Isolation, enzymatic characterization and antiedematogenic activity of the first reported rattlesnake hyaluronidase from Crotalus durissus terrificus venom

A hyaluronidase (CdtHya1) from Crotalus durissus terrificus snake venom (CdtV) was isolated and showed to exhibit a high activity on hyaluronan cleavage. However, surveys on this enzyme are still limited. This study aimed at its isolation, functional/structural characterization and the evaluation of its effect on the spreading of crotoxin and phospholipase A(2) (PLA(2)). The enzyme was purified through cation exchange, gel filtration and hydrophobic chromatography. After that, it was submitted to a reverse-phase fast protein liquid chromatography (RP-FPLC) and Edman degradation sequencing, which showed the first N-terminal 44 amino acid residues whose sequence evidenced identity with other snake venom hyaluronidases. CdtHya1 is a monomeric glycoprotein of 64.5 kDa estimated by SDS-PAGE under reducing conditions. It exhibited maximum activity in the presence of 0.2 M NaCl, at 37 degrees C, pH 5.5 and a specificity to hyaluronan higher than that to chondroitin-4-sulphate, chondroitin-6-sulphate or dermatan. Divalent cations (Ca2+ and Mg2+) and 1 M NaCl significantly reduced the enzyme activity. The specific activity of CdtHya1 was 5066 turbidity reducing units (TRU)/mg, against 145 TRU/mg for the soluble venom, representing a 34.9-fold purification. The pure enzyme increased the diffusion of crotoxin and PLA (2) through mice tissues. CdtHya1 (32 TRU/40 mu L) potentiated crotoxin action, as evidenced by mice death, and it decreased the oedema caused by subplantar injections of buffer, crotoxin or PLA(2), thus evidencing the relevance of hyaluronidase in the crotalic envenoming. This work yielded a highly active antiedematogenic hyaluronidase from CdtV, the first one isolated from rattlesnake venoms. (C) 2012 Elsevier Masson SAS. All rights reserved.

Source of slow lithium atoms from Ne or H-2 matrix isolation sublimation

We have studied, via laser absorption spectroscopy, the velocity distribution of Li-7 atoms released from cryogenic matrices of solid neon or molecular hydrogen. The Li atoms are implanted into the Ne or H-2 matrices - grown onto a sapphire substrate - by laser ablation of a solid Li or LiH precursor. A heat pulse is then applied to the sapphire substrate sublimating the matrix together with the isolated atoms. With a NiCr film resistor deposited directly onto the sapphire substrate we are able to transfer high instantaneous power to the matrix, thus reaching a fast sublimation regime. In this regime the Li atoms can get entrained in the released matrix gas, and we were also able to achieve matrix sublimation times down to 10 mu s for both H-2 or Ne matrix, enabling us to proceed with the trapping of the species of our interest such as atomic hydrogen, lithium, and molecules. The sublimation of the H-2 matrix, with its large center-of-mass velocity, provides evidence for a new regime of one-dimensional thermalization. The laser ablated Li seems to penetrate the H-2 matrix deeper than it does in Ne. (C) 2012 American Institute of Physics. [http://dx.doi.org/10.1063/1.4704125]

Panic-like defensive behavior but not fear-induced antinociception is differently organized by dorsomedial and posterior hypothalamic nuclei of Rattus norvegicus (Rodentia, Muridae)

The hypothalamus is a forebrain structure critically involved in the organization of defensive responses to aversive stimuli. Gamma-aminobutyric acid (GABA)ergic dysfunction in dorsomedial and posterior hypothalamic nuclei is implicated in the origin of panic-like defensive behavior, as well as in pain modulation. The present study was conducted to test the difference between these two hypothalamic nuclei regarding defensive and antinociceptive mechanisms. Thus, the GABA A antagonist bicuculline (40 ng/0.2 µL) or saline (0.9% NaCl) was microinjected into the dorsomedial or posterior hypothalamus in independent groups. Innate fear-induced responses characterized by defensive attention, defensive immobility and elaborate escape behavior were evoked by hypothalamic blockade of GABA A receptors. Fear-induced defensive behavior organized by the posterior hypothalamus was more intense than that organized by dorsomedial hypothalamic nuclei. Escape behavior elicited by GABA A receptor blockade in both the dorsomedial and posterior hypothalamus was followed by an increase in nociceptive threshold. Interestingly, there was no difference in the intensity or in the duration of fear-induced antinociception shown by each hypothalamic division presently investigated. The present study showed that GABAergic dysfunction in nuclei of both the dorsomedial and posterior hypothalamus elicit panic attack-like defensive responses followed by fear-induced antinociception, although the innate fear-induced behavior originates differently in the posterior hypothalamus in comparison to the activity of medial hypothalamic subdivisions.

Evaluating methods for the isolation of marine-derived fungal strains and production of bioactive secondary metabolites

In the present investigation we evaluate methods for the isolation and growth of marine-derived fungal strains in artificial media for the production of secondary metabolites. Inoculation of marine macroorganisms fragments in Petri dishes proved to be the most convenient procedure for the isolation of the largest number of strains. Among the growth media used, 3% malt extract showed the best result for strains isolation and growth, and yielded the largest number of strains from marine macroorganisms. The percentage of strains isolated using each of the growth media which yielded cytotoxic and/or antibiotic extracts was in the range of 23-35%, regardless of the growth media used. Further investigation of extracts obtained from different marine-derived fungal strains yielded several bioactive secondary metabolites, among which (E)-4-methoxy-5-(3-methoxybut-1-enyl)-6-methyl-2H-pyran-2-one is a new metabolite isolated from the Penicillium paxilli strain Ma(G)K.

Enterobacteria isolation in ostrich eggs (Struthio Camelus)

This study was conducted to determine the presence of enterobacteria in the eggs of ostriches reared on a farm with a history of reproductive failure. Ninety samples from twenty eggs were submitted to bacteriological tests. The results showed Enterobacteria growth in 100% of the eggs. The microorganisms isolated were Hafnia alvei in 50% (10/20), Serratia spp. in 20% (4/20), Escherichia coli in 15% (3/20), and Citrobacter freundii in 15% (3/20). All eggs presented poor eggshell quality, which favored enterobacteria contamination. Hafnia alvei was present only in the internal egg structures (albumen and yolk sac), suggesting the possibility of vertical infection.

Isolation of Microsporum gypseum in soil samples from different geographical regions of Brazil, evaluation of the extracellular proteolytic enzymes activities (keratinase and elastase) and molecular sequencing of selected strains

A survey of Microsporum gypseum was conducted in soil samples in different geographical regions of Brazil. The isolation of dermatophyte from soil samples was performed by hair baiting technique and the species were identified by morphology studies. We analyzed 692 soil samples and the recuperating rate was 19.2%. The activities of keratinase and elastase were quantitatively performed in 138 samples. The sequencing of the ITS region of rDNA was performed in representatives samples. M. gypseum isolates showed significant quantitative differences in the expression of both keratinase and elastase, but no significant correlation was observed between these enzymes. The sequencing of the representative samples revealed the presence of two teleomorphic species of M. gypseum (Arthroderma gypseum and A. incurvatum). The enzymatic activities may play an important role in the pathogenicity and a probable adaptation of this fungus to the animal parasitism. Using the phenotypical and molecular analysis, the Microsporum identification and their teleomorphic states will provide a useful and reliable identification system.

Isolation of Sporothrix schenckii from the claws of domestic cats (indoor and outdoor) and in captivity in São Paulo (Brazil)

Sporotrichosis is a subcutaneous mycosis and is also a zoonosis (sapro- and anthropozoonosis). The objective of the present study was to determine the occurrence of sporotrichosis in domestic cats and in wild or exotic felines in captivity through the isolation of Sporothrix spp. from claw impressions in a culture medium. The samples included 132 felines, of which 120 (91.0 %) were domestic cats, 11 (8.3 %) were wild felines, and one (0.7 %) was an exotic felid. Twenty-one (17.5 %) were outdoor cats. Of the total, 89 (67.4 %) had contact with other animals of the same species. It was possible to isolate Sporothrix schenckii from the claws of one (0.7 %) of the felids probed; this animal exhibited generalised sporotrichosis and had infected a female veterinarian. The potential pathogenic agents Microsporum canis and Malassezia pachydermatis were isolated in 12.1 and 5.3 % of the animals, respectively. The following anemophilous fungi, which were considered to be contaminants, were also isolated: Penicillium sp. (28 or 21.2 %), Aspergillus sp. (13 or 9.8 %), Rhodotorula sp. (5 or 3.8 %), Candida sp. (5 or 3.8 %), Trichoderma sp. (1 or 0.7 %), and Acremonium sp. (1 or 0.7 %). Due to the low magnitude of occurrence (0.7 %) of Sporothrix in feline claws, the potential of the cats evaluated in this study to be sources of infection in the city of São Paulo is considerably low.

Isolation and functional characterization of Hrp65-binding proteins in Chironomus tentans

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

Isolation and characterization of microsatellite loci in Sorbus aria (Rosaceae)

[EN] Sorbus aria (L) Crantz (Common Whitebeam) is native to Europe, east of the Balkans and in North Africa; it is also present in the Canary Islands. To evaluate the genetic diversity in natural populations of this vulnerable species, nine novel polymorphic microsatellite markers were isolated from enriched libraries. Microsatellite loci were screened in 97 individuals from La Palma (Canary Islands) and Sierra Nevada (Granada, Spain). Examination of the microsatellite profiles shows that S. aria individuals have up to three alleles per locus. The cloned sequences in microsatellite loci confirmed the polyploidy status of the plants. The number of alleles ranged from 5 to 14 per locus. The phenotype diversities across loci (H0 T) ranging from 0.653 to 0.847

The complement system of the goat: haemolytic assays and isolation of major proteins

[EN] Background: The aim of the present study was to develop a haemolytic assay for the study of the complement system in dairy goats (Capra aegagrus hircus) and to characterize the major goat complement system proteins. Results: The commonly used sheep erythrocyte sensitized with rabbit antibodies were not sensitive to lysis by goat serum, but the combination of human red blood cells (RBC) plus rabbit antibodies was the best option found for goat complement assay. A buffer based on HEPES instead of the classical veronal (barbitone) was developed. Three proteins were isolated: factor H, C1q and C3 and these were compared with the corresponding human proteins. A novel affinity chromatography technique was developed for isolation of factor H. Conclusions: Human RBC plus rabbit antibodies were a suitable option for haemolytic assays. The isolated proteins are similar to the human counterparts.

Diencephalic asymmetries: morphological, immunohistochemical and ultrastructural analysis of habenular nuclei in elasmobranchs and teleosts

The habenular nuclei are diencephalic structures present in Vertebrates and they form, with the associated fiber systems, a part of the system that connects the telencephalon to the ventral mesencephalon (Concha M. L. and Wilson S. W., 2001). In representative species of almost all classes of Vertebrates the habenular nuclei are asymmetric, both in terms of size and of neuronal and neurochemical organization, although different types of asymmetry follow different evolutionary courses. Previous studies have analyzed the spread and diversity of the asymmetry in species for which data are not clear (Kemali M. et al., 1980). Notwithstanding that, it’s still not totally understood the evolution of the phenomenon, and the ontogenetic mechanisms that have led to the habenular asymmetry development are not clear (Smeets W.J. et al., 1983). For the present study 14 species of Elasmobranchs and 15 species of Teleostean have been used. Brains removed from the animals have been fixed using 4% paraformaldehyde in phosphate buffer; brains have been analyzed with different tecniques, and I used histological, immunohistochemical and ultrastructural analysis to describe this asymmetry. My results confirm data previously obtained studying other Elasmobranchs species, in which the left habenula is larger than the right one; the Teleostean show some slightly differences regarding the size of the habenular ganglia, in some species, in which the left habenular nucleus is larger than the right. In the course of studies, a correlation between the habits of life and the diencephalic asymmetry seems to emerge: among the Teleostean analyzed, the species with benthic life (like Lepidorhombus boscii, Platichthys flesus, Solea vulgaris) seem to possess a slight asymmetry, analogous to the one of the Elasmobranchs, while in the other species (like Liza aurata, Anguilla anguilla, Trisopterus minutus) the habenulae are symmetrical. However, various aspects of the neuroanatomical asymmetries of the epithalamus have not been deepened in order to obtain a complete picture of the evolution of this phenomenon, and new searches are needed to examine the species without clear asymmetry, in order to understand the spread and the diversity of the asymmetry among the habenulae between the Vertebrates.

Two-dimensional numerical approach for the vibration isolation analysis on thin walled wave barriers in poroelastic soils.

[EN]This paper is concerned with the vibration isolation efficiency analysis of total or partially buried thin walled wave barriers in poroelastic soils. A two-dimensional time harmonic model that treats soils and structures in a direct way by combining appropriately the conventional Boundary Element Method (BEM), the Dual BEM (DBEM) and the Finite Element Method es developed to this aim.

Isolation and identification of molecular partners of the proteins encoded by the Drosophila tumor suppressor gene lethal(2)tumorous imaginal discs

Ziel dieser Arbeit war es, die funktionelle Bedeutung des Drosophila melanogaster tumor suppressor Gens lethal(2)tumorous imaginal discs (l(2)tid) durch die Identifikation von molekularen Partnern der vom Gen kodierten Proteine zu etablieren. Mit dem Screening einer Expressionsbibliothek mittels des Hefe-Di-Hybrid-Systems wurde das Protein Patched (Ptc) als ein neues Tid-bindendes Protein identifiziert. Ptc ist ein Zentralregulator der Hedhehog-Signalkette. Diese ist in der Entwicklung konserviert und in manchen humanen Krebsarten verwickelt. Die Tid/Ptc-Interaktion wurde mittels unabhängigen biochemischen Methoden wie dem GST-pulldown-Test oder der Immunopräzipitation überprüft. Außerdem ergaben funktionelle Studien in tumorosen Imaginalscheiben einen möglichen inhibitorischen Effekt von Tid über die Hh Signaltransduktion.Im letzten Teil dieser Arbeit wurde die Interaktion zwischen Tid und dem E-APC-Protein (Adenomatous polyposis coli) bewiesen. Polakis und seine Gruppe zeigten durch Studien mit dem Hefe-Di-Hybrid-System und in vitro, dass das hTid mit dem APC-Protein interagiert. Um dies auch auf Drosophila-Ebene zu überprüfen, wurden Immunopräzipitation-Studien mit den Drosophila-Gegenstücken durchgeführt. Diese Studien zeigen zum ersten Mal eine direkte Interaktion beider Proteine in vivo.