Serum biochemistry profile of laying hens fed diets with fish waste oil / [Parâmetros bioquímicos sanguíneos de poedeiras leves alimentadas com rações contendo óleo do resíduo de pescado]

The present study aimed to evaluate increasing levels of fish waste oil in diets for laying hens on serum biochemistry profile. 192 Hisex White laying hens at 29 weeks of age were used, with water and food ad libitum. The experimental design was completely randomized consisting of eight treatments corresponding to the inclusion levels of fish waste oil (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5%) in the diets, with four replicates of six birds each. Data collected were subjected to polynomial regression at 5% of significance. Significant differences (P<0.05) were observed in triglycerides, glucose, total cholesterol, and uric acid. These parameters presented a decrease when hens fed diets with higher level of fish waste oil. The results of the present study indicated that the inclusion of fish waste oil caused a significant effect in the serum biochemical profile of laying hens, especially in glucose, triglycerides, total cholesterol, and uric acid concentrations. The inclusion level of 3.5% of fish waste oil caused larger disequilibrium in the serum biochemical profile of laying hens.(AU)

O presente estudo objetivou avaliar os níveis crescentes de óleo de resíduo de pescado em dietas para poedeiras leves sobre o perfil bioquímico sérico. Foram utilizadas poedeiras Hisex White com 29 semanas, com água e ração ad libitum. O delineamento experimental foi inteiramente ao acaso, consistindo de oito tratamentos correspondentes aos níveis de inclusão de óleo de resíduo de pescado (0; 0,5; 1,0; 1,5; 2,0; 2,5; 3,0 e 3,5%) nas dietas, com quatro repetições de seis aves cada. Os dados coletados foram submetidos à regressão polinomial a 5% de significância. Diferenças significativas (P<0,05) foram observadas nas concentrações de triglicerídeos, glicose, colesterol total e ácido úrico. Esses parâmetros apresentaram uma diminuição quando as aves se alimentaram com rações contendo maior nível de óleo do resíduo de pescado. Os resultados do presente estudo indicaram que a inclusão de óleo do resíduo de pescado acarretou um efeito significativo no perfil bioquímico sérico de poedeiras, principalmente nas concentrações de glicose, triglicerídeos, colesterol total e ácido úrico. O nível de inclusão de 3,5% do óleo do resíduo de pescado acarretou maior desequilíbrio no perfil bioquímico sérico das poedeiras.(AU)

Insects Associated to Crime Scenes in the Northeast of Brazil: Consolidation of Collaboration Between Entomologists and Criminal Investigation Institutes.

In Brazil, many studies on Forensic Entomology analyze the activity and succession of flies in animal models. Data on human corpses are always collected and evaluated in isolated cases. This study aimed to list the insect species associated with crime scenes investigated by the Technical-Scientific Institute of criminal expertise of the State of Rio Grande do Norte (ITEP-RN), in the Northeast of Brazil, a region exposed to high homicide rates. In total, 10 cases were investigated, of which 50% were in the initial stage of decomposition. The examined bodies were colonized by species of three orders of insects, Diptera, Coleoptera and Hymenoptera. The order Diptera represented 96% of the total insects, being represented by the following species: Chrysomya albiceps (Wiedemann) (Diptera: Calliphoridae), Chrysomya megacephala (Fabricius) (Diptera: Calliphoridae), Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae), and Cochliomyia macellaria, (Fabricius) (Diptera: Calliphoridae); Musca domestica (Linnaeus) (Diptera: Muscidae); and unidentified females of the Sarcophagidae family. Among beetles, the occurrence of Dermestes maculatus (DeGeer) (Coleoptera: Dermestidae), Necrobia rufipes (De Geer) (Coleoptera: Cleridae), and Onthophagus sp. (Scarabaeidae), as well as unidentified specimens of the families Tenebrionidae and Histeridae were recorded. In addition, specimens of Ectatomma sp. (Formicidae) were also recorded. Considering that the Rio Grande do Norte state presents a high homicide rate and the last cadaver study was conducted over a decade ago, these records update the list of species associated with cadaveric decomposition and contribute to consolidate forensic entomology in the Northeast region.

A new species of Parapharyngodon Chatterji, 1933 (Oxyuroidea: Pharyngodonidae), parasitic in Osteocephalus taurinus (Anura: Hylidae) from Brazil.

Parapharyngodon politoedi n. sp. is described here, based on specimens found in the large intestines of Osteocephalus taurinus from the Caxiuanã National Forest, State of Pará, Brazil. The new species is assigned to Parapharyngodon based on the presence of non-embryonated eggs with sub-terminal opercula, when in the ovijector. Parapharyngodon politoedi belongs to a group of species with three pairs of cloacal papillae and differs from its congeners by morphometric aspects, such as the length of the spicule, and the combination of the following morphological characters: ovaries never encircling the oesophagus, tail shape in females, cloacal lips, sharply pointed spicule and presence of genital cone. This is the second species of nematode reported to parasitize O. taurinus and the eleventh species of Parapharyngodon from hylids in the Neotropical region.

IL-1 beta- and IL-4-induced down-regulation of autotaxin mRNA and PC-1 in fibroblast-like synoviocytes of patients with rheumatoid arthritis (RA).

Autotaxin (ATX) is a 125-kD ectonucleotide pyrophosphate/phosphodiesterase, which was initially isolated and cloned from human melanoma cells as a potent stimulator of tumour cell motility. ATX shows 44% identity to the plasma cell membrane marker PC-1. Recently, we described the decreased expression of ATX mRNA in cultured fibroblast-like synoviocytes (SFC) of patients with RA by interferon-gamma. In this study using a competitive reverse transcriptase-polymerase chain reaction, we show an increased ATX mRNA expression in SFC from patients with RA in comparison with synoviocytes from non-RA patients. The median ATX mRNA amount in SFC of RA patients (440 pg/microg total RNA) was five-fold higher than the expression in synoviocytes from non-RA patients (80 pg/microg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/microg total RNA). In contrast to the elevated ATX mRNA expression in SFC of patients with RA, we did not measure increased mRNA amounts of PC-1 in these cells. Both the ATX mRNA amount and the 5'-nucleotide phosphodiesterase (PDE) activity of SFC lysate were reduced after treatment of SFC with the cytokines IL-1beta or IL-4. IL-1beta and IL-4 induced a down-regulation of PC-1 mRNA and protein expression in SFC. In SFC treated with transforming growth factor-beta the expression of PC-1 mRNA and protein was increased, whereas no significant effect on ATX mRNA expression was detectable. Pharmacological drugs used in therapy for RA, such as dexamethasone, cyclosporin, methotrexate and indomethacin, did not show a statistically significant effect on either ATX mRNA or PC-1 mRNA expression. Only pentoxifylline suppressed ATX mRNA as well as PC-1 mRNA expression. In conclusion, we show a tight regulation of ATX and PC-1 gene expression by cytokines detectable in the inflamed tissue of RA. Further investigations will deal with the regulation of ATX protein expression as well as with the function of ATX in RA.

Caveolae/lipid rafts in fibroblast-like synoviocytes: ectopeptidase-rich membrane microdomains.

Membrane peptidases play important roles in cell activation, proliferation and communication. Human fibroblast-like synoviocytes express considerable amounts of aminopeptidase N/CD13, dipeptidyl peptidase IV/CD26, and neprilysin/CD10, transmembrane proteins previously proposed to be involved in the regulation of intra-articular levels of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report these peptidases in synoviocytes to be localized predominantly in glycolipid- and cholesterol-rich membrane microdomains known as 'rafts'. At the ultrastructural level, aminopeptidase N/CD13 and dipeptidyl peptidase IV/CD26 were found in caveolae, in particular in intracellular yet surface-connected vesicle-like structures and 'rosettes' made up of several caveolae. In addition, clusters of peptidases were seen at the cell surface in flat patches ranging in size from about 60 to 160 nm. Cholesterol depletion of synoviocytes by methyl-beta-cyclodextrin disrupted >90% of the caveolae and reduced the raft localization of aminopeptidase N/CD13 without affecting Ala-p-nitroanilide-cleaving activity of confluent cell cultures. In co-culture experiments with T-lymphocytes, cholesterol depletion of synoviocytes greatly reduced their capability to induce an early lymphocytic expression of aminopeptidase N/CD13. We propose caveolae/rafts to be peptidase-rich 'hot-spot' regions of the synoviocyte plasma membrane required for functional cell-cell interactions with lymphocytes. The peptidases may act in concert with other types of proteins such as receptors and signal transducers localized in these specialized membrane domains.

Aminopeptidase N/CD13 is directly linked to signal transduction pathways in monocytes.

In the present study, we characterized in monocytes the rise in [Ca(2+)](i) evoked by monoclonal antibodies (mAbs) to aminopeptidase N (APN)/CD13, showing a two-phase calcium increase with a small-belled [Ca(2+)](i) rise due to the release of calcium from intracellular stores and a more sustained plateau due to the influx of calcium from the extracellular environment. Tyrosine kinase inhibitors were able to inhibit the rise in [Ca(2+)](i) induced by ligation APN/CD13, as were inhibitors of the phosphatidylinositol 3-kinase. For the first time we can show that mAbs to APN/CD13 provoke phosphorylation of the mitogen-activated protein kinases ERK1/2, JNK, and p38. Furthermore, we show that mRNA of the chemotactic cytokine IL-8 is upregulated under the influence of APN/CD13 ligation. Although the in vivo ligand as well as possible cooperating membrane molecules remains to be identified, our results suggest that the membrane ectoenzyme APN/CD13 is a novel signal transduction molecule in monocytes.

Oxygen stress increases prolyl cis/trans isomerase activity and expression of cyclophilin 18 in rabbit blastocysts.

The peptidyl-prolyl cis/trans isomerase (PPIase) activity and the expression of cyclophilins were studied in 6-day-old rabbit preimplantation embryos cultured under physiological and increased oxygen concentrations of 5% and 20% O(2), respectively. The PPIase activity was completely inhibited by cyclosporin A (CsA). The inhibitor of FK506-binding proteins, rapamycin, had no effect on the PPIase activity, indicating that the PPIase activity in rabbit blastocysts originates from cyclophilins. Using CsA affinity chromatography, only one cyclophilin with a molecular mass of about 17.8 kDa was separated. The cDNA of rabbit cyclophilin was cloned and sequenced. Analysis of the 682-base pair cDNA revealed an open reading frame coding for a polypeptide of 164 amino acid residues with a molecular weight of 17.83 kDa. Homologies of 90% and 96% for the cDNA and amino acid sequence, respectively, to the human CyP18 were found, suggesting that the novel rabbit cyclophilin is a member of the CyP18 family (rabCyP18). The transcription level of rabCyP18 mRNA was 8.3 +/- 0.6 pg in 100 ng total RNA in noncultured blastocysts. In vitro culture with moderate oxygen stress (20% O(2)) resulted in a 1.5-fold increase in rabCyP18 transcription and an increased PPIase activity compared to that of blastocysts cultured with 5% O(2). Increase in transcription rate and PPIase activity by oxygen stress suggests an involvement of CyP18 in oxygen defense in rabbit preimplantation embryos.

Transcriptional organization of the czc heavy-metal homeostasis determinant from Alcaligenes eutrophus.

The Czc system of Alcaligenes eutrophus mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB2A cation-proton antiporter. DNA sequencing of the region upstream of the czcNICBADRS determinant located on megaplasmid pMOL30 revealed the 5' end of czcN and a gene for a MgtC-like protein which is transcribed in the orientation opposite that of czc. Additional open reading frames upstream of czc had no homologs in the current databases. Using oligonucleotide-probed Northern blotting experiments, a 500-nucleotide czcN message and a 400-nucleotide czcI message were found, and the presence of 6, 200-nucleotide czcCBA message (D. Van der Lelie et al., Mol. Microbiol. 23:493-503, 1997) was confirmed. Induction of czcN, czcI, czcCBA, and czcDRS followed a similar pattern: transcription was induced best by 300 microM zinc, less by 300 microM cobalt, and only slightly by 300 microM cadmium. Reverse transcription-PCR gave evidence for additional continuous transcription from czcN to czcC and from czcD to czcS, but not between czcA and czcD nor between czcS and a 131-amino-acid open reading frame following czcS. The CzcR putative response regulator was purified and shown to bind in the 5' region of czcN. A reporter strain carrying a czcNIC-lacZ-czcBADRS determinant on plasmid pMOL30 was constructed, as were DeltaczcR and DeltaczcS mutants of this strain and of AE128(pMOL30) wild type. Experiments on (i) growth of these strains in liquid culture containing 5 mM Zn2+, (ii) induction of the beta-galactosidase in the reporter strains by zinc, cobalt, and cadmium, and (iii) cDNA analysis of czcCBA mRNA synthesis under inducing and noninducing conditions showed that the CzcRS two-component regulatory system is involved in Czc regulation.

Increased expression of NADH-ubiquinone oxidoreductase chain 2 (ND2) in preimplantation rabbit embryos cultured with 20% oxygen concentration.

In vitro culture of mammalian preimplantation embryos is associated with various developmental disorders such as retardation in development and cell damage. The molecular mechanisms underlying these processes are poorly understood. One of the possible reasons may be the unphysiologically high oxygen concentration used for culture. Four-day-old rabbit blastocysts were cultured with 5% O2 (physiologic oxygen concentration) or 20% O2 (usually used for in vitro culture) for 4 hr. Differences in gene expression were analysed by differential display reverse-transcriptase polymerase chain reaction (DD RT-PCR). Thirty-two differentially expressed RNA bands were found. Two of them revealed a high sequence homology to the equine NADH-ubiquinone oxidoreductase chain 2 (ND2), a subunit of complex I of the respiratory chain. mRNA expression of ND2 was increased in blastocysts cultured with the higher oxygen concentration. Increased expression of ND2 was confirmed by semiquantitative and semiquantitative competitive RT-PCR.

Treatment of fibroblast-like synoviocytes with IFN-gamma results in the down-regulation of autotaxin mRNA.

In an effort to isolate genes that change expression at the mRNA level during treatment of fibroblast-like synoviocytes (SFC) with IFN-gama, we performed a differential display analysis. Here, we report the isolation of a cDNA clone corresponding to a 3.1 kb mRNA species that is reduced in synoviocytes after culture with IFN-gama. Sequence analysis revealed the 211 bp length cDNA clone to be identical to the motility-stimulating 125 kDa protein autotaxin (ATX). The down-regulation of ATX mRNA was confirmed by Northern blot analysis as well as competitive RT-PCR. SFC express 1 ng ATX mRNA/microgram total RNA. IFN-gama down-regulated ATX mRNA up to 50% as compared to control. Our results add a new finding to the manifold functions described for IFN-gama in rheumatoid arthritis.