Molecular characterization of the Aspergillus fumigatus NCS-1 homologue, NcsA

Here, we characterize the Aspergillus homologue ncsA Neuronal Calcium Sensor. We that ncsA is not an essential gene and Delta ncsA growth decreased in the presence of EGTA and SDS. the Delta ncsA mutant is more resistant to calcium NcsA: mRFP localizes to the cytoplasm and its localization is not affected by the cellular response to calcium chloride or EGTA. The Delta ncsA mutant strain more sensitive to voriconazole, itraconazole, and Polar growth in the Delta ncsA mutant was also more aVected by lovastatin than in the wild type The Spitzenkorper can be visualized in both strains although the vacuolar system does not seem to be very different, there is an increase in the staining intensity on the germling surface of the Delta ncsA strain. NcsA promotes pmcA and pmcB expression and therefore there is a reduced expression of these ion pumps in the Delta ncsA mutant background, and also of other genes involved in the response to calcium in A. fumigatus. The ncsA inactivation mutation is not causing loss of virulence in a low dose murine infection when compared to the corresponding wild type strain.

Molecular dynamics, density functional, ADMET predictions, virtual screening, and molecular interaction field studies for identification and evaluation of novel potential CDK2 inhibitors in cancer therapy

In this work, we have used molecular dynamics, density functional theory, virtual screening, ADMET predictions, and molecular interaction field studies to design and propose eight novel potential inhibitors of CDK2. The eight molecules proposed showed interesting structural characteristics that are required for inhibiting the CDK2 activity and show potential as drug candidates for the treatment of cancer. The parameters related to the Rule of Five were calculated, and only one of the molecules violated more than one parameter. One of the proposals and one of the drug-like compounds selected by virtual screening indicated to be promising candidates for CDK2-based cancer therapy.

Use of virtual screening, flexible docking, and molecular interaction fields to design novel HMG-CoA reductase inhibitors for the treatment of hypercholesterolemia

Dietary changes associated with drug therapy can reduce high serum cholesterol levels and dramatically decrease the risk of coronary artery disease, stroke, and overall mortality. Statins are hypolipemic drugs that are effective in the reduction of cholesterol serum levels, attenuating cholesterol synthesis in liver by competitive inhibition regarding the substrate or molecular target HMG-CoA reductase. We have herewith used computer-aided molecular design tools, i.e., flexible docking, virtual screening in large data bases, molecular interaction fields to propose novel potential HMG-CoA reductase inhibitors that are promising for the treatment of hypercholesterolemia.

Microparticles as a Strategy for Low-Molecular-Weight Heparin Delivery

The aims of this work were preparation and physical-chemical characterization of a microparticulate release system for delivery of enoxaparin sodium (ENX), a low-molecular-weight heparin, as a potential vehicle for optimization of deep venous thrombosis therapy. Microparticles (MPs) containing ENX were prepared from polylactide-co-glycolic acid [PLGA; (50: 50)] by a double emulsification/solvent evaporation method. The preparation parameters, such as proportion ENX/PLGA, surfactant concentration, type, time, and speed of stirring, were evaluated. The encapsulation efficiency and yield process were determined and optimized, and the in vitro release profile was analysed at 35 days. The MPs showed a spherical shape with smooth and regular surfaces. The size distribution showed a unimodal profile with an average size of 2.0 +/- 0.9 mu m. The low encapsulation efficiency (< 30%), characteristic of hydrophilic macromolecules was improved, reaching 50.2% with a procedure yield of 71.3%. The in vitro profile of ENX release from the MPs was evaluated and showed pseudo-zero-order kinetics. This indicated that diffusion was the main drug release mechanism. (C) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:1783-1792, 2011

SOLID STATE STUDIES ON MOLECULAR INCLUSIONS OF LIPPIA SIDOIDES ESSENTIAL OIL OBTAINED BY SPRAY DRYING

Inclusion complexes of Lippia sidoides essential oil and beta-cyclodextrin were obtained by slurry method and its solid powdered form was prepared using spray drying. The influence of the spray drying, as well as the different essential oil:beta-cyclodextrin ratio on the characteristics of the final product was investigated. With regard to the total oil retention 1:10 mass/mass ratio as optimal was found between the essential oil and beta-cyclodextrin. Thermoanalytical techniques (TG, EGD, TG-MS) were used to support the formation of inclusion complex and to examine their physicochemical properties after accelerated storage conditions. It may be assumed that the thermal properties of the complexes were influenced not only by the different essential oil/beta-cyclodextrin ratio but also by the storage conditions. In the aspect of their thermal stabilities, complex prepared with 1:10 m/m ratio (essential oil: beta-cyclodextrin) was the most stable one.

Synthesis, antichagasic in vitro evaluation, cytotoxicity assays, molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO(2), pIC(50) = 4.55 M) and 6l (X = F, Y = p-CN, pIC(50) = 4.27 M) as the most potent derivatives compared to crystal violet (pIC(50) = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising pro. le with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it. (C) 2008 Elsevier Ltd. All rights reserved.

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M-r = 61,000, pI similar to 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696 bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans, to improve thrombin-like activity of BjussuSP-I toxin. (c) 2007 Elsevier Masson SAS. All rights reserved.

Evaluation of the Experimental Inoculation of Cryptococcus albidus and Cryptococcus laurentii in Normal Mice: Virulence Factors and Molecular Profile Before and After Animal Passage

The genus Cryptococcus includes free-developing species, a few of which are of medical importance. Some, such as C. neoformans and C. gattii, cause infections in man frequently and C. albidus and C. laurentii cause less so. The aims of this study were to evaluate organ colonization after inoculation of C. albidus and C. laurentii isolates in normal BALB/c mice, the virulence factors (growth at 37A degrees C, capsule, melanin, proteinase, and phospholipase production) and the molecular profile (PCR-fingerprinting) of the yeasts before and after infection. The importance of different profiles (virulence and molecular) was considered in relation to the distribution in different organs and to the time intervals of isolation from organs. C. albidus was isolated from animal organs 2 to 10 days after inoculation and C. laurentii from 2 to 120 days. Most isolates of the two species kept the virulence factors showed before inoculation. The high homogeneity of the molecular profile of C. albidus and the high heterogeneity of C. laurentii were kept through the passages in animals. It is concluded that most isolates of both species were recovered from the animal organs after 5 or more days, and phenotypes were not altered by inoculation. No molecular alteration was detected and the virulence factors were not related to the time intervals before isolation from organs.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

Molecular dynamics, flexible docking, virtual screening, ADMET predictions, and molecular interaction field studies to design novel potential MAO-B inhibitors

Monoamine oxidase is a flavoenzyme bound to the mitochondrial outer membranes of the cells, which is responsible for the oxidative deamination of neurotransmitter and dietary amines. It has two distinct isozymic forms, designated MAO-A and MAO-B, each displaying different substrate and inhibitor specificities. They are the well-known targets for antidepressant, Parkinson`s disease, and neuroprotective drugs. Elucidation of the x-ray crystallographic structure of MAO-B has opened the way for the molecular modeling studies. In this work we have used molecular modeling, density functional theory with correlation, virtual screening, flexible docking, molecular dynamics, ADMET predictions, and molecular interaction field studies in order to design new molecules with potential higher selectivity and enzymatic inhibitory activity over MAO-B.

Regulation of xylanase in Aspergillus phoenicis: a physiological and molecular approach

Microbial xylanolytic enzymes have a promising biotechnological potential, and are extensively applied in industries. In this study, induction of xylanolytic activity was examined in Aspergillus phoenicis. Xylanase activity induced by xylan, xylose or beta-methylxyloside was predominantly extracellular (93-97%). Addition of 1% glucose to media supplemented with xylan or xylose repressed xylanase production. Glucose repression was alleviated by addition of cAMP or dibutyryl-cAMP. These physiological observations were supported by a Northern analysis using part of the xylanase gene ApXLN as a probe. Gene transcription was shown to be induced by xylan, xylose, and beta-methylxyloside, and was repressed by the addition of 1% glucose. Glucose repression was partially relieved by addition of cAMP or dibutyryl cAMP.

Molecular phylogeny of the freshwater prawn genus Macrobrachium (Decapoda, Palaemonidae), with emphasis on the relationships among selected American species

The genus Macrobrachium Bate, 1868 is one of the best examples of widespread crustacean genera distributed globally throughout tropical and subtropical waters. Previous investigators have noted the systematic complexity of the group, and have suggested rearrangements within the family Palaemonidae. Our phylogenetic analysis of new mitochondrial DNA sequences of 58 species of Macrobrachium distributed mainly in America support the hypothesis of monophyly of this genus, if Cryphiops Dana, 1852 is accepted as a generic synonym. We concluded that the independent evolution of different types of life cycle (abbreviated larval development-ALD and extended larval development-ELD) must have occurred more than once in the history of the group. Similarly, we also concluded that the current type species of the genus, Macrobrachium americanum Bate, 1868, should not be considered valid, as previously proposed. The synonymy of two members of the `olfersi` species complex (M. birai Lobao, Melo&Fernandes, 1986 and M. holthuisi Genofre&Lobao, 1978) with M. olfersi (Wiegmann, 1836) was confirmed. Similar results were found in comparing M. petronioi Melo, Lobao&Fernandes, 1986 and M. potiuna (Muller, 1880), in which the genetic divergence placed M. petronioi within the level of intraspecific variation of M. potiuna. The taxonomic status of the genus Cryphiops, as well as theories on the origin of Macrobrachium, is also called into question.

Taxonomic re-examination of the hermit crab species Pagurus forceps and Pagurus comptus (Decapoda: Paguridae) by molecular analysis

The current taxonomy of two poorly known hermit crab species Pagurus forceps H. Milne Edwards, 1836 and Pagurus comptus White, 1847 from temperate Pacific and Atlantic coastlines of South America is based only on adult morphology. Past studies have questioned the separation of these two very similar species, which occur sympatrically. We included specimens morphologically assignable to P. forceps and P. comptus in a phylogenetic analysis, along with other selected anomuran decapods, based on 16S ribosomal gene sequences. Differences between samples putatively assigned to either P. forceps and P. comptus were moderate, with sequence similarity ranging from 98.2 to 99.4% for the fragments analyzed. Our comparison of mitochondrial DNA sequences (16S rRNA) revealed diagnostic differences between the two putative species, suggesting that P. forceps and P. comptus are indeed phylogenetically close but different species, with no genetic justification to support their synonymization. The polyphyly of Pagurus is not corroborated here among the represented Atlantic species, despite obviously complex relationships among the members of the genus.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Study of the antimicrobial peptide indolicidin and mutants in eukaryotic modelled membrane by molecular dynamics simulations

In this work the interaction of the antimicrobial peptide indolicidin (IND) and its mutants CP10A and CP11 with a eukaryotic membrane model was examined by molecular dynamics simulations. The aim was to analyse the behaviour of these antimicrobial peptides when they interact with a eukaryotic modelled membrane, thereby obtaining atomic detailed observations that are not experimentally available. In the simulations, the widely studied dipalmitoylphosphatidylcholine hydrated bilayer was used as a eukaryotic membrane model. In agreement with experimental observations, the peptides IND, CP10A, and CP11 insert into the bilayer differently; the peptides that insert more deeply present the major hemolytic activities. The hydrophobic residues are responsible for the insertion, but some Trp residues of the peptides remain at the bilayer/water interface because they interact with the bilayer choline groups by cation-pi interactions that should be important for recognition of eukaryotic membrane by the three studied peptides.