Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

In this investigation, the anterior and posterior regions of the midgut of resistant (RL) and non-resistant (SL) Anticarsia gemmatalis larvae were analyzed morphometrically to characterize different regions along their length. Also, this investigation compares the results between SL and RL to improve the understanding of the resistance mechanisms to the virus. Histological sections were analyzed in a computerized system and the data were statistically analyzed by the Kruskal-Wallis test and by multivariate analysis. The midguts are morphometrically different in the two larval populations; we observed higher values in RL. The morphometric analysis of the epithelial cells showed that only columnar and goblet cells were distinct along the midgut, in both larvae, with the higher values found in the anterior region. Comparing the results between the two larval populations, all the epithelial cells presented significant differences, with RL showing the higher morphometric values. We concluded that there are regional differences along the length of midgut in SL and RL that confirm the idea of two morpho-functional distinct regions. The consistently morphometric superior values in RL indicate that this variability can be related with the resistance of A. gemmatalis to its AgMNPV.     

Mutations in methylenetetrahydrofolate reductase and in cysthationine beta synthase: is there a link to homocysteine levels in peripheral arterial disease?

Peripheral arterial disease (PAD) is an atherosclerotic disturbance characterized by a progressive obstruction of lower limb arteries. Many risk factors associated with PAD development have being reported in the literature. The present study aimed to investigate whether mutations in the methylenetetrahydrofolate reductase (MTHFR) or in the cystathionine beta synthase (CBS) genes are associated with higher levels of homocysteine and the risk of PAD in patients from Brazil. This study analyzed 39 patients with PAD and 32 without PAD in whom risk factors and C677T mutations in the MTHFR gene and both 844ins68 and T833C mutations in the CBS gene were investigated. Although higher levels of homocysteine could be observed in patients with PAD compared to controls, no association between the increase of homocysteine and the frequency of C677T, 844ins68, and T833C mutations could be observed. The results suggest that these mutations do not appear to be related to either homocysteine levels or the development of the disease. However, hyperhomocysteinemia and smoking are important factors in PAD development.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

VOCAL REPERTOIRE OF WILD BREEDING ORANGE-WINGED PARROTS AMAZONA AMAZONICA IN AMAZONIA

ABSTRACT

The vocal repertoire of Amazona amazonica during its breeding season has been recorded from wild individuals in Santa Bárbara do Pará, Pará State, Brazil. At individual nests, we continuously recorded vocalizations and behaviour for four hours in the early morning and three hours in the late afternoon, three times a week throughout the breeding season. We identified nine vocalizations that we classified in three behavioural categories: (1) Flight call—emitted when parrots arrive in the nest area; (2) Perched contact calls—two different vocalizations, one of them related to feeding, were emitted when the pair was perched in the nest area and interacted socially between themselves or with other individuals; (3) Aggressive calls—emitted when birds were in a dangerous situation, i.e. alarm (three types of calls), agonistic contact and distress calls (two types of call). The Orange-winged Parrot is a highly social species and the complexity of its social interactions is reflected in the diversity of its vocal repertoire.     

Reassembly of a large multisubunit protein promoted by nonprotein factors. Effects of calcium and glycerol on the association of extracellular hemoglobin

The effects of cations and glycerol on the dissociation induced by pressure and on the reassembly of Glossoscolex paulistus hemoglobin were examined by light scattering, gel filtration, and electron microscopy. Calcium stabilized the quaternary structure of the hemoglobin against pressure dissociation. In the presence of 50 mM Ca2+, the half-dissociation pressure (p 1/2) increased by 400 bar, which corresponds to an average stabilization of -0.62 kcal/mol of dissociating subunit. Calcium also promoted a large increase in the yield of recovery of fully assembled hemoglobin at the expense of the partially dissociated (one-twelfth subunit) and fully dissociated forms. Glycerol protected the hemoglobin from pressure dissociation, increasing the half-dissociation pressure (p 1/2) and promoted an increase in the yield of recovery of fully assembled hemoglobin by about 40%. Addition of calcium after return to atmospheric pressure increased recovery of the fully associated form only in a long time scale (many days). The existence of time-dependent changes in the conformation of the dissociated subunits is suggested to explain the partial association to one-twelfth subaggregates (drifted forms) that lack the ability to reassemble to native hemoglobin. The promotion of reassembly by nonprotein factors (calcium and glycerol) is suggested to occur by preventing the formation of wrong intermediate forms (drifted one-twelfth subunits).     

Synthesis and stability in biological media of 1H-imidazole-1-carboxylates of ROS203, an antagonist of the histamine H3 receptor

A series of carbamate derivatives of the H(3) antagonist ROS203 (1) were prepared, and their lipophilicity and steric hindrance were modulated by introducing linear or branched alkyl chains of various lengths. In vitro stability studies were conducted to evaluate how structural modulations affect the intrinsic reactivity of the carbamoyl moiety and its recognition by metabolic enzymes. Linear alkyl carbamates were the most susceptible to enzymatic hydrolysis, with bioconversion rates being higher in rat liver and plasma. Chain ramification significantly enhanced the enzymatic stability of the set, with two derivatives (1g and 1h) being more stable by a factor of 8-40 than the ethyl carbamate 1a. Incubation with bovine serum albumin (BSA) showed a protective role of proteins on chemical and porcine-liver esterase (PLE)-catalyzed hydrolysis. Ex vivo binding data after i.v. administration of 1h revealed prolonged displacement of the labeled ligand [(3)H]-(R)-alpha-methylhistamine ([(3)H]RAMHA) from rat-brain cortical membranes, when compared to 1. However, the high rates of bioconversion in liver, as well as the chemical instability of 1h, suggest that further work is needed to optimize the enzymatic and chemical stability of these compounds.     

Association between IGF2 and CYP21 gene polymorphisms and characteristics of economic interest in Nellore cattle

We analyzed two single nucleotide polymorphisms (SNPs) of the IGF2 and CYP21 genes in Nellore cattle participating in the Brazilian Animal Breeding Program. The SNPs were found in exon 6 of the IGF2 (insulin-like growth factor 2) gene (RFLP/MboII) as well as in the promoter region of the CYP21 (steroid 21-hydroxylase) gene (RFLP/HpaII) of these animals. The TC heterozygotes were significantly more frequent than CC and TT homozygotes in the RFLP/MboII polymorphism. The T allele was significantly more frequent than the C allele in RFLP/HpaII polymorphism. This population was found to be in Hardy-Weinberg equilibrium for these SNPs. Association of these polymorphisms with expected progeny differences of reproductive and productive traits was investigated, but proved to be significant only for DP550 (expected progeny differenced for weight at 365 days - IGF2 - RFLP/MboII) and DP450 (expected progeny differenced for weight at 450 days - CYP21 - RFLP/HpaII). This is the first study on the occurrence of these two polymorphisms in this Zebu breed of cattle. A total of 147 Nellore animals participating in the Breeding Program of the Nellore Breed (PMGRN) under the management of the National Association of Breeders and Researchers (ANCP) in the city of Ribeir?o Preto were analyzed.     

Piezoelectric biosensors for biorecognition analysis: application to the kinetic study of HIV-1 Vif protein binding to recombinant antibodies

In this work three piezoelectric sensors modified with anti-HIV-1 Vif (virion infectivity factor) single fragment antibodies (4BL scFV), single domains (VH) and camelized single domains (VHD) were constructed and used to detect HIV1 Vif in liquid samples. Dithio-bis-succinimidyl-undecanoate (DSU) and 11-hydroxy-1-undecanethiol (HUT) mixed self assembled monolayers (SAM) were generated at the sensors surface onto which the antibodies were immobilized. All sensors detected specifically the target HIV1-Vif antigen in solution and no unspecific binding was monitored. Impedance analysis was performed to quantify electroacoustic and viscoelastic interferences during antibody immobilization and antigen recognition. The elimination of such interferences enabled the quantitative use of the piezoelectric immunosensors to estimate the antibody surface density as well as antigen binding and equilibrium constants. In spite of the possible limitation regarding mass transport and other related molecular phenomena, which were not considered in the binding model used, this work demonstrates the usefulness of piezoelectric biosensors in biorecognition analysis and evidences the advantages on using simultaneous impedance analysis to bring analytical significance to measured data, and thus to improve piezoelectric sensors sensitivity and applicability.     

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA‐deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.