Age influence on quality of shell and penetration in bacterial laying lightweight eggs / Influência da idade na qualidade da casca e na penetração bacteriana em ovos de poedeiras leves

This study evaluated the influence of the bird's age on the quality of the shell and percentage of bacterial penetration in commercial eggs. White-shelled commercial eggs were used, laid by light laying hens in their first laying cycle at 21, 39, 51, and 62 weeks of age. Shell quality evaluations comprised: egg weight, specific gravity, percentage and thickness of shell, number and size of pores. For evaluations regarding bacterial penetration, strains of several enterobacterias and one salmonella were used, all of which resistant to Nalidixic acid (100µg/ml). The method employed for evaluation of bacterial penetration was filling the eggs with growth medium. The data were subjected to variance analysis with 5% of probability using SAS (Education Analytical Software, 2013). Eveb though increase in the laying hen's age caused reduction of the quality of eggshells, it failed to affect the percentage of penetration of the bacterial samples evaluated.(AU)

O presente estudo avaliou a influência da idade da ave sobre a qualidade da casca e na porcentagem de penetração bacteriana em ovos comerciais. Foram utilizados ovos brancos comerciais provenientes de poedeiras leves em primeiro ciclo de postura com 21, 39, 51 e 62 semanas de idade. As avaliações de qualidade da casca realizadas foram: peso do ovo, gravidade específica, porcentagem e espessura da casca, número e tamanho dos poros. Para as avaliações da penetração bacteriana, foram utilizadas cepas de diversas enterobactérias e uma salmonela, sendo todas resistentes ao ácido nalidíxico (100µg/mL). O método utilizado para a avaliação da penetração bacteriana foi por meio do preenchimento dos ovos com meio de cultura. Os dados foram submetidos à análise de variância com 5% de probabilidade utilizando-se o programa SAS - Statistical Analisys System (Education Analytical Software, 2013). O aumento da idade da poedeira promoveu a redução da qualidade da casca dos ovos, porém não foi capaz de influenciar a porcentagem de penetração das amostras bacterianas avaliadas.(AU)

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.

Clinical significance of MUC1, MUC2 and CK17 expression patterns for diagnosis of pancreatobiliary arcinoma.

Pancreatic cancer is characterized by aggressive growth and resistance to treatment. Identification of unique biomarkers for diagnosis and prognosis is important for treatment of this disease. We investigated the expression patterns of mucin 1 (MUC1), mucin 2 (MUC2) and cytokeratin 17 (CK17) in both normal tissues and metastatic adenocarcinomas using immunohistochemistry (IHC). We have shown that MUC1 (pan-epithelial membrane mucin), MUC2 (intestinal-type secretory mucin) and CK17 can be used as a panel of markers to distinguish collectively pancreatobiliary carcinoma from other primary site carcinomas. Tumors originating in the pancreatobiliary system showed an expression pattern of MUC1 (+), MUC2 (-) and CK17 (+). By contrast, tumors arising from the colorectal region were MUC1 (-), MUC2 (+) and CK17 (-), while tumors originating from non-pancreatobiliary system tissue expressed a MUC1 (+), MUC2 (-) and CK17 (-) profile. More importantly, the MUC1 (+), MUC2 (-) and CK17 (+) result showed greater sensitivity than CA19-9 by IHC, which is the currently accepted and widely used pancreatic tumor marker for diagnosing pancreatic cancer. Thirteen of 51 cases (25%) of pancreatobiliary adenocarcinomas with the pattern MUC1 (+), MUC2 (-) and CK17 (+) showed no immunoreactivity for CA19-9, while 34/51 (67%) cases having MUC1 (+), MUC2 (-) and CK17 (+) were correlated with positive CA19-9 staining. Our data support using an antibody panel of MUC1, MUC2 and CK17 to enhance current methods for pancreatic cancer diagnosis by identifying specifically the primary tissue of origin.

Dynamin isoform-specific interaction with the shank/ProSAP scaffolding proteins of the postsynaptic density and actin cytoskeleton.

Dynamin is a GTPase involved in endocytosis and other aspects of membrane trafficking. A critical function in the presynaptic compartment attributed to the brain-specific dynamin isoform, dynamin-1, is in synaptic vesicle recycling. We report that dynamin-2 specifically interacts with members of the Shank/ProSAP family of postsynaptic density scaffolding proteins and present evidence that dynamin-2 is specifically associated with the postsynaptic density. These data are consistent with a role for this otherwise broadly distributed form of dynamin in glutamate receptor down-regulation and other aspects of postsynaptic membrane turnover.

Multiple distinct coiled-coils are involved in dynamin self-assembly.

Dynamin, a 100-kDa GTPase, has been implicated to be involved in synaptic vesicle recycling, receptor-mediated endocytosis, and other membrane sorting processes. Dynamin self-assembles into helical collars around the necks of coated pits and other membrane invaginations and mediates membrane scission. In vitro, dynamin has been reported to exist as dimers, tetramers, ring-shaped oligomers, and helical polymers. In this study we sought to define self-assembly regions in dynamin. Deletion of two closely spaced sequences near the dynamin-1 C terminus abolished self-association as assayed by co-immunoprecipitation and the yeast interaction trap, and reduced the sedimentation coefficient from 7.5 to 4.5 S. Circular dichroism spectroscopy and equilibrium ultracentrifugation of synthetic peptides revealed coiled-coil formation within the C-terminal assembly domain and at a third, centrally located site. Two of the peptides formed tetramers, supporting a role for each in the monomer-tetramer transition and providing novel insight into the organization of the tetramer. Partial deletions of the C-terminal assembly domain reversed the dominant inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association was also observed between different dynamin isoforms. Taken altogether, our results reveal two distinct coiled-coil-containing assembly domains that can recognize other dynamin isoforms and mediate endocytic inhibition. In addition, our data strongly suggests a parallel model for dynamin subunit self-association.

Role of the basic, proline-rich region of dynamin in Src homology 3 domain binding and endocytosis.

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor platelet-derived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

The regulation of endocytosis: identifying dynamin's binding partners.

Dynamin is a large GTPase implicated in controlling the initial stages of endocytosis. Its mechanism of action remains uncertain, but it is expected to interact cyclically with components of the endocytic machinery. Dynamin binds to a number of different macromolecules, including microtubules, SH3-domain-containing proteins, and acidic phospholipids. We interpret these findings in terms of a cooperative interaction between dynamin and components of coated and non-coated pits.

Cloning and characterization of human tissue inhibitor of metalloproteinases-3.

The tissue inhibitors of metalloproteinases (TIMPs) comprise a family of proteins, of which two members have so far been described in humans. We have cloned and sequenced a third human TIMP (hTIMP-3) from phorbol ester-differentiated THP-1 cells stimulated with bacterial lipopolysaccharide. The open reading frame encodes a 211-amino-acid precursor including a 23-residue secretion signal. The mature polypeptide has a calculated molecular weight of 21.6 kD and includes an N-linked glycosylation site near the carboxyl terminus. The protein is quite basic, having a predicted isoelectric point of 9.04. We have mapped the single gene encoding human TIMP-3 to chromosome 22. By Northern analysis, transcripts for TIMP-3 were identified in a broad cross-section of tissues examined from both embryonic and adult origin. In all tissues except the placenta, the predominant transcript was 5.0 kb in size, with minor bands around 2.4 and 2.6 kb comprising no more than about 10% of the signal. In the placenta, the smaller bands accounted for close to 50% of the signal. Human TIMP-3 shows slightly closer amino acid sequence similarity to TIMP-2 (44.3%) than to TIMP-1 (38.4%), but is most closely related to a recently reported chicken TIMP, chIMP-3 (80.8% amino acid; 77.7% nucleic acid similarity.

Nitrate reductase of Neurospora crassa: the functional role of individual amino acids in the heme domain as examined by site-directed mutagenesis.

The enzyme nitrate reductase, which catalyzes the reduction of nitrate to nitrite, is a multi-redox center homodimeric protein. Each polypeptide subunit is approximately 100 kDa in size and contains three separate domains, one each for a flavin, a heme-iron, and a molybdopterin cofactor. The heme-iron domain of nitrate reductase has homology with the simple redox protein, cytochrome b5, whose crystal structure was used to predict a three-dimensional structure for the heme domain. Two histidine residues have been identified that appear to coordinate the iron of the heme moiety, while other residues may be important in the folding or the function of the heme pocket. Site-directed mutagenesis was employed to obtain mutants that encode nitrate reductase derivatives with eight different single amino acid substitutions within the heme domain, including the two central histidine residues. Replacement of one of these histidines by alanine resulted in a completely nonfunctional enzyme whereas replacement of the other histidine resulted in a stable and functional enzyme with a lower affinity for heme. Certain amino acid substitutions appeared to cause a rapid turnover of the heme domain, whereas other substitutions were tolerated and yielded a stable and fully active enzyme. Three different single amino acid replacements within the heme domain led to a dramatic change in regulation of nitrate reductase synthesis, with significant expression of the enzyme even in the absence of nitrate induction.

Molecular characterization of conventional and new repeat-induced mutants of nit-3, the structural gene that encodes nitrate reductase in Neurospora crassa.

Nitrate reductase of Neurospora crassa is a dimeric protein composed of two identical subunits, each possessing three separate domains, with flavin, heme, and molybdenum-containing cofactors. A number of mutants of nit-3, the structural gene that encodes Neurospora nitrate reductase, have been characterized at the molecular level. Amber nonsense mutants of nit-3 were found to possess a truncated protein detected by a specific antibody, whereas Ssu-1-suppressed nonsense mutants showed restoration of the wild-type, full-length nitrate reductase monomer. The mutants show constitutive expression of the truncated nitrate reductase protein; however normal control, which requires nitrate induction, was restored in the suppressed mutant strains. Three conventional nit-3 mutants were isolated by the polymerase chain reaction and sequenced; two of these mutants were due to the deletion of a single base in the coding region for the flavin domain, the third mutant was a nonsense mutation within the amino-terminal molybdenum-containing domain. Homologous recombination was shown to occur when a deleted nit-3 gene was introduced by transformation into a host strain with a single point mutation in the resident nit-3 gene. New, severely damaged, null nit-3 mutants were created by repeat-induced point mutation and demonstrated to be useful as host strains for transformation experiments.

Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover.

The nit-3 gene of the filamentous fungus Neurospora crassa encodes the enzyme nitrate reductase, which catalyzes the first reductive step in the highly regulated nitrate assimilatory pathway. The nucleotide sequence of nit-3 was determined and translates to a protein of 982 amino acid residues with a molecular weight of approximately 108 kDa. Comparison of the deduced nit-3 protein sequence with the nitrate reductase protein sequences of other fungi and higher plants revealed that a significant amount of homology exists, particularly within the three cofactor-binding domains for molybdenum, heme and FAD. The synthesis and turnover of the nit-3 mRNA were also examined and found to occur rapidly and efficiently under changing metabolic conditions.