters various biologically relevant mechanical properties of wood, thus expanding the possibilities of wood as a model material, which could include e.g. scaffold applications, bulk bone applications and serving as a tool for both mechanical testing and for further development of synthetic fiber reinforced composites.

Alpha2-Adrenoceptor-mediated vascular responses induced by dexmedetomidine

Alpha2-Adrenoceptors are cell-surface G protein coupled receptors that mediate many of the effects of the catecholamines noradrenaline and adrenaline. The three human α2-adrenoceptor subtypes are widely expressed in different tissues and organs, and they mediate many different physiological and pharmacological effects in the central and peripheral nervous system and as postsynaptic receptors in target organs. Previous studies have demonstrated that α2-adrenoceptors mediate both vascular constriction and dilatation in humans. Large inter-individual variation has been observed in the vascular responses to α2-adrenoceptor activation in clinical studies. All three receptor subtypes are potential drug targets. It was therefore considered important to further elucidate the details of adrenergic vascular regulation and its genetic variation, since such knowledge may help to improve the development of future cardiovascular drugs and intensive care therapies. Dexmedetomidine is the most selective and potent α2-adrenoceptor agonist currently available for clinical use. When given systemically, dexmedetomidine induces nearly complete sympatholysis already at low concentrations, and postsynaptic effects, such vasoconstriction, can be observed with increasing concentrations. Thus, local infusions of small doses of dexmedetomidine into dorsal hand veins and the application of pharmacological sympathectomy with brachial plexus block provide a means to assess drug-induced peripheral vascular responses without interference from systemic pharmacological effects and autonomic nervous system regulation. Dexmedetomidine was observed to have biphasic effects on haemodynamics, with an initial decrease in blood pressure at low concentrations followed by substantial increases in blood pressure and coronary vascular resistance at high concentrations. Plasma concentrations of dexmedetomidine that significantly exceeded the recommended therapeutic level did not reduce myocardial blood flow below the level that is observed with the usual therapeutic concentrations and did not induce any evident myocardial ischaemia in healthy subjects. Further, it was demonstrated that dexmedetomidine also had significant vasodilatory effects through activation of endothelial nitric oxide synthesis, and thus when the endothelial component of the blood vessel response to dexmedetomidine was inhibited, peripheral vasoconstriction was augmented. Hand vein constriction responses to α2-adrenoceptor activation by dexmedetomidine were only weakly associated with the constriction responses to α1-adrenoceptor activation, pointing to independent cellular regulation by these two adrenoceptor classes. Substantial inter-individual variation was noted in the venous constriction elicited by activation of α2-adrenoceptors by dexmedetomidine. In two study populations from two different continents, a single nucleotide polymorphism in the PRKCB gene was found to be associated with the dorsal hand vein constriction response to dexmedetomidine, suggesting that protein kinase C beta may have an important role in the vascular α2-adrenoceptor signalling pathways activated by dexmedetomidine.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Auscultatory and electrocardiographic characteristics of Crioulo horses

In order to determine auscultatory and electrocardiographic characteristics of Crioulo horses, one hundred animals ranging between one and twenty-six years of age (21 stallions, nine geldings, 27 pregnant mares e 43 not pregnant mares) were evaluated. The cardiac auscultation was performed during the clinical examination of the cardiovascular system, evaluating frequency, rate, normal and abnormal heart sounds (heart murmurs). The electrocardiographic examination followed the bipolar base-apex derivative system with animals at rest, by using an ECG-PC TEB equipment. The cardiac frequency, heart rate, morphology, duration, wave and complex amplitudes and interval durations were determined. The results were submitted to ANOVA and Tukey tests with an error probability of 5%. The cardiac auscultation revealed presence of functional systolic and diastolic murmur (10.00%) and systolic murmur compatible with tricuspid regurgitation besides normal heart sounds S1 (100.0%), S2 (100.0%), S3 (19.0%) and S4 (34.0%). The cardiac frequency obtained the average of 43.64 bpm, observing significative differences in relation to sexual and age factors and training level. The sinus rhythm was the most frequent (57.00%), followed by sinus tachycardia (38.00%) and sinus arrhythmia (5.00%), being observed rhythm disturbances in 16% of tracings. The P and T waves were observed more frequently in their forms P bifida positive (95.00%) and biphasic T (91.00%), being variable at tracing. There were also observed Q waves in 12.00% of the tracings. Thus, it was concluded that the auscultatory characteristics of Crioulo horses are according to the described in the literature for the species and the sexual factor, category, age factor and training level can influence some electrocardiographic parameters.

Characterization of nerve-induced relaxation of gastrointestinal sphincteric smooth muscle in a South American opossum (Didelphis albiventris)

The presence of inhibitory nonadrenergic noncholinergic (NANC) intrinsic innervation of the circular muscle of the gastrointestinal sphincters of the South American (SA) opossum was investigated in vitro. Isolated circular muscle strips from the esophagogastric and ileocolonic junctions but not from the gastroduodenal (pylorus) region developed spontaneous tension. Tetrodotoxin (TTX, 1 µM) augmented the spontaneous tension only in the ileocolonic junction strips. Electrical field stimulation of esophagogastric and ileocolonic junction strips caused frequency-dependent responses consisting of a relaxation at lower frequencies (<1 Hz) and a biphasic response or contraction at higher frequencies. In the strips from the pyloric region electrical field stimulation abolished the spontaneous activity at lower frequencies and induced contractions at higher frequencies. The responses elicited by electrical field stimulation in the three sphincters were abolished by TTX (1 µM). Electrical field-induced contractions were reduced while relaxations were enhanced by atropine (1 µM). In the presence of atropine (1 µM) and guanethidine (3 µM), electrical field stimulation, nicotine and ATP induced frequency- or concentration-dependent relaxations of the three sphincters that were abolished by TTX (1 µM). Isoproterenol and sodium nitroprusside caused concentration-dependent relaxations which were TTX-resistant. These findings indicate that the sphincteric circular muscle of the SA opossum gastrointestinal tract is relaxed by the activation of intrinsic NANC nerves and therefore can be used as a model for the study of the mechanisms involved in these responses

Hepatic damage during acute pancreatitis in the rat

We studied the alterations in the metabolism of liver mitochondria in rats with acute pancreatitis. Male Wistar rats were allocated to a control group (group I) and to five other groups corresponding to 2, 4, 12, 24 and 48 h after the induction of acute pancreatitis by the injection of 5% sodium taurocholate into the pancreatic duct. Sham-operated animals were submitted to the same surgical steps except for the induction of acute pancreatitis. Mitochondrial oxidation and phosphorylation were measured polarographically by determining oxygen consumption without ADP (basal respiration, state 4) and in the presence of ADP (activated respiration, state 3). Serum amylase, transaminases (ALT and AST) and protein were also determined. Ascitic fluid, contents of amylase, trypsin and total protein were also determined and arterial blood pressure was measured in all groups. In ascitic fluid, trypsin and amylase increased reaching a maximum at 2 and 4 h, respectively. Serum amylase increased at 2 h reaching a maximum at 4 h. Serum transaminase levels increased at 12 and 24 h. After 2 h (and also 4 h) there was an increase in state 4 respiration (45.65 ± 1.79 vs 28.96 ± 1.50) and a decrease in respiration control rate (3.53 ± 0.09 vs 4.45 ± 0.08) and in the ADP/O ratio (1.77 ± 0.02 vs 1.91 ± 0.01) compared to controls (P<0.05). These results indicate a disruption of mitochondrial function, which recovered after 12 h. In the 48-h groups there was mitochondrial damage similar to that occurring in ischemic lesion. Beat-to-beat analysis (30 min) showed that arterial blood pressure remained normal up to 24 h (111 ± 3 mmHg) while a significant decrease occurred in the 48-h group (91 ± 4 mmHg). These data suggest biphasic damage in mitochondrial function in acute pancreatitis: an initial uncoupled phase, possibly secondary to enzyme activity, followed by a temporary recovery and then a late and final dysfunction, associated with arterial hypotension, possibly related to ischemic damage.

Behavioral profiles displayed by rats in an elevated asymmetric plus-maze: effects of diazepam

When rats are exposed to unknown environments where novelty and fear-inducing characteristics are present (conflictive environments), some specific behaviors are induced and exploration is apparently modulated by fear. In our laboratory, a new type of plus-maze was designed as a model of conflictive exploration. The maze is composed of four arms with different geometrical characteristics, differing from each other by the presence or absence of walls. The degree of asymmetry was as follows: NW, no wall arm; SW, a single high wall present; HL, a low and a high wall present, and HH, two high walls present. The four arms were arranged at 90o angles and the apparatus was called the elevated asymmetric plus-maze (APM). The purpose of the present study was to assess the behavioral profile of rats exposed for a single time to the APM with or without treatment with benzodiazepine. Increasing doses of diazepam were injected intraperitoneally in several groups of male, 90-day-old Holtzman rats. Distilled water was injected in control animals. Thirty minutes after treatment all rats were exposed singly to a 5-min test in the APM. Diazepam induced a biphasic modification of exploration in the NW and SW arms. The increase in the exploration score was evident at low doses of diazepam (0.25-1.0 mg/kg body weight) and the decrease in exploration was found with the higher doses of diazepam (2.0-3.0 mg/kg body weight). Non-exploratory behaviors (permanency) were not affected by benzodiazepine treatment. In the HL arm, exploration was not modified but permanency was increased in a dose-dependent manner. In the HH arm, exploration and permanency were not affected. Results are compatible with the idea that exploration-processing mechanisms in conflictive environments are modulated by fear-processing mechanisms of the brain.

Circadian rhythms in blood pressure in free-ranging three-toed sloths (Bradypus variegatus)

Blood pressure (BP) profiles were monitored in nine free-ranging sloths (Bradypus variegatus) by coupling one common carotid artery to a BP telemetry transmitter. Animals moved freely in an isolated and temperature-controlled room (24ºC) with 12/12-h artificial light-dark cycles and behaviors were observed during resting, eating and moving. Systolic (SBP) and diastolic (DBP) blood pressures were sampled for 1 min every 15 min for 24 h. BP rhythm over 24 h was analyzed by the cosinor method and the mesor, amplitude, acrophase and percent rhythm were calculated. A total of 764 measurements were made in the light cycle and 721 in the dark cycle. Twenty-four-hour values (mean ± SD) were obtained for SBP (121 ± 22 mmHg), DBP (86 ± 17 mmHg), mean BP (MBP, 98 ± 18 mmHg) and heart rate (73 ± 16 bpm). The SBP, DBP and MBP were significantly higher (unpaired Student t-test) during the light period (125 ± 21, 88 ± 15 and 100 ± 17 mmHg, respectively) than during the dark period (120 ± 21, 85 ± 17 and 97 ± 17 mmHg, respectively) and the acrophase occurred between 16:00 and 17:45 h. This circadian variation is similar to that observed in cats, dogs and marmosets. The BP decreased during "behavioral sleep" (MBP down from 110 ± 19 to 90 ± 19 mmHg at 21:00 to 8:00 h). Both feeding and moving induced an increase in MBP (96 ± 17 to 119 ± 17 mmHg at 17:00 h and 97 ± 19 to 105 ± 12 mmHg at 15:00 h, respectively). The results show that conscious sloths present biphasic circadian fluctuations in BP levels, which are higher during the light period and are mainly synchronized with feeding.

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 µmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Modulation of hormone secretion by functional electrical stimulation of the intact and incompletely dysfunctional dog pancreas

The purpose of the present study was to modulate the secretion of insulin and glucagon in Beagle dogs by stimulation of nerves innervating the intact and partly dysfunctional pancreas. Three 33-electrode spiral cuffs were implanted on the vagus, splanchnic and pancreatic nerves in each of two animals. Partial dysfunction of the pancreas was induced with alloxan. The nerves were stimulated using rectangular, charge-balanced, biphasic, and constant current pulses (200 µs, 1 mA, 20 Hz, with a 100-µs delay between biphasic phases). Blood samples from the femoral artery were drawn before the experiment, at the beginning of stimulation, after 5 min of stimulation, and 5 min after the end of stimulation. Radioimmunoassay data showed that in the intact pancreas stimulation of the vagal nerve increased insulin (+99.2 µU/ml) and glucagon (+18.7 pg/ml) secretion and decreased C-peptide secretion (-0.15 ng/ml). Splanchnic nerve stimulation increased insulin (+1.7 µU/ml), C-peptide (+0.01 ng/ml), and glucagon (+50 pg/ml) secretion, whereas pancreatic nerve stimulation did not cause a marked change in any of the three hormones. In the partly dysfunctional pancreas, vagus nerve stimulation increased insulin (+15.5 µU/ml), glucagon (+11 pg/ml), and C-peptide (+0.03 ng/ml) secretion. Splanchnic nerve stimulation reduced insulin secretion (-2.5 µU/ml) and increased glucagon (+58.7 pg/ml) and C-peptide (+0.39 ng/ml) secretion, and pancreatic nerve stimulation increased insulin (+0.2 µU/ml), glucagon (+5.2 pg/ml), and C-peptide (+0.08 ng/ml) secretion. It was concluded that vagal nerve stimulation can significantly increase insulin secretion for a prolonged period of time in intact and in partly dysfunctional pancreas.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22ºC) and non-thermal hot-plate stress (30ºC) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50ºC) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50ºC; N = 9 in each group). The results (means ± SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 ± 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 ± 3.19 s), 5.0 mg/kg (11.53 ± 1.64 s) and 7.5 mg/kg naloxone (7.38 ± 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 ± 8.53 s) and saline groups (29.1 ± 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.