Apoptosis and production of TNF-α by tumor-associated inflammatory cells in histological grade III breast cancer

Tumor necrosis factor alpha (TNF-) is a cytokine that acts as an important mediator of the apoptotic process that also demonstrates selective citotoxicity against malignant breast tumor cells. In the present study, the presence of apoptotic tumor cells and the synthesis of TNF- by inflammatory cells were investigated in tissue samples from grade III invasive breast cancer with long-term follow-up. In situ detection of tumor apoptotic cells was investigated by direct immuno-peroxidase of digoxigenin-labeled genomic DNA. The production of TNF- and tumor cell proliferation were investigated by immunohistochemical procedures. Our data demonstrated that patients with a clinical history of cancer recurrence and metastasis presented a lower number of cancerous apoptotic cells, higher tumor proliferation rates, and lower TNF- expression rates by inflammatory cells than what is observed among patients diagnosed with the same histopathological breast cancer type but in the absence of tumor recurrence and metastasis.     

Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.     

Endoparasites observed within invertebrates used as live food items for captive wild birds: Overview and potential risks

To monitor and evaluate potential risks to birds’ health, invertebrate species that have been used as live food items had their body contents searched for endoparasites. The contents of approximately 10,000 invertebrates were analyzed. A principal component analysis was performed to study the relationship between the presence/absence of endoparasites and the characteristics of the invertebrates. In most of them, including the species preferred by birds such as caterpillars, waxworms, mealworms, most grasshoppers, and spiders, no organism was identified. Such findings suggest a low potential for parasite transmission associated with its consumption by birds. Although they had unknown or even unlikely implications for the birds’ health, gregarines, oxyurides Leidynema sp., and digenetic trematodes Monolecithotrema sp. were found in samples from woodlice, cockroaches, and centipedes, respectively. The only avian parasites observed in this study were Heterakis gallinarum in samples from earthworms and Acuaria spiralis from woodlice. Suggestively, soil invertebrates showed a higher prevalence of endoparasites and may represent a higher potential risk in comparison to the other categories of invertebrates sampled herein. Detritivory and collected origin were also explanatory variables related to the presence of endoparasites in the current study.     

Evolutionary history of the Pectoral Sparrow Arremon taciturnus: evidence for diversification during the Late Pleistocene

We focus on reconstructing a spatiotemporal scenario of diversification of a widespread South American species, the Pectoral Sparrow Arremon taciturnus (Aves: Passerellidae). This species is widely distributed in both the humid and the dry forests of South America and therefore provides an interesting model for understanding the connection between different biomes of South America. We examined nucleotide sequences of the mitochondrial genes Cytochrome b (cyt-b) and NADH subunit 2 (ND2) from 107 specimens, and one nuclear marker (intron 7 of the β-fibrinogen gene) from a subset of samples collected across the distribution ranges of A. t. taciturnus and A. t. nigrirostris. Six major lineages were recovered in the phylogenies that displayed high levels of variance of allele frequencies and corresponded to distinct geographical locations. The estimation of divergence times provided evidence that diversification of the six lineages of the Pectoral Sparrow occurred throughout the Late Pleistocene across major cis-Andean biomes and Amazonian interfluves. Our dataset for A. taciturnus provides further evidence that rivers in Amazonia constitute barriers promoting allopatric speciation, with occasional sharing of alleles among lineages, particularly those with adjacent distributions.     

Transgene transmission in South American catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>) larvae by sperm-mediated gene transfer

The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E, INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (P>0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology.     

Anti-inflammatory effects of the products from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice.

Wilbrandia ebracteata Cogn. (Cucurbitaceae) is commonly known in Brazil as "Taiuia". The roots are employed in folk medicine for the treatment of several diseases, such as rheumatic disease. This study has evaluated the anti-inflammatory action of dicloromethane fraction (F-DCM), purified fraction (PFIII) and Cucurbitacin B extracted from crude extract of W. ebracteata in experimental models in vivo. The F-DCM (0.3 to 10 mg.kg(-1), i.p. or 3 to 30 mg.kg(-1) p.o.) produced significant but not dose-dependent inhibition of the carrageenan-induced cell influx and exsudate leakage in the pleural cavity of mice. The F-DCM 0.01 to 10 mg.kg(-1), i.p. or 0.1 to 10 mg.kg(-1) p.o.) decreased the levels of PGE2 in the exsudate leakage induced by carrageenan in the pleural cavity after 4 h with a calculated ID50 of 0.01 (0.002-0.09, i.p.) and 0.29 (0.05-1.45, p.o.) mg.kg(-1). The PFIII (3 mg.kg(-1), i.p.) inhibited 80% of cell migration (1.50 +/- 0.09 x 10(6) cells/cavity) and exsudate leakage by about 50% (3.09 +/- 0.71 microg/ml) in relation to the control group. Cucurbitacin B (0.1 mg.kg(-1), i.p.), the main compound of PFIII, reduced significantly the levels of PGE2 in the exsudate leakage by 40.7% (10.41 +/- 2.67 ng.ml(-1)). These data show that the active principle(s) present in the F-DCM of W. ebracteata elicited pronounced anti-inflammatory effects when assessed by i.p. or p.o. routes, as well as PFIII. The F-DCM was also able to prevent PGE2 formation in exsudate leakage induced by carrageenan, as well as Cucurbitacin B, its active principle. These results indicate that the anti-inflammatory activity of Wilbrandia ebracteata can be related with the inhibition of the production of PGE2.     

Structural composition and differential anticoagulant activities of dermatan sulfates from the skin of four species of rays, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and Potamotrygon motoro

We compared the disaccharide composition of dermatan sulfate (DS) purified from the ventral skin of three species of rays from the Brazilian seacoast, Dasyatis americana, Dasyatis gutatta, Aetobatus narinari and of Potamotrygon motoro, a fresh water species that habits the Amazon River. DS obtained from the four species were composed of non-sulfated, mono-sulfated disaccharides bearing esterified sulfate groups at positions C-4 or C-6 of N-acetyl galactosamine (GalNAc), and disulfated disaccharides bearing esterified sulfate groups at positions C-2 of the uronic acid and at position C-4 or C-6 of GalNAc. However, DS from the skin of P. motoro presented a very low content of the disulfated disaccharides. The anticoagulant actions of ray skin DS, measured by both APTT clotting and HCII-mediated inhibition of thrombin assays, were compared to that of mammalian DS. DS from D. americana had both high APTT and HCII activities, whereas DS from D. gutatta showed activity profiles similar to those of mammalian DS. In contrast, DS from both A. narinari and P. motoro had no measurable activity in the APTT assay. Thus, the anticoagulant activity of ray skin DS is not merely a consequence of their charge density. We speculate that the differences among the anticoagulant activities of these three DS may be related to both different composition and arrangements of the disulfated disaccharide units within their polysaccharide chains.     

Melanophore appearance in wild and red tilapia embryos

Red tilapia has aroused interest in many countries for its commercial potential. This tilapia strain combines a desirable coloration and appearance with other advantageous farming characteristics. To study the early appearance of melanophore pigmentation in tilapia, a red tilapia strain originating from Thailand and a wild type coloration of Oreochromis niloticus were used as broodstock to produce artificially wild x wild and red x red progenies. The larvae were assessed periodically up to the first feeding and were recorded. Wild type fish showed a regular appearance of stellate melanophores. In the red strain, the pattern of chromatophores varies from total absence of black spotting to different degrees of macromelanophore distribution. Comparison between red and wild types showed that these two tilapia can be easily scored at day 7. Further, we present indications that the pigmentation over the body develops independently of the initial degree of pigmentation.     

Purification and characterization of a new lectin from the red marine alga Hypnea musciformis

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.