Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

Bed nucleus of the stria terminalis alpha(1)-adrenoceptor modulates baroreflex cardiac component in unanesthetized rats

The bed nucleus of stria terminalis (BST) has a tonic modulating role on the baroreflex parasympathetic component. In the present study, we verified that local BST-adrenoceptors modulate baroreflex-evoked bradycardiac responses in unanesthetized rats. Bilateral microinjection of the selective alpha(1)-adrenoceptor antagonist WB4101 (15 nmol/100 nL) into the BST increased the gain of reflex bradycardia in response to mean arterial pressure increases caused by intravenous (i.v.) infusion of phenylephrine, suggesting that BST alpha(1)-adrenoceptors modulate baroreflex bradycardiac response. Bilateral microinjection of either the selective alpha(2)-adrenoceptor antagonist RX821002 (15 nmol/100 nL) or the non-selective beta-adrenoceptor antagonist propranolol (15 nmol/100 nL) into the BST had not affected baroreflex bradycardia. Animals were pretreated intravenously with the cholinergic muscarinic receptor antagonist homatropine methyl bromide (HMB, 1.5 mg/Kg) to test the hypothesis that activation of alpha(1)-adrenoceptors in the BST would modulate the baroreflex parasympathetic component. Baroreflex bradycardiac responses evoked before and after BST treatment with WB4101 were no longer different when rats were pretreated with HMB. These results suggest that parasympathetic activation accounts for the effects saw after BST pharmacological manipulation and ruling out the possibility of a sympathetic withdraw. In conclusion, our data point out that local alpha(1)-adrenoceptors mediate the BST tonic influence on the baroreflex bradycardiac response modulating parasympathetic cardiac activity. (C) 2008 Elsevier B.V. All rights reserved.

alpha(1)-Adrenoceptors in the lateral septal area modulate food intake behaviour in rats

Background and purpose: Control of food intake is a complex behaviour which involves many interconnected brain structures. The present work assessed if the noradrenergic system in the lateral septum (LS) was involved in the feeding behaviour of rats. Experimental approach: In the first protocol, the food intake of rats was measured. Then non-food-deprived animals received either 100 nL of 21 nmol of noradrenaline or vehicle unilaterally in the LS 10 min after local 10 nmol of WB4101, an alpha(1)-adrenoceptor antagonist, or vehicle. In the second protocol, different doses of WB4101 (1, 10 or 20 nmol in 100 nL) were microinjected bilaterally into the LS of rats, deprived of food for 18 h and food intake was compared to that of satiated animals. Key results: One-sided microinjection of noradrenaline into the LS of normal-fed rats evoked food intake, compared with vehicle-injected control animals, which was significantly reduced by alpha(1)-adrenoceptor antagonism. In a further investigation, food intake was significantly higher in food-deprived animals, compared to satiated controls. Pretreatment of the LS with WB4101 reduced food intake in only food-deprived animals in a dose-related manner, suggesting that the LS noradrenergic system was involved in the control of food intake. Conclusion and implications: Activation by local microinjection of noradrenaline of alpha(1)-adrenoceptors in the LS evoked food intake behaviour in rats. In addition, blockade of the LS alpha(1)-adrenoceptors inhibited food intake in food-deprived animals, suggesting that the LS noradrenergic system modulated food intake behaviour and satiation.

Both alpha 1 and alpha 2-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the bed nucleus of the stria terminal of rats

Background and purpose: We have previously shown that noradrenaline microinjected into the bed nucleus of stria terminalis (BST) elicited pressor and bradycardiac responses in unanaesthetized rats. In the present study, we investigated the subtype of adrenoceptors that mediates the cardiovascular response to noradrenaline microinjection into the BST. Experimental approach: Cardiovascular responses following noradrenaline microinjection into the BST of male Wistar rats were studied before and after BST pretreatment with different doses of the selective alpha(1)-adrenoceptor antagonist WB4101, the alpha(2)-adrenoceptor antagonist RX821002, the combination of WB4101 and RX821002, the non-selective beta-adrenoceptor antagonist propranolol, the selective beta(1)-adrenoceptor antagonist CGP20712 or the selective beta(2)-adrenoceptor antagonist ICI118,551. Key results: Noradrenaline microinjected into the BST of unanaesthetized rats caused pressor and bradycardiac responses. Pretreatment of the BST with different doses of either WB4101 or RX821002 only partially reduced the response to noradrenaline. However, the response to noradrenaline was blocked when WB4101 and RX821002 were combined. Pretreatment with this combination also shifted the resulting dose-effect curve to the left, clearly showing a potentiating effect of this antagonist combination. Pretreatment with different doses of either propranolol or CGP20712 increased the cardiovascular responses to noradrenaline microinjected into the BST. Pretreatment with ICI118,551 did not affect cardiovascular responses to noradrenaline. Conclusion and implications: The present results indicate that alpha(1) and alpha(2)-adrenoceptors mediate the cardiovascular responses to noradrenaline microinjected into the BST. In addition, they point to an inhibitory role played by the activation of local beta(1)-adrenoceptors in the cardiovascular response to noradrenaline microinjected into the BST.

BED NUCLEUS OF THE STRIA TERMINALIS alpha(1)- AND alpha(2)-ADRENOCEPTORS DIFFERENTIALLY MODULATE THE CARDIOVASCULAR RESPONSES TO EXERCISE IN RATS

Dynamic exercise evokes sustained blood pressure and heart rate (HR) increases. Although it is well accepted that there is a CNS mediation of cardiovascular adjustments during dynamic exercise, information on the role of specific CNS structures is still limited. The bed nucleus of the stria terminalis (BST) is involved in exercise-evoked cardiovascular responses in rats. However, the specific neurotransmitter involved in BST-related modulation of cardiovascular responses to dynamic exercise is still unclear. In the present study, we investigated the role of local BST adrenoceptors in the cardiovascular responses evoked when rats are submitted to an acute bout of exercise on a rodent treadmill. We observed that bilateral microinjection of the selective alpha 1-adrenoceptor antagonist WB4101 into the BST enhanced the HR increase evoked by dynamic exercise without affecting the mean arterial pressure (MAP) increase. Bilateral microinjection of the selective alpha 2-adrenoceptor antagonist RX821002 reduced exercise-evoked pressor response without changing the tachycardiac response. BST pretreatment with the nonselective beta-adrenoceptor antagonist propranolol did not affect exercise-related cardiovascular responses. BST treatment with either WB4101 or RX821002 did not affect motor performance in the open-field test, which indicates that effects of BST adrenoceptor antagonism in exercise-evoked cardiovascular responses were not due to changes in motor activity. The present findings are the first evidence showing the involvement of CNS adrenoceptors in cardiovascular responses during dynamic exercise. Our results indicate an inhibitory influence of BST alpha 1-adrenoceptor on the exercise-evoked HR response. Data also point to a facilitatory role played by the activation of BST alpha 2-adrenoceptor on the pressor response to dynamic exercise. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Insular cortex alpha(1)-adrenoceptors modulate the parasympathetic component of the baroreflex in unanesthetized rats

The insular cortex (IC) has been reported to modulate the cardiac parasympathetic activity of the baroreflex in unanesthetized rats. However, which neurotransmitters are involved in this modulation is still unclear. In the present study, we evaluated the possible involvement of local IC-noradrenergic neurotransmission in modulating reflex bradycardiac responses. Bilateral microinjection of the selective alpha(1)-adrenoceptor antagonist WB4101 (15 nmol/100 nL), into the IC of male Wistar rats, increased the gain of reflex bradycardia in response to mean arterial pressure (MAP) increases evoked by intravenous infusion of phenylephrine. However, bilateral microinjection of equimolar doses of either the selective alpha(2)-adrenoceptor antagonist RX821002 or the non-selective beta-adrenoceptor antagonist propranolol into the IC did not affect the baroreflex response. No effects were observed in basal MAP or heart rate values after bilateral microinjection of noradrenergic antagonists into the IC, thus suggesting no tonic influence of IC-noradrenergic neurotransmission on resting cardiovascular parameters. In conclusion, these data provide evidence that local IC-noradrenergic neurotransmission has an inhibitory influence on baroreflex responses to blood pressure increase evoked by phenylephrine infusion through activation of alpha(1)-adrenoceptors. (C) 2009 Elsevier B.V. All rights reserved.

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.

Suppression of keratinocyte growth and differentiation by transforming growth factor beta 1 involves multiple signaling pathways

Transforming growth factor beta1 treatment of keratinocytes results in a suppression of differentiation, an induction of extracellular matrix production, and a suppression of growth. In this study we utilized markers specific for each of these functions to explore the signaling pathways involved in mediating these transforming-growth-factor-beta1-induced activities. In the first instance, we found that the induction of extracellular matrix production (characterized by 3TP-Lux reporter activity) was induced in both keratinocytes and a keratinocyte-derived carcinoma cell line, SCC25, in a dose-dependent manner. Furthermore, transforming growth factor beta1 also suppressed the differentiation-specific marker gene, transglutaminase type 1, in both keratinocytes and SCC25 cells. In contrast, transforming growth factor beta1 inhibited proliferation of keratinocytes but did not cause growth inhibition in the SCC25 cells. Transforming-growth-factor-beta1-induced growth inhibition of keratinocytes was characterized by decreases in DNA synthesis, accumulation of hypophosphorylated Rb, and the inhibition of the E2F:Rb-responsive promoter, cdc2, and an induction of the p21 promoter. When the negative regulator of transforming growth factor beta1 signaling, SMAD7, was overexpressed in keratinocytes it could prevent transforming-growth-factor-beta1-induced activation of the 3TP-Lux and the p21 promoter. SMAD7 could also prevent the suppression of the transglutaminase type 1 by transforming growth factor beta1 but it could not inhibit the repression of the cdc2 promoter. These data indicate that the induction of 3TP-Lux and p21 and the suppression of transglutaminase type 1 are mediated by a different proximate signaling pathway to that regulating the suppression of the cdc2 gene. Combined, these data indicate that the regulation of transforming growth factor beta1 actions are complex and involve multiple signaling pathways.

Early Australian experience with infliximab, a chimeric antibody against tumour necrosis factor-alpha, in the treatment of Crohn's disease: is its efficacy augmented by steroid-sparing immunosuppressive therapy?

Background: Tumour necrosis factor-alpha (TNF-alpha) plays an important role in the pathology of Crohn's disease. Infliximab, a chimeric antibody against TNF-alpha, has been shown in controlled clinical trials to be effective in two-thirds of patients with refractory or fistulating Crohn's disease. The factors that determine a clinical response in some patients but not others are unknown. Aims: To document the early Australian experience with infliximab treatment for Crohn's disease and to identify factors that may determine a beneficial clinical response. Methods: Gastroenterologists known to have used infliximab for Crohn's disease according to a compassionate use protocol were asked to complete a spreadsheet that included demographic information, Crohn's disease site, severity, other medical or surgical treatments and a global clinical assessment of Crohn's disease outcome, judged by participating physicians as complete and sustained (remission for the duration of the study), complete but unsustained (remission at 4 weeks but not for the whole study) or partial clinical improvement (sustained or unsustained). Results: Fifty-seven patients were able to be evaluated, with a median follow-up time of 16.4 (4-70) weeks, including 23 patients with fistulae. There were 21 adverse events, including four serious events. Fifty-one patients (89%) had a positive clinical response for a median duration (range) of 11 (2-70) weeks. Thirty patients (52%) had a remission at 4 weeks, 10 of whom had remission for longer than 12 weeks. Forty-two per cent of fistulae closed. Sustained remission (P = 0.065), remission at 4 weeks (P = 0.033) and a positive clinical response of any sort (P = 0.004) were more likely in patients on immunosuppressive therapy, despite there being more smelters in this group. Conclusion: This review of the first Australian experience with infliximab corroborates the reported speed and efficacy of this treatment for Crohn's disease. The excellent response appears enhanced by the concomitant use of conventional steroid-sparing immunosuppressive therapy.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) TNF-alpha is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-alpha in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-alpha in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro-TNF-alpha. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-alpha in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-alpha accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-alpha. Tracking a bolus of TNF-alpha over time in cycloheximide-treated cells confirmed that uncleaved TNF-alpha is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-alpha were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-alpha provides a mechanism for modulating the quantity of biologically active 26 kd TNF-alpha expressed on macrophages, allowing regulation of paracrine and autocrine responses.

The surface accessibility of the glycine receptor M2-M3 loop is increased in the channel open state

Mutations in the extracellular M2-M3 loop of the glycine receptor (GlyR) alpha1 subunit have been shown previously to affect channel gating. In this study, the substituted cysteine accessibility method was used to investigate whether a structural rearrangement of the M2-M3 loop accompanies GlyR activation. All residues from R271C to V277C were covalently modified by both positively charged methanethiosulfonate ethyltrimethylammonium (MTSET) and negatively charged methanethiosulfonate ethylsulfonate (MTSES), implying that these residues form an irregular surface loop. The MTSET modification rate of all residues from R271C to K276C was faster in the glycine-bound state than in the unliganded state. MTSES modification of A272C, L274C, and V277C was also faster in the glycine-bound state. These results demonstrate that the surface accessibility of the M2-M3 loop is increased as the channel transitions from the closed to the open state, implying that either the loop itself or an overlying domain moves during channel activation.

Two new classes of conopeptides inhibit the alpha 1-adrenoceptor and noradrenaline transporter

Cone snails use venom containing a cocktail of peptides ('conopeptides') to capture their prey. Many of these peptides also target mammalian receptors, often with exquisite selectivity. Here we report the discovery of two new classes of conopeptides. One class targets alpha (1)-adrenoceptors (rho -TIA from the fish-hunting Conus tulipa), and the second class targets the neuronal noradrenaline transporter (chi -MrIA and chi -MrIB from the mollusk-hunting C. marmoreus). rho -TIA and chi -MrIA selectively modulate these important membrane-bound proteins. Both peptides act as reversible non-competitive inhibitors and provide alternative avenues for the identification of inhibitor drugs.

Insights into the structural basis for zinc inhibition of the glycine receptor

Histidines 107 and 109 in the glycine receptor ( GlyR) alpha(1) subunit have previously been identified as determinants of the inhibitory zinc-binding site. Based on modeling of the GlyR alpha(1) subunit extracellular domain by homology to the acetylcholine-binding protein crystal structure, we hypothesized that inhibitory zinc is bound within the vestibule lumen at subunit interfaces, where it is ligated by His(107) from one subunit and His(109) from an adjacent subunit. This was tested by co-expressing alpha(1) subunits containing the H107A mutation with alpha(1) subunits containing the H109A mutation. Although sensitivity to zinc inhibition is markedly reduced when either mutation is individually incorporated into all five subunits, the GlyRs formed by the co-expression of H107A mutant subunits with H109A mutant subunits exhibited an inhibitory zinc sensitivity similar to that of the wild type alpha(1) homomeric GlyR. This constitutes strong evidence that inhibitory zinc is coordinated at the interface between adjacent alpha(1) subunits. No evidence was found for beta subunit involvement in the coordination of inhibitory zinc, indicating that a maximum of two zinc-binding sites per alpha(1)beta receptor is sufficient for maximal zinc inhibition. Our data also show that two zinc-binding sites are sufficient for significant inhibition of alpha(1) homomers. The binding of zinc at the interface between adjacent alpha(1) subunits could restrict intersubunit movements, providing a feasible mechanism for the inhibition of channel activation by zinc.

Anti-Tumor Necrosis Factor Alpha-Induced Drug Eruptions: One Patient, More Than a Pattern

Background: Tumor necrosis factor alpha (TNFα) antagonists are effective in treating several immune-inflammatory diseases, including psoriasis and inflammatory bowel disease. The paradoxical and unpredictable induction of psoriasis and psoriasiform skin lesions is a recognized adverse event, although of unclear aetiology. However, histological analysis of these eruptions remains insufficient, yet suggesting that some might constitute a new pattern of adverse drug reaction, rather than true psoriasis. Case report: The authors report the case of a 43-year-old woman with severe recalcitrant Crohn disease who started treatment with infliximab. There was also a personal history of mild plaque psoriasis without clinical expression for the past eight years. She developed a heterogeneous cutaneous eruption of psoriasiform morphology with pustules and crusts after the third infliximab infusion. The histopathological diagnosis was of a Sweet-like dermatosis. The patient was successfully treated with cyclosporine in association with both topical corticosteroid and vitamin D3 analogue. Three weeks after switching to adalimumab a new psoriasiform eruption was observed, histologically compatible with a psoriasiform drug eruption. Despite this, and considering the beneficial effect on the inflammatory bowel disease, it was decided to maintain treatment with adalimumab and to treat through with topicals, with progressive control of skin disease. Discussion: Not much is known about the pathogenesis of psoriasiform eruptions induced by biological therapies, but genetic predisposition and Koebner phenomenon may contribute to it. Histopathology can add new facets to the comprehension of psoriasiform reactions. In fact, histopathologic patterns of such skin lesions appear to be varied, in a clear asymmetry with clinical findings. Conclusion: The sequential identification in the same patient of two clinical and histopathologic patterns of drug reaction to TNFα antagonists is rare. Additionally, to the authors’ knowledge, there is only one other description in literature of a TNFα antagonist-induced Sweet-like dermatosis, emphasizing the singularity of this case report.