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.

Association of a homozygous nonsense caveolin-1 mutation with Berardinelli-Seip congenital lipodystrophy

Context: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue, resulting in severe dyslipidemia and insulin resistance. In most reported cases, BSCL is due to alterations in either seipin, of unknown function, or 1-acylglycerol-3- phosphate acyltransferase-beta (AGPAT2), which catalyzes the formation of phosphatidic acid. Objective: We sought to determine the genetic origin of the unexplained cases of BSCL. We thus sequenced CAV1, encoding caveolin-1, as a candidate gene involved in insulin signaling and lipid homeostasis. CAV1 is a key structural component of plasma membrane caveolae, and Cav1-deficient mice display progressive loss of adipose tissue and insulin resistance. Design: We undertook phenotyping studies and molecular screening of CAV1 in four patients with BSCL with no mutation in the genes encoding either seipin or AGPAT2. Results: A homozygous nonsense mutation (p.Glu38X) was identified in CAV1 in a patient with BSCL born from a consanguineous union. This mutation affects both the alpha-and beta-CAV1 isoforms and ablates CAV1 expression in skin fibroblasts. Detailed magnetic resonance imaging of the proband confirmed near total absence of both sc and visceral adipose tissue, with only vestigial amounts in the dorsal sc regions. In keeping with the lack of adipose tissue, the proband was also severely insulin resistant and dyslipidemic. In addition, the proband had mild hypocalcemia likely due to vitamin D resistance. Conclusions: These findings identify CAV1 as a new BSCL-related gene and support a critical role for caveolins in human adipocyte function.

High numbers of human skin cancers express MMP2 and several integrin genes

Background: Basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and cutaneous malignant melanoma (MM) are solid skin cancers derived from different cell types, with different ability to metastasize. Several subtypes of integrins and matrix metalloproteinases (MMP) have been related to malignization and metastasis processes. This work aimed at a quantitative evaluation of skin cancers expressing eight integrins and MMP2 genes. Methods: Expression of integrins and MMP2 genes was evaluated on fresh tumor biopsies from BCC, SCC and MM, and respective controls, by the reverse transcriptase polychain reaction (RT-PCR) technique. Results: More than 90% tumors expressed alpha 6a, beta 1, beta 3 and beta 6 (non-melanoma), and alpha 5a, alpha 6a and MMP2 (MM). Up to 100% controls also expressed beta 1 and beta 3. The results were significant for alpha 6a in BCC (p = 0.026), alpha 6b in SCC (p = 0.035), alpha 2a in BCC (p = 0.003), beta 5 and beta 6 in BCC (p = 0.005). MMP2 was expressed in 100% MM (p = 0.0004). Conclusion: Integrin subunits alpha 2a and alpha 6a would be interesting targets for BCC anti-tumor therapy, as well as alpha 6b in case of SCC. The elevated number of BCC expressing alpha 2 and alpha 6, and of MM expressing alpha v and MMP2, corroborate literature data.

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.

NF-kappa b expression predicts clinical outcome for nasal polyposis

Objective: To correlate clinical prognosis of patients with nasal polyps to the expression of p65, c-Fos, GR alpha and GR beta. Methods: A biopsy was obtained at the first evaluation of patients with nasal polyps, and at rhinoplasty for control mucosa. Patients with nasal polyps were treated with glucocorticoids and followed for at least 60 months. The expression of p65, c-Fos, GR alpha and GR beta was determined by Real Time-PCR and correlated to clinical outcome. The end-point of resistance to glucocorticoid therapy was considered when surgery was indicated. Results: Patients with nasal polyps presented a higher expression of p65, a lower expression of GR alpha, and a lower GR alpha/GR beta ratio than control mucosa. The patients with nasal polyps who had a higher expression of p65 correlated with a poorer response to glucocorticoids, with a 3.5-fold higher risk for surgery. Conclusion: Patients with a higher p65 (NF-kappa B) expression at diagnosis were associated to a worse response to clinical treatment, suggesting one of the mechanisms of cell resistance to glucocorticoid treatment in patients with nasal polyps.

Evaluating budesonide efficacy in nasal polyposis and predicting the resistance to treatment

Cell resistance to glucocorticoids is a major problem in the treatment of nasal polyposis (NP). The objectives of this study were to observe the effect of budesonide on the expression of IL-1 beta, TNF-alpha, granulocyte macrophage-colony stimulating factor, intercellular adhesion molecule (ICAM)-1, basic fibroblast growth factor, eotaxin-2, glucocorticoid receptor (GR)-alpha, GR-beta, c-Fos and p65 in nasal polyps and to correlate their expression to clinical response. Biopsies from nasal polyps were obtained from 20 patients before and after treatment with topical budesonide. Clinical response to treatment was monitored by a questionnaire and nasal endoscopy. The mRNA levels of the studied genes were measured by real-time quantitative (RQ)-PCR. There was a significant decrease in the expression of TNF-alpha (P < 0.05), eotaxin-2 (P < 0.05) and p65 (P < 0.05) in NP after treatment. Poor responders to glucocorticoids showed higher expression of IL-1 beta (3.74 vs. 0.14; P < 0.005), ICAM-1 (1.91 vs. 0.29; P < 0.05) and p65 (0.70 vs. 0.16; P < 0.05) before treatment. Following treatment, IL-1 beta (4.18 vs. 0.42; P < 0.005) and GR-beta (0.95 vs. 0.28; P < 0.05) mRNA expression was higher in this group. Topical budesonide reduced the expression of TNF-alpha, eotaxin-2 and p65. Poor responders to topical budesonide exhibit higher levels of IL-1 beta, ICAM-1 and nuclear factor (NF)-kappa B at diagnosis and higher expression of both IL-1 beta and GR-beta after treatment. These results emphasize the anti-inflammatory action of topical budesonide at the molecular level and its importance in the treatment of NP. Nevertheless, IL-1 beta, ICAM-1 and NF-kappa B may be associated with primary resistance to glucocorticoids in NP, whereas higher expression of GR-beta in poor responders only after glucocorticoid treatment may represent a secondary drug resistance mechanism in this disease.

Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Crystallization of the FAD-independent acetolactate synthase of Klebsiella pneumoniae

Leucine and valine are formed in a common pathway from pyruvate in which the first intermediate is 2-acetolactate. In some bacteria, this compound also has a catabolic fate as the starting point for the butanediol fermentation. The enzyme (EC 4.1.3.18) that forms 2-acetolactate is known as either acetohydroxyacid synthase (AHAS) or acetolactate synthase (ALS), with the latter name preferred for the catabolic enzyme. A significant difference between AHAS and ALS is that the former requires FAD for catalytic activity, although the reason for this requirement is not well understood. Both enzymes require the cofactor thiamine diphosphate. Here, the crystallization and preliminary X-ray diffraction analysis of the Klebsiella pneumoniae ALS is reported. Data to 2.6 Angstrom resolution have been collected at 100 K using a rotating-anode generator and an R-AXIS IV++ detector. Crystals have unit-cell parameters a = 137.4, b = 143.9, c = 134.4 Angstrom, alpha = 90, beta = 108.4, gamma = 90degrees and belong to space group C2. Preliminary analysis indicates that there are four monomers located in each asymmetric unit.

Linear ketenimines. Variable structures of C,C-dicyanoketenimines and C,C-bis-sulfonylketenimines

C,C-Dicyanoketenimines 10a-c were generated by flash vacuum thermolysis of ketene NS-acetals 9a-c or by thermal or photochemical decomposition of alpha-azido-,beta-cyanocinnamonitrile 11. In the latter reaction, 3,3-dicyano-2-phenyl-1-azirine 12 is also formed. IR spectroscopy of the keteniminines isolated in Ar matrixes or as neat films, NMR spectroscopy of 10c, and theoretical calculations (B3LYP/6-31G*) demonstrate that these ketenimines have variable geometry, being essentially linear along the CCN-R framework in polar media (neat films and solution), but in the gas phase or Ar matrix they are bent, as is usual for ketenimines. Experiments and calculations agree that a single CN substituent as in 13 is not enough to enforce linearity, and sulfonyl groups are less effective that cyano groups in causing linearity. C,C-Bis(methylsulfonyl)ketenimines 4-5 and a C-cyano-C-(methylsulfonyl)ketenimine 15 are not linear. The compound p-O2NC6H4N=C= C(COOMe)2 previously reported in the literature is probably somewhat linearized along the CCNR moiety. A computational survey (B3LYP/6-31G*) of the inversion barrier at nitrogen indicates that electronegative C-substituents dramatically lower the barrier; this is also true of N-acyl substituents. Increasing polarity causes lower barriers. Although N-alkylbis(methylsulfonyl)ketenimines are not calculated to be linear, the barriers are so low that crystal lattice forces can induce planarity in N-methylbis(methylsulfonyl)ketenimine 3.

Ozothamnus diosmifolius (Vent.) DC flower and leaf essential oils

Essential oils of rice flower, Ozothamnus diosmifolius, were analyzed by capillary gas chromatograplay-mass spectrometry. Flower oil contained beta-pinene (28.4%) and 1,8-cineole (28.2%), while the leaf oil contained a-pinene (26.0%), beta-pinene (11.6%) and 1,8-cineole (22.2%). Both oils had small amounts of spathulenol (4.1% and 5.2%, respectively).

INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index). Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

Saprotrophic fungi Hypholoma fasciculare effect on the fungal community associated to Castanea sativa

Dissertação de mestrado em Biologia Molecular, Biotecnologia e Bioempreendedorismo em Plantas

Distribuição da xantina oxidase no fígado e no sôro de rato

The localization of the xanthine oxidase (X.O.) and xanthine dehydrogenase (X.D.) activities in rat liver have been studied using separation of cytoplasmic particles into fractions by differential centrifugation. The results clearly demonstrate that practically all the enzymic activity is present in the supernatant fluid corresponding to the cell sap containing the soluble proteins of the cell. No activity could be detected for the nuclear, mitocondrial and microsomal fractions. The enzymatic activity of the mixture of the four factions was 102 per cent of that of the original homogenate. The distribution of the xanthine dehydrogenase in the protein fractions of the rat serum was accomplished in preliminary experiments by means of 50% ammonium sulphate precipitation and subsequent dialysis against water. All enzymatic activity was confined to the globulin fractions of the serum. Paper electrophoresis was performed and the protein and lipoprotein fractions determined. A method for the localization of the X.D. activity in the protein fractions separated by paper electrophoresis was developed. The results obtained suggest that xanthine dehydrogenase is localized in the globulin fractions possessing mobilities of [alpha 1], [beta] and [gamma] globulins and are probably bound to the lipoproteins.