Inhibition of Eph/ephrin interaction with the small molecule UniPR500 improves glucose tolerance in healthy and insulin-resistant mice.

Eph/ephrin interactions and their bidirectional signaling are integral part of the complex communication system between ß-cells, essential for glucose homeostasis. Indeed, Eph/ephrin system was shown to be directly involved in the glucose-stimulated insulin secretion (GSIS) process occurring in the pancreatic islets. Here we tested the Eph antagonist UniPR500 as GSIS enhancer. UniPR500 was validated as EphA5-ephrin-A5 inhibitor in vitro and its efficacy as GSIS enhancer was assessed on EndoC-ßH1 cells. The selectivity of UniPR500 was evaluated by testing this compound on a panel of well-known molecular targets responsible for the regulation of glucose homeostasis. Plasmatic levels of UniPR500 were measured by HPLC/MS approach after oral administration. Finally, UniPR500 was tested as hypoglycemic agent in healthy mice, in a non-genetic mouse model of insulin resistance (IR) and in a non-genetic mouse model of type 1 diabetes (T1D). The compound is an orally bioavailable and selective Eph antagonist, able to increase GSIS from EndoC-ßH1 cells. When tested in vivo UniPR500 showed to improve glucose tolerance in healthy and IR mice. As expected by a GSIS enhancer acting on healthy ß-cells, UniPR500 was ineffective when tested on a non-genetic mouse model of type 1 diabetes, where pancreatic function was severely compromised. In conclusion our findings suggest that Eph targeting is a new and valuable pharmacological strategy in the search of new hypoglycemic agents.

Sluggish dorsally-driven inhibition of return during orthographic processing in adults with dyslexia.

Dyslexia (D) is a neurodevelopmental reading disorder characterized by phonological and orthographic deficits. Before phonological decoding, reading requires a specialized orthographic system for parallel letter processing that assigns letter identities to different spatial locations. The magnocellular-dorsal (MD) stream rapidly process the spatial location of visual stimuli controlling visuo-spatial attention. To investigate the visuo-spatial attention efficiency during orthographic processing, inhibition of return (IOR) was measured in adults with and without D in a lexical decision task. IOR is the delay in responding to stimuli displayed in a cued location after a long cue-target interval. Only adults with D did not showed IOR effect during letter-string recognition, despite the typical left-hemisphere specialization for word identification. A specific deficit in coherent-dot-motion perception confirmed an MD-stream disorder in adults with D. Our results suggest that adults with D might develop an efficient visual word form area, but a dorsal-attentional dysfunction impairs their reading fluency.

The balance between rRNA and ribosomal protein synthesis up- and downregulates the tumour suppressor p53 in mammalian cells.

Data on the relationship between ribosome biogenesis and p53 function indicate that the tumour suppressor can be activated by either nucleolar disruption or ribosomal protein defects. However, there is increasing evidence that the induction of p53 does not always require these severe cellular changes, and data are still lacking on a possible role of ribosome biogenesis in the downregulation of p53. Here, we studied the effect of the up- and downregulation of the rRNA transcription rate on p53 induction in mammalian cells. We found that a downregulation of rRNA synthesis, induced by silencing the POLR1A gene coding for the RNA polymerase I catalytic subunit, stabilised p53 without altering the nucleolar integrity in human cancer cells. p53 stabilisation was due to the inactivation of the MDM2-mediated p53 degradation by the binding of ribosomal proteins no longer used for ribosome building. p53 stabilisation did not occur when rRNA synthesis downregulation was associated with a contemporary reduction of protein synthesis. Furthermore, we demonstrated that in three different experimental models characterised by an upregulation of rRNA synthesis, cancer cells treated with insulin or exposed to the insulin-like growth factor 1, rat liver stimulated by cortisol and regenerating rat liver after partial hepatectomy, the p53 protein level was reduced due to a lowered ribosomal protein availability for MDM2 binding. It is worth noting that the upregulation of rRNA synthesis was responsible for a decreased p53-mediated response to cytotoxic stresses. These findings demonstrated that the balance between rRNA and ribosomal protein synthesis controls the function of p53 in mammalian cells, that p53 can be induced without the occurrence of severe changes of the cellular components controlling ribosome biogenesis, and that conditions characterised by an upregulated rRNA synthesis are associated with a reduced p53 response.

Intestinal chronic obstruction affects motor responsiveness of rat hypertrophic longitudinal and circular muscles.

Extensive morphological and neurochemical changes have been experimentally and clinically documented in the hypertrophied intestine located orally to a chronic partial stenosis of the lumen. Functional studies revealed not only disruption of the interdigestive motor complex in vivo and decreased efficiency of contraction but also preservation of the peristaltic reflex in vitro. Given the critical role played in intestinal peristalsis by the coordinated activity of the longitudinal (LM) and circular muscle (CM), this work focuses on the motor responses of LM and CM isolated from rat hypertrophied ileum following mechanical obstruction. Maximal contractions to both receptor (acetylcholine and substance P) and non-receptor (K+) mediated stimuli were up to 10-fold increased in hypertrophic CM rings compared with control tissues, while a higher potency of substance P was revealed in both hypertrophied muscle layers. Relaxations to vasoactive intestinal polypeptide and 8-Br-cGMP were more intense on prostaglandin F(2alpha)-contracted hypertrophic LM strips compared with control tissues and a general tendency towards increased relaxation was shared also by hypertrophic CM basal tone. The present results collectively suggest that hypertrophic growth leads to hyperresponsiveness to contractile agents, particularly evident in the CM, and to increased sensitivity to relaxing mediators, especially exhibited by the LM. In this regard, the complementary role exerted by each muscle layer and the plasticity of the intestinal tissue could both come into play to preserve the intestinal functions in a changing environment.

Is alexithymia a personality trait increasing the risk of depression? A prospective study evaluating alexithymia before, during and after a depressive episode.

BACKGROUND: Whether alexithymia is a personality trait that increases the risk of major depression (MD) is still debated. In this prospective study, alexithymic levels were evaluated before, during and after a depressive episode. METHOD: The alexithymic levels, the presence of MD and the severity of anxious-depressive symptoms were evaluated at intervals of about 1 month in pregnant women attending the Centers for Prenatal Care, using the Toronto Alexithymia Scale (TAS), the Primary Care Evaluation of Mental Disorders (PRIME-MD) and the Hospital Anxiety and Depression Scale (HADS). RESULTS: Sixteen women affected by MD, 21 affected by subthreshold depression and 112 non-depressed women were included in the study. Women who developed depression, compared to non-depressed women, showed similar TAS and HADS scores during the pre-morbid phase, a significant increase in the scores during depression and a significant decrease after remission, whereas no change was observed in non-depressed women. CONCLUSIONS: Our data suggest that in pregnant women alexithymia does not represent a personality trait that increases the risk of developing a depressive episode, and they support the hypothesis that alexithymia is a state-dependent phenomenon in depressed pregnant women.

Plasticity of rat small intestine after removal of a chronic mechanical obstruction.

Chronic intestinal obstruction is associated with morphological changes and functional disorders clinically reported and experimentally documented in laboratory animals. In contrast, little is known about the properties of the hypertrophied intestine after removal of the obstruction. In the present study, we removed the ileal obstruction previously applied to the ileum of rats and, after 1 or 2 weeks, studied in vitro the motor responses of de-obstructed segments of intestine to pharmacological or electrical field stimulation (EFS). By 2 weeks after de-obstruction, maximal contractile responses to receptor (acetylcholine) and non-receptor (K(+)) mediated stimuli were comparable in operated and control tissues; furthermore, the loss of sensitivity to nitric oxide (NO) unmasked in obstructed tissues was, after de-obstruction, replaced by supersensitivity to exogenous NO and vasoactive intestinal polypeptide, probably acting through cyclic nucleotide-independent pathways. Despite the complete recovery of smooth muscle responses, neurogenic contractions remained impaired in de-obstructed tissue; however, the equal contribution of cholinergic/peptidergic components to EFS responses could represent a sign of gradual but delayed recovery of enteric neurotransmission.

Effect of N-methyl-d-aspartate receptor blockade on neuronal plasticity and gastrointestinal transit delay induced by ischemia/reperfusion in rats.

Intestinal ischemia impairs gastrointestinal motility. The aims of this study were to investigate the effect of intestinal ischemia on gastrointestinal transit and on the expression of enteric transmitters in the rat, and whether the glutamate N-methyl-d-aspartate receptors influence these effects. Ischemia (1 h), induced by occluding the superior mesenteric artery, was followed by 0 or 24 h of reperfusion. Normal and sham-operated rats served as controls. Serosal blood flow was measured with laser Doppler flow meter. Gastrointestinal transit was measured as time of appearance of a marker in fecal pellets. Immunohistochemistry was used to evaluate the number of neurons immunoreactive for neuronal nitric oxide synthase (NOS) or vasoactive intestinal polypeptide and the density of substance P immunoreactive fibers in the myenteric plexus. The N-methyl-d-aspartate receptors antagonist, (+)-5-methyl-10,11-dihydro-5HT-[a,b] cyclohepten-5,10-imine (MK-801) (1 mg/kg i.v.) or the NOS inhibitor, N-nitro-l-arginine (10 mg/kg i.v.) was administered prior to ischemia. Serosal blood flow was decreased by 70% during ischemia, but it was not altered in sham-operated rats. Gastrointestinal transit was significantly prolonged in ischemic/reperfused rats compared with controls. There was a significant increase in the number of vasoactive intestinal polypeptide and neuronal nitric oxide synthase immunoreactive neurons, and a marked decrease of substance P immunoreactive fibers in ischemia followed by 24 h of reperfusion animals compared with controls. These alterations were not observed in ischemia without reperfusion. A significant delay of gastrointestinal transit and increase of vasoactive intestinal polypeptide neurons were also observed in sham-operated rats. The changes in transmitter expression and gastrointestinal transit in ischemic/reperfused rats were prevented by pre-treatment with the NOS inhibitor, N-nitro-l-arginine or the N-methyl-d-aspartate receptors antagonist, MK-801. This study suggests an involvement of the glutamatergic system and its interaction with nitric oxide in intestinal ischemia/reperfusion. Ischemia/reperfusion might induce local release of glutamate that activates N-methyl-d-aspartate receptors leading to increased production of nitric oxide and adaptive changes in enteric transmitters that might contribute to gastrointestinal dysmotility.

Motor responses of rat hypertrophic intestine following chronic obstruction.

The present work aims at investigating the changes in motor responsiveness of rat intestine hypertrophied by chronic mechanical obstruction. Motor responses to pharmacological agents and electrical field stimulation (EFS) were studied in hypertrophic ileal segments excised from rats subjected to experimental stenosis (n = 20) and compared with responses of control tissues from sham-operated animals (n = 20). Spontaneous motility and contractile responses to exogenous agents (KCl, acetylcholine and substance P) and EFS (10-s trains every minute, 120 mA, 0.5 ms, 1-10 Hz) were increased in hypertrophic longitudinal segments; however, normalization of motor responses to tissue wet weight revealed a remarkable reduction of contractile efficiency in hypertrophied tissues coupled with a loss of sensitivity to nitric oxide-mediated relaxation. Furthermore, EFS under non-adrenergic non-cholinergic (NANC) conditions unveiled a major role of the cholinergic component over the peptidergic one in the neurogenic contraction of hypertrophic intestine. On the whole, hypertrophic intestinal growth emerges as a dynamic process entailing adaptation of smooth muscle and neuronal structures to the increased functional load imposed by lumen obstruction.

Different role of the histamine H3-receptor in vagal-, betanechol-, pentagastrin-induced gastric acid secretion in anaesthetized rats.

BACKGROUND: To date, involvement of the histamine H3-receptor in the control of gastric acid secretion in rats is not conclusively defined because of the variability of experimental results. This study was therefore aimed at investigating the role of H3-receptors in acid secretion produced by nervous or pharmacological stimulation in anaesthetized rats. METHODS: Gastric acid output was measured by flushing the rat stomach lumen with 5 ml saline and titrating the flushed perfusate. Hypersecretory responses were evoked through direct vagal stimulation (0.5 msec, 10 Hz, 50 V for 30 min every 30 min) or by stimulation with pentagastrin (20, 40, 100, 250 microg/kg/h i.v.) or betanechol (100, 250, 500 microg/kg/h i.v.). The selective H3 ligands (R)-alpha-methylhistamine and thioperamide (100 microg/kg i.v.) were tested alone or in combination on both basal and electrically/pharmacologically induced secretion. RESULTS: Vagally-induced response was significantly reduced by the agonist R-alpha-methylhistamine and this effect was antagonized by the antagonist thioperamide at a dose unable by itself to modify vagal response. Thioperamide significantly increased acid response only on pentagastrin low dose (20 microg/kg/h) and this effect was counteracted by R-alpha-methylhistamine, which was ineffective when administered alone. Betanechol-induced hypersecretion was substantially unaffected by the H3 ligands, which were also inactive on basal acid output. CONCLUSIONS: Although this functional study confirms the presence of histamine H3-receptors in the rat stomach, they appear to have minor weight in regulation of the acid secretion in this species.

New polycyclic pyrimidine derivatives with antiplatelet in vitro activity: synthesis and pharmacological screening.

The preparation and the pharmacological screening of novel anti-aggregatory/antiphlogistic polycyclic pyrimidine derivatives are described. The compounds were developed starting from bioactive 2-aminobenzopyranopyrimidine derivatives in order to assess the importance of the benzopyrano[4,3-d]pyrimidine structure and the role of an amino basic moiety in position 2. Antiplatelet activity was assessed in vitro against ADP and arachidonic acid-induced aggregation in guinea-pig plasma. Anti-inflammatory/analgesic/antipyretic activities were studied in rat paw oedema, mouse writhing test and E. coli-induced rat fever. Ulcerogenic and gastroprotective effects were also investigated in vivo on rat gastric mucosa. Among the tested compounds, the 5-substituted benzopyranopyrimidine derivatives 3d and 4d proved to be the most active antiplatelet agents as potent as acetylsalicylic acid against arachidonic acid-stimulated aggregation. Furthermore the 2-methylthio derivative 4d was endowed with greater efficacy against ADP aggregation suggesting that additional non-TXA2 dependent mechanisms are involved in its biological activity. Orally administered at 100 mg kg(-1) in rats this latter compound displayed antiphlogistic acitivity comparable to indomethacin (10 mg kg(-1)) coupled with an unusual gastroprotective effect on ethanol-induced ulcers. In conclusion, these findings indicate that the 5-pyrrolidino-2-methylthiobenzopyrano[4,3-d]pyrimidine 4d fulfils the chemical requirements to exhibit antiplatelet activity associated with gastroprotective effect.

Evidence for specific analgesic activity of a muscarinic agonist selected among a new series of acetylenic derivatives.

The central and peripheral effects of a series of Oxotremorine/Oxotremorine-M derivatives, previously characterized as muscarinic agonists in isolated preparations, were investigated in in vivo experiments. The molecules were tested for their antinociceptive activity (formalin licking and acetic acid writhing tests) and for their ability to induce tremor in mice. Peripheral cholinergic effects such as salivation, bradycardia, hypotension and intestinal hypermotility were studied in anaesthetized rats. All of the acetylenic compounds acted as muscarinic analgesics displaying the same order of potency shown in in vitro studies. The Oxotremorine-like subset showed a clearer distinction between doses producing antinociception and doses exerting undesirable central/peripheral side effects compared to the Oxotremorine-M derivatives. The most promising profile was displayed by the isoxazolin-3-one Oxotremorine-like derivative (compound 1a), which was characterized by a wider therapeutic window than that of the parent molecule Oxotremorine. Indeed, it produced atropine-sensitive analgesia (ID50 about 0.1 mg/kg i.p.) in the absence of tremorogenic (EC50 2.73 mg/kg i.p.) and cardiovascular effects while lethality occurred only at higher doses (LD50 19 mg/kg i.p.). These results suggest that such a derivative could be a candidate for further development of selective muscarinic analgesics.

Hypertrophy of mucosa and serosa in the obstructed intestine of rats.

After a surgically induced partial obstruction of the small intestine (ileum) in adult rats there is an accumulation of ingesta and a progressive enlargement of the lumen accompanied by wall thickening: over a period of 2-3 wk the circumference of the hypertrophic intestine increases by a factor of 2.7 and the thickness of the musculature increases more than threefold, while the length of the ileum (measured at the mesenteric attachment) remains unchanged. The villi become markedly larger and more elongated in the circumferential direction, and have a greater separation between one another. The number of villi per unit surface is markedly reduced but the number of villi per unit length of ileum, whilst appearing to show a small increase, was not significantly altered. The component epithelial cells (absorptive cells) appear unchanged in morphology and size (height). The microvilli of the epithelial cells have the same appearance, size (height) and packing density in the control and the hypertrophic ileum. Glands of Lieberkühn, Peyer's patches and single lymphatic follicles constituting the Peyer's patches are significantly increased in size in the hypertrophic intestine. The serosal surface of the hypertrophic ileum, in spite of the great expansion, remains regularly covered by mesothelial cells; these are much larger than in the controls and have an altered distribution of their microvilli.

Synthesis and pharmacological screening of novel non-acidic gastroprotective antipyretic anti-inflammatory agents with anti-platelet properties. 5-Alkyl/cycloalkylamino substituted 2-amino-5H-[1]benzopyrano[4,3-d] pyrimidines.

The preparation and screening of antipyretic, anti-inflammatory, analgesic, gastroprotective and antiplatelet activities of original non-acidic aminobenzo-pyranopyrimidine derivatives are described. Major and dose-dependent analgesic and antipyretic properties were detected in all the compounds after oral administration (6.25-100 mg kg-1) in the mouse writhing test and in the E. coli lipopolysaccharide-induced fever in the rat, respectively. Unlike the reference drug indometacin (ED50 ulcer = 9.1 mg kg-1), no gastrolesivity was displayed by all the new compounds up to 150 mg kg-1 so that therapeutical indices equal or higher than that of indometacin were calculated. Furthermore, at 100 mg kg-1 they were able to prevent ethanol-induced gastric mucosal injury in rats. At a 1 mmol/l concentration the compounds under study were as effective as acetylsalicylic acid in inhibiting in vitro platelet aggregation induced by adenosine diphosphate.

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Interactions of new and conventional H3-antagonists with non-histaminergic receptors involved in neurogenic and myogenic contractile responses of guinea-pig ileum.

1. Possible effects of new and conventional H3-receptor antagonists towards various non-histaminergic receptors (alpha2-adrenergic, 5-HT3-serotonin, mu-opiate, A1-adenosine, M1-and M3-muscarinic) involved in neurogenic and myogenic contractile responses of guinea-pig ileum are investigated. 2. When the isolated ileum was contracted by the 5-HT3 receptor agonist, 2-methyl-5-HT (5 x 10(-7)-8 x 10(-6) M), acetylcholine (1 x 10(-9)-1 x 10(-7) M), KCl (3 x 10(-2) M) or electrical stimulation some of the drugs, included thioperamide and clobenpropit, reduced the contractile response when tested at micromolar concentrations (1-3 x 10(-5) M) (only compound IV exhibited an M3 competitive antagonism with a pK(B) = 5.49 +/- 0.18). 3. Ileal twitch responses to electrical stimulation were dose-dependently inhibited by the selective agonists clonidine (3 x 10(-10)-1 x 10(-7) M), dermorphin (1 x 10(-11)-1 x 10(-8) M), R-N6-(2-phenylisopropyl)-adenosine (1 x 10(-9)-3 x 10(-8) M) and McN-A-343 (1 x 10(-7)-1 x 10(-5) M) with different potencies and comparable efficacy (spike amplitude reduction > 85%). All the H3 antagonists under study (up to 1 x 10(-5) M) showed no or minor interactions at the neuronal sites except the compound V which behaved as a weak competitive antagonist at alpha2-adrenoreceptors (pK(B) = 5.96 +/- 0.06). 4. In conclusion, both new and conventional H3-blockers interacted at the enteric neuronal sites here studied with a 1000-30,000 fold lower antagonistic potency than that previously reported for the ileal H3 histamine receptors. Their spasmolytic activity precludes firm conclusions about the non-competitive interaction with 5-HT3 ileal receptor which requires further investigations.

In vitro characterization of potency, affinity and selectivity of H3-antagonists: from thioperamide to thioperamide unrelated imidazole derivatives.

This paper summarizes the findings obtained for three different series of original compounds designed as potential H3-antagonists starting from thioperamide structure. The compounds were tested in functional and binding assays to estimate their potency, affinity and selectivity for histamine H3 receptors. Among them, many non-thiourea/isothiourea derivatives acted as selective H3 competitive antagonists and, particularly, 4(5)-[2-[4(5)-cyclohexylimidazol-2-ylthio]ethyl] imidazole (dIII) proved to be the most potent H3 blocker vs (R)-alpha-methylhistamine in electrically-stimulated ileum. This imidazole derivative, devoid of thiourea dependent toxic effects, with high affinity displaced biphasically [3H]-N alpha-methylhistamine bound to rat brain H3 sites. Thus, such compound could be proposed as the prototype molecule for the development of new non-thiourea/isothiourea H3-antagonists and as experimental tool to explore the intriguing question of H3 receptor heterogeneity.

[Cholesterol in beef, pork, chicken and their products commercialized in Maringá, Paraná, Brazil]. / Colesterol em carnes bovinas, suínas, frangos e derivados de carnes comercializados em Maringá, Paraná, Brasil.

A rapid method developed by Al-Hasani et al. with modifications for cholesterol determination was applied in samples of beef, pork, chicken and meat products. Analyses of cholesterol were performed by capillary gas chromatography. The obtained results showed that chicken presented higher concentration (126.96 to 188.29 mg/100 g) of cholesterol in comparison to beef and pork and similar to meat products, except bovine liver that presented 265.03 mg/100 g. Cholesterol values of beef and pork samples were considered low due to the absence of superficial fat in meat samples.