In Silico Design of an Oseltamivir Derivative with Increased Affinity against Wild-Type and Mutant Variants of Neuraminidase and Hemagglutinin of Influenza A H1N1 Virus

Antiviral resistance has turned into a world concern nowadays. Influenza A H1N1 emerged as a problem at the world level due to the neuraminidase (NA) mutations. The NA mutants conferred resistance to oseltamivir and zanamivir. Several efforts were conducted to develop better anti-influenza A H1N1 drugs. Our research group combined in silico methods to create a compound derived from oseltamivir to be tested in vitro against influenza A H1N1. Here we show the results of a new compound derived from oseltamivir but with specific chemical modifications, with significant affinity either on NA (in silico and in vitro assays) or HA (in silico) from influenza A H1N1 strain. We include docking and molecular dynamics (MD) simulations of the oseltamivir derivative at the binding site onto NA and HA of influenza A H1N1. Additionally, the biological experimental results show that oseltamivir derivative decreases the lytic-plaque formation on viral susceptibility assays, and it does not show cytotoxicity. Finally, oseltamivir derivative assayed on viral NA showed a concentration-dependent inhibition behavior at nM, depicting a high affinity of the compound for the enzyme, corroborated with the MD simulations results, placing our designed oseltamivir derivative as a potential antiviral against influenza A H1N1.     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Comparative neurochemical features of the innervation patterns of the gut of the basal actinopterygian,Lepisosteus oculatus,and the euteleost,Clarias batrachus

The structure and physiology of enteric system are very similar in all classes of vertebrates, although they have been investigated only occasionally in non‐mammalian vertebrates. Very little is known about the distribution of the neurotransmitters in the gut of actinopterygian fishes. Anatomical and physiological studies of enteric nervous systems in the spotted gar (Lepisosteus oculatus) and airbreathing catfish (Clarias batrachus), a non‐teleost and teleost actinopterygian, respectively, have not been undertaken. This study provides the first comprehensive characterization of the range of neurochemical coding in the enteric nervous system of these two species, including the chemical diversity of the mucosal endocrine cells in the pyloric stomach of Clarias. Autonomic innervation of the secretory glands is also described and reported herein for the first time for fishes. We also report splanchnic (spinal) innervation of the stomach, submucosal ganglia (that also colocalize with nNOS) and caudal intestine of Clarias. In both fish species, numerous 5HT, ChAT, nNOS and TH‐positive nerve fibres have been observed. These discoveries demonstrate that much more physiological and pharmacological data are needed before a comprehensive model of enteric nervous system control in vertebrates can be developed.     

Kinetics of reaction of (nitrotyrosyl)cytochrome c with ligands.

The reaction of [nitrotyrosyl]cytochrome c with ligands was studied by stopped-flow techniques. At pH 7.0 the reaction with imidazole shows two distinct phases, one fast phase being concentration-dependent and a slow phase being concentration-independent. The results are consistent with the existence of two forms of [nitrotyrosyl]cytochrome c in solutions [Schejter et al. (1970) Biochemistry 9, 5118-5122]; form I, the smaller fraction, seems to be responsible for the slow first-order process.     

Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai

The secreted form of mouse meprin A is a homooligomer of meprin alpha subunits that contain a prosequence, a catalytic domain, and three domains designated as MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (AfterMath). Previous studies indicated that wild-type mouse meprin alpha is predominantly a secreted protein, while the MAM deletion mutant (DeltaMAM) is degraded intracellularly. The work herein indicates that the DeltaMAM mutant is ubiquitinated and degraded via the proteasomal pathway. Both wild-type meprin alpha and the DeltaMAM mutant interact with the molecular chaperones calnexin and calreticulin in the endoplasmic reticulum. The interactions of the chaperones with the DeltaMAM mutant were significantly prolonged in the presence of lactacystin, a specific inhibitor of the proteasome, whereas those with the wild type were not affected by this inhibitor. Trimming of the Asn-linked core oligosaccharides of meprin subunits was required for interactions with the chaperones. The data indicated that folding of the wild-type protein was accelerated by chaperones, whereas the rate of dimerization was unaffected. Thus, calnexin and calreticulin are intimately involved in the correct folding and transport of meprin to the plasma membrane, as well as in retrograde transport of the DeltaMAM mutant to the ubiquitin-dependent proteasomal degradative pathway in the cytosol.     

Co-aggregation of penicillin g acylase and polyionic polymers: an easy methodology to prepare enzyme biocatalysts stable in organic media

A novel type of biocatalyst that combines the good properties of cross-linked enzyme aggregates (CLEAs) and hydrophilic microenvironments has been developed. Dextran sulfate- and polyethyleneimine-coated CLEAs of penicillin acylase (CLEA-GDP) were prepared by adding the polymers of different sizes before the precipitation stage of the enzyme. This study presents the development and optimization of a protocol to produce such a biocatalyst using penicillin acylase as a model. Experiments show that CLEA-GDPs have a highly increased stability in organic media. The average half-life of the preparations was much higher than standard CLEA without a microenvironment (CLEA-G), (e.g., more than 25-fold) in the presence of dioxane. However, their thermal stability was not increased, which leads to the conclusion that the stability of CLEA-GDPs in organic media is due to the hydrophilic microenvironment that surrounds the protein enzyme more than to a conformational stiffening effect. This is further supported by solvation experiments that show a preferential hydration of CLEA when polymers are used to coat the enzyme. CLEA-GDPs are clearly better than other biocatalysts in terms of solvent stability.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.     

Stereoselective convergent synthesis of 24-substituted metabolites and analogues of vitamin D

The synthesis of vitamin D(3) active metabolites [24R,25-(OH)(2)-D(3), 24S,25-(OH)(2)-D(3) and 1alpha,24R,25-(OH)(3)-D(3)] and the first 24-aminovitamin D(3) derivatives [24S-benzoylamino-25-OH-D(3) and 24S-benzoylamino-1alpha,25-(OH)(2)-D(3)] are reported. The stereogenic center at C-24 was generated through ultrasonically induced aqueous conjugate addition of iodide 8 to dioxolanone 6 or oxazolidinone 7. The vitamin D triene system was constructed using the Lythgoe approach. The synthetic route, which is both short (6 or 7 steps from iodide 8) and efficient (32-45% overall yield), constitutes a practical method for the preparation of 24-functionalized metabolites and analogues of vitamin D(3). The ultrasonically induced conjugate addition in the key step provides a novel example of a highly stereoselective reaction promoted by the zinc-copper couple in aqueous media.     

Toxic effects of zinc on anaerobic microbiota from Zimapán Reservoir (Mexico)

The toxic effects of heavy metals have been extensively documented in different organisms. Nevertheless, a lack of information exists with regard to this topic in the case of autochthonous microorganism communities. The aim of this study was to evaluate the toxic effects of zinc on the anaerobic microorganisms present in the sediment and anoxic water of Zimapán Reservoir (Mexico), with particular focus on dissimilatory sulphate reducing bacteria. In the laboratory, a system of enrichment microcosms was set up with sediment and water from the reservoir. ATP, protein, carbohydrates and lactate and alcohol dehydrogenase activity were determined. The physicochemical parameters of the reservoir were evaluated over the course of one year. Sulphate reduction occurred in the reservoir throughout the year, but was most pronounced at the end of the wet season and during winter. In the field, increases in the rate of sulphate reduction coincided with the lowest levels of total phosphorus and hydrosoluble organic carbon. Zinc enrichment was observed to modify protein and carbohydrate content as well as to affect lactate and alcohol dehydrogenase activity. All responses followed a zinc concentration-response relationship and were dependent on reservoir physicochemical parameters. ATP content was used as a biomarker to evaluate the sublethal toxic effects of zinc. The acceptable threshold concentration of zinc in the aquatic and sediment enrichment microcosms was determined to be 0.06mgZn/L and 711.1mgZn/kg, respectively.