Diagrammatical scale to quantify herbivory severity on feijoa leaf caused by Paraulaca dives (Coleoptera: Chrysomelidae).

The aims of this work were to characterize the damage caused by Paraulaca dives in feijoa (Acca sellowiana) and develop a diagrammatic scale to evaluate the severity of herbivory. The evaluations were carried out in a feijoa progenies orchard with eight-year-old. The beetles caused damage mainly on leaves, from October to December (spring season). The distribution of beetles in the orchard occurred randomly, not following a defined pattern of incidence. The diagrammatic scale of herbivory severity was represented by seven levels (1%, 3%, 5%, 7%, 15%, 32%, and 55% of the leaf area consumed). The use of this diagrammatic scale significantly increased the precision and accuracy of severity estimates by inexperienced evaluators. This can help in strategies to control this pest to expand the cultivation of feijoa in Brazil.

Targeted deletion of Insm2 in mice result in reduced insulin secretion and glucose intolerance.

BACKGROUND: Neurogenin3 (Ngn3) and neurogenic differentiation 1 (NeuroD1), two crucial transcriptional factors involved in human diabetes (OMIM: 601724) and islet development, have been previously found to directly target to the E-boxes of the insulinoma-associated 2 (Insm2) gene promoter, thereby activating the expression of Insm2 in insulin-secretion cells. However, little is known about the function of Insm2 in pancreatic islets and glucose metabolisms. METHODS: Homozygous Insm2-/- mice were generated by using the CRISPR-Cas9 method. Glucose-stimulated insulin secretion and islet morphology were analyzed by ELISA and immunostainings. Expression levels of Insm2-associated molecules were measured using quantitative RT-PCR and Western blots. RESULTS: Fasting blood glucose levels of Insm2-/- mice were higher than wild-type counterparts. Insm2-/- mice also showed reduction in glucose tolerance and insulin/C-peptide levels when compared to the wild-type mice. RT-PCR and Western blot analysis revealed that expression of Insm1 was significantly increased in Insm2-/- mice, suggesting a compensatory response of the homolog gene Insm1. Similarly, transcriptional levels of Ngn3 and NeuroD1 were also increased in Insm2-/- mice. Moreover, Insm2-/- female mice showed a significantly decreased reproductive capacity. CONCLUSIONS: Our findings suggest that Insm2 is important in glucose-stimulated insulin secretion and is involved in the development pathway of neuroendocrine tissues which are regulated by the transcription factors Ngn3, NeuroD1 and Insm1.

Small cell lung cancer growth is inhibited by miR-342 through its effect of the target gene IA-2.

BACKGROUND: Small cell lung cancers (SCLC) are tumors of neuroendocrine origin. Previous in vitro studies from our laboratory showed that SCLC expresses high levels of the transmembrane dense core vesicle protein IA-2 (islet cell antigen-2) as compared to normal lung cells. IA-2, through its effect on dense core vesicles (DCVs), is known to be involved in the secretion of hormones and neurotransmitters. It is believed that the dysregulated release of the neurotransmitter Acetylcholine (ACh) by DCVs has an autocrine effect on SCLC cell growth. Recently, we found that IA-2 is a target of the microRNA miR-342 and that miR-342 mimics suppress the expression of IA-2. The present experiments were initiated to see whether IA-2 and/or miR-342 affect the growth of SCLC. METHODS: SCLC cell growth was evaluated following the knockdown of endogenous IA-2 with RNAi or by overexpressing miR-342 with a mimic. The secretion and content of ACh in SCLC cells was analyzed using a human acetylcholine ELISA (enzyme-linked immunosorbent assay) kit. RESULTS: The knockdown of endogenous IA-2 by RNAi reduced SCLC cell growth within 4 days by 40 % or more. Similar results were obtained when these cell lines were transfected with a miR-342 mimic. The knockdown of IA-2 by RNAi or miR-342 with a mimic also resulted in a significant decrease in the secretion of ACh, one of the autocrine hormones secreted by SCLC. Further studies revealed that the growth of SCLC cell lines that had been treated with the miR-342 mimic was restored to nearly normal levels by treatment with ACh. CONCLUSION: Our studies show for the first time that both miR-342 and its target gene IA-2 are involved in the growth process of SCLC cells and act by their effect on autocrine secretion. These findings point to possible new therapeutic approaches for the treatment of autocrine-induced tumor proliferation.

The Ia-2ß intronic miRNA, miR-153, is a negative regulator of insulin and dopamine secretion through its effect on the Cacna1c gene in mice.

AIMS/HYPOTHESIS: miR-153 is an intronic miRNA embedded in the genes that encode IA-2 (also known as PTPRN) and IA-2ß (also known as PTPRN2). Islet antigen (IA)-2 and IA-2ß are major autoantigens in type 1 diabetes and are important transmembrane proteins in dense core and synaptic vesicles. miR-153 and its host genes are co-regulated in pancreas and brain. The present experiments were initiated to decipher the regulatory network between miR-153 and its host gene Ia-2ß (also known as Ptprn2). METHODS: Insulin secretion was determined by ELISA. Identification of miRNA targets was assessed using luciferase assays and by quantitative real-time PCR and western blots in vitro and in vivo. Target protector was also employed to evaluate miRNA target function. RESULTS: Functional studies revealed that miR-153 mimic suppresses both glucose- and potassium-induced insulin secretion (GSIS and PSIS, respectively), whereas miR-153 inhibitor enhances both GSIS and PSIS. A similar effect on dopamine secretion also was observed. Using miRNA target prediction software, we found that miR-153 is predicted to target the 3'UTR region of the calcium channel gene, Cacna1c. Further studies confirmed that Cacna1c mRNA and protein are downregulated by miR-153 mimics and upregulated by miR-153 inhibitors in insulin-secreting freshly isolated mouse islets, in the insulin-secreting mouse cell line MIN6 and in the dopamine-secreting cell line PC12. CONCLUSIONS/INTERPRETATION: miR-153 is a negative regulator of both insulin and dopamine secretion through its effect on Cacna1c expression, which suggests that IA-2ß and miR-153 have opposite functional effects on the secretory pathway.

Protein Tyrosine Phosphatase Receptors N and N2 Control Pituitary Melanotroph Development and POMC Expression.

The neuroendocrine marker genes Ptprn and Ptprn2 encode protein tyrosine phosphatase receptors N and N2, 2 members of protein tyrosine phosphatase receptors void of enzymatic activity, and whose function and mechanism of action have not been elucidated. To explore the role(s) of Ptprn and Ptprn2 on the hypothalamic-pituitary-adrenal axis, we used mice in which both genes were knocked out (DKO). The focus in this study was on corticotrophs and melanotrophs from the anterior and intermediate lobes of the pituitary gland, respectively. In both sexes, DKO caused an increase in the expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-melanocyte-stimulating hormone, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased the expression of other melanotroph-specific genes, but not corticotroph-specific genes. The dopaminergic pathway in the hypothalamus and dopaminergic receptors in melanotrophs were not affected in DKO mice. However, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased proopiomelanocortin immunoreactivity per cell. These results indicate that protein tyrosine phosphatase receptor type N contributes to hypothalamic-pituitary-adrenal function by being involved in processes governing postnatal melanotroph development and Pomc expression.

CSF catecholamine profile in subarachnoid hemorrhage patients with neurogenic cardiomyopathy.

BACKGROUND: Patients experiencing apoplectic intracranial processes may develop neurogenic cardiomyopathy (NC). The purpose of this research is to determine whether cerebrospinal fluid (CSF) catecholamine levels are elevated in subarachnoid hemorrhage (SAH) patients with NC when compared to those without NC. METHODS: Observational study of consecutive grades 3-5 SAH patients requiring ventriculostomy. All patients underwent CSF sampling for catecholamine levels, and transthoracic echocardiography (TTE) to assess for NC, within 48 h of SAH onset. Univariate analyses were performed to identify clinical and laboratory variables associated with NC. Clinical variables associated with NC in the univariate analysis were entered into logistic regression models along with the candidate catecholamine variables to identify predictors of NC. RESULTS: The study group contained 100 patients--mean age of study subjects was 58 years, 73% were female, and 15% developed NC. NC patients were more likely to have a worse clinical grade than patients without NC (80 vs. 34%, P = 0.001). NC patients possessed greater DOPA levels (5.83 vs. 4.60 nmol/l, P = 0.044), and a trend toward greater noradrenergic activity as determined by NE/DHPG ratio (0.3799 vs. 0.2519, P = 0.073). Multivariate analysis identified worse clinical grade (OR 7.09, P = 0.005) and possibly NE levels (OR 1.005, P = 0.057) as independent predictors of NC. Bivariate analysis reinforced the findings for NE (OR 1.006, P = 0.022), and also identified DOPA levels (OR 1.001, P = 0.034) and NE/DHPG (OR 22.18, P = 0.019) as predictors of NC. CONCLUSIONS: SAH patients with NC tend to have greater CSF catecholamine levels than those without NC. However, the development of NC may also be related to factors not evaluated by our study.

Kinin B1 receptors contributes to acute pain following minor surgery in humans.

BACKGROUND: Kinins play an important role in regulation of pain and hyperalgesia after tissue injury and inflammation by activating two types of G-protein-coupled receptors, the kinin B1 and B2 receptors. It is generally accepted that the B2 receptor is constitutively expressed, whereas the B1 receptor is induced in response to inflammation. However, little is known about the regulatory effects of kinin receptors on the onset of acute inflammation and inflammatory pain in humans. The present study investigated the changes in gene expression of kinin receptors and the levels of their endogenous ligands at an early time point following tissue injury and their relation to clinical pain, as well as the effect of COX-inhibition on their expression levels. RESULTS: Tissue injury resulted in a significant up-regulation in the gene expression of B1 and B2 receptors at 3 hours post-surgery, the onset of acute inflammatory pain. Interestingly, the up-regulation in the gene expression of B1 and B2 receptors was positively correlated to pain intensity only after ketorolac treatment, signifying an interaction between prostaglandins and kinins in the inflammatory pain process. Further, the gene expression of both B1 and B2 receptors were correlated. Following tissue injury, B1 ligands des-Arg9-BK and des-Arg10-KD were significantly lower at the third hour compared to the first 2 hours in both the placebo and the ketorolac treatment groups but did not differ significantly between groups. Tissue injury also resulted in the down-regulation of TRPV1 gene expression at 3 hours post-surgery with no significant effect by ketorolac treatment. Interestingly, the change in gene expression of TRPV1 was correlated to the change in gene expression of B1 receptor but not B2 receptor. CONCLUSIONS: These results provide evidence at the transcriptional level in a clinical model of tissue injury that up-regulation of kinin receptors are involved in the development of the early phase of inflammation and inflammatory pain. The up-regulation of B1 receptors may contribute to acute inflammatory pain through TRPV1 activation.

Intravenous butyrylcholinesterase administration and plasma and brain levels of cocaine and metabolites in rats.

Butyrylcholinesterase is a major cocaine-metabolizing enzyme in humans and other primates, catalyzing hydrolysis to ecgonine methylester. Increasing butyrylcholinesterase activity may be a treatment for cocaine addiction. We evaluated the effect of 30-min pretreatment with horse-derived butyrylcholinesterase (5-15,000 U i.v.) or with the selective butyrylcholinesterase inhibitor cymserine (10 mg/kg i.v.) on the metabolism of cocaine (17 mg/kg i.p.) in anesthetized rats. Venous blood samples were collected for two hours after cocaine administration and later assayed for cocaine and metabolites by gas chromatography/mass spectroscopy. Whole brains were collected after the last blood sample and similarly assayed. Butyrylcholinesterase significantly increased plasma and brain ecgonine methylester levels and decreased cocaine plasma half-life from 26.2 min (saline) to 16.4 min (15,000 U). Butyrylcholinesterase had no significant effect on plasma or brain cocaine or benzoylecgonine levels. Cymserine had no effect on any variable. These findings suggest that butyrylcholinesterase treatment may have benefits in enhancing cocaine metabolism and in increasing levels of ecgonine methylester, which may have a protective action against cocaine.

Effects of dopamine agonists and antagonists on locomotor activity in male and female rats.

Male and female Sprague-Dawley rats were treated with cocaine, the specific dopamine uptake inhibitor GBR 12909, the dopamine D1 agonist SKF 82958 or the dopamine D2 agonist quinpirole. After treatment, the rats were placed in an activity chamber for 30 min and locomotor activity was monitored. Cocaine, GBR 12909 and SKF 82958 all increased locomotor activity in both males and females, but greater increases were observed in females. In contrast, quinpirole produced decreases in activity, with males showing greater decreases than females. Separate groups of animals were given SCH 23390 or eticlopride prior to cocaine. The D1 antagonist SCH 23390 reduced the locomotor activating effects of cocaine in both males and females, with females showing greater sensitivity to SCH 23390. The D2 antagonist eticlopride also reduced the locomotor activating effects of cocaine, with no clear differences between males and females. These results suggest that the differences between males and females in their locomotor response to cocaine can be at least partially attributed to differences in the function of dopamine D1 and D2 receptors.

Central dopamine deficiency in pure autonomic failure.

OBJECTIVE: Pure autonomic failure (PAF) and Parkinson's disease (PD) share several clinical laboratory abnormalities; however, PAF is not associated with parkinsonism. In this study, we tested the hypothesis that preservation of nigrostriatal dopaminergic innervation explains the absence of motor dysfunction in PAF. MMETHODS: Patients with PAF (N = 5) or PD (N = 21) and control subjects (N= 14) had brain 6-[18F]fluorodopa positron emission tomographic scanning and cerebrospinal fluid catechol measurements. A patient with PAF and another with PD had rapid postmortem striatal, nigral, and sympathetic ganglion sampling, with assays of catechols and tyrosine hydroxylase activity. RESULTS: The PAF and PD groups had similarly low mean substantia nigra (SN):occipital (OCC) ratios of 6-[18F]fluorodopa-derived radioactivity and similarly low cerebrospinal fluid dihydroxyphenylacetic acid and DOPA levels. Only the PD group, however, had low PUT:OCC, caudate:OCC, or PUT:SN ratios. The PAF and PD cases had similarly low SN tissue concentrations of dopamine and tyrosine hydroxylase activity, but the PD patient had tenfold lower PUT dopamine and the PAF patient 15-fold lower myocardial norepinephrine concentrations. CONCLUSIONS: Surprisingly, PAF and PD entail similarly severe nigral and overall central dopaminergic denervation. There is more severe loss of striatal dopaminergic terminals in PD than in PAF and more severe loss of sympathetic noradrenergic terminals in PAF than in PD. These differences explain the distinctive clinical manifestations of the two Lewy body diseases. Parkinsonism appears to reflect striatal dopamine deficiency rather than loss of nigral dopaminergic neurons per se.

Lead exposure and (n-3) fatty acid deficiency during rat neonatal development affect subsequent spatial task performance and olfactory discrimination.

Docosahexaenoic acid [22:6(n-3), DHA] is important for optimal infant central nervous system development, and lead (Pb) exposure during development can produce neurological deficits. Long-Evans strain rats were fed either an (n-3) deficient [(n-3) Def] diet to produce brain DHA deficiency, or an adequate [(n-3) Adq] diet through 2 generations. At the birth of the 2nd generation, the dams were subdivided into 4 groups and supplied drinking water containing either 5.27 mmol/L (Pb) or sodium (Na) acetate until weaning. Rats were killed at 3 wk (weaning) and 11 wk (maturity) for brain Pb and fatty acid analysis. Spatial task and olfactory-cued behavioral assessments were initiated at 9 wk. Rats in the (n-3) Def group had a 79% lower concentration of brain DHA compared with the (n-3) Adq group with no effect of Pb exposure. At weaning, Pb concentrations were 7.17 +/- 0.47 nmol Pb/g of brain (wet weight) in the (n-3) Adq-Pb group and 6.49 +/- 0.63 nmol Pb/g of brain (wet weight) in the (n-3) Def-Pb group. At maturity, the brains contained 1.30 +/- 0.22 and 1.07 +/- 0.12 nmol Pb/g (wet weight), respectively. In behavioral testing, significant effects of both Pb and DHA deficiency were observed in the Morris water maze probe trial and in 2-odor olfactory discrimination acquisition and olfactory-based reversal learning tasks. Both lactational Pb exposure and (n-3) fatty acid deficiency led to behavioral deficits with additive effects observed only in the acquisition of 2-odor discriminations.

Mixed cocaine agonist/antagonist properties of (+)-methyl 4beta-(4-chlorophenyl)-1-methylpiperidine-3alpha-carboxylate, a piperidine-based analog of cocaine.

The present study investigated the pharmacological properties of a piperidine-based novel cocaine analog, namely, (+)-methyl 4beta-(4-chlorophenyl)-1-methylpiperidine-3alpha-carboxylic acid [(+)-CPCA]. Like cocaine, (+)-CPCA inhibited rat synaptosomal dopamine and norepinephrine uptake with high affinity, but was 33-fold less potent than cocaine in inhibiting serotonin uptake. Like cocaine, (+)-CPCA is a locomotor stimulant, although it was less potent and efficacious than cocaine. Importantly, pretreatment with (+)-CPCA dose dependently blocked the locomotor stimulant effects of cocaine in rats. (+)-CPCA completely substituted for cocaine in drug discrimination tests, although it was about 3 times less potent than cocaine. It was also self-administered by rats. Unexpectedly, (+)-CPCA did not enhance cocaine-induced convulsions in mice. As expected from rodent studies, rhesus monkeys readily self-administered (+)-CPCA. However, compared with cocaine, (+)-CPCA showed limited reinforcing properties in rats as assessed by both fixed and progressive ratio intravenous drug self-administration tests. These results collectively suggest that (+)-CPCA has an atypical pharmacological profile having both cocaine-like "agonist" and some cocaine "antagonist" properties. These properties of (+)-CPCA suggest that it may have utility in the treatment of cocaine craving and dependence.