The GM2 ganglioside inhibits iNKT cell responses in a CD1d-dependent manner.

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid antigens presented on CD1d molecules at the surface of antigen-presenting cells. GM2 is a glycosphingolipid abundant in cellular membranes and known to bind CD1d molecules, but the functional consequences of this binding are not completely clarified. Herein, we analyzed the effect of GM2 in iNKT cell activation. We found that culturing antigen-presenting cells or total peripheral blood mononuclear cells with GM2 did not induce activation of human iNKT cells, implying that this lipid is not antigenic for human iNKT cells. To investigate if this lipid could inhibit iNKT cell activation, we simultaneously incubated antigen-presenting cells with GM2 and the iNKT cell antigen α-Galactosylceramide (α-GalCer) and used them to stimulate iNKT cells. We found that GM2 reduced human iNKT cell activation in a dose-dependent manner. An explanation for this effect could be a direct competition of GM2 with antigenic lipids for CD1d binding. This was demonstrated by the use of an antibody (L363) that stains mouse CD1d:α-GalCer complexes, as in the presence of GM2 the amount of CD1d:α-GalCer complexes are reduced. We further explored the consequences of chronic GM2 overload on human iNKT cells by analyzing iNKT cells in patients diagnosed with GM2 gangliosidoses. We found that pediatric patients present a higher frequency of circulating CD4+ iNKT cells and concomitant lower frequency of CD4-CD8- iNKTs. A lower percentage of iNKT cells expressing the NK marker CD161 was also observed in these patients. In contrast, in two adult patients studied, no differences on iNKT cell phenotype were observed. Altogether, this study uncovers a new role for GM2 in the modulation of iNKT cell activation, thus strengthening the central role of lipid metabolism in iNKT cell biology.

Genetic diversity of native populations of Croton tetradenius Baill. using ISSR markers.

Brazil has about 300 Croton species in different types of vegetation. Croton tetradenius Baill., which is endemic to the Northeast region and predominant in the Caatinga vegetation, stands out among the several species of this genus. Considering the importance of knowing the genetic variability of a species, the objective of this study was to analyze the genetic diversity of the genotypes of natural populations of C. tetradenius in the State of Sergipe, using ISSR molecular markers. Forty individuals were sampled in four natural populations of the State of Sergipe, Brazil. Thirteen primers were used for DNA amplification using ISSR-PCR, totaling 77 amplified fragments, of which 94.8% were polymorphic. Results of the cluster analysis obtained by the Jaccard's similarity index, using the UPGMA method, resulted in the formation of six distinct clusters. Analysis of molecular variance (AMOVA), used to estimate the genetic variability among populations, revealed significant genetic variance (P < 0.01) between and within the studied populations, and most of the genetic diversity was found (87%) within populations. According to the Jaccard's similarity index, none of the studied plants was genetically identical. CTE210 and CTE305 presented high similarity index (0.76), while CTE105 presented low similarity index (<0.16) with all related individuals. ISSR markers were efficient and allowed the formation of a molecular profile, and had sufficient polymorphism to estimate the genetic variability between the accessions of the studied populations.

Human lactoferrin triggers a mitochondrial- and caspase-dependent regulated cell death in Saccharomyces cerevisiae.

We have previously shown that the antifungal activity of human lactoferrin (hLf) against Candida albicans relies on its ability to induce cell death associated with apoptotic markers. To gain a deeper understanding of the mechanisms underlying hLf-induced apoptosis, we characterized this cell death process in the well-established Saccharomyces cerevisiae model. Our results indicate that hLf induces cell death in S. cerevisiae in a manner that requires energy and de novo protein synthesis. Cell death is associated with nuclear chromatin condensation, preservation of plasma membrane integrity, and is Yca1p metacaspase-dependent. Lactoferrin also caused mitochondrial dysfunction associated with ROS accumulation and release of cytochrome c. Pre-incubation with oligomycin, an oxidative phosphorylation inhibitor, increased resistance to hLf and, accordingly, mutants deficient in the F1F0-ATP synthase complex were more resistant to death induced by hLf. This indicates that mitochondrial energetic metabolism plays a key role in the killing effect of hLf, though a direct role of F1F0-ATP synthase cannot be precluded. Overexpression of the anti-apoptotic protein Bcl-xL or pre-incubation with N-acetyl cysteine reduced the intracellular level of ROS and increased resistance to hLf, confirming a ROS-mediated mitochondrial cell death process. Mitochondrial involvement was further reinforced by the higher resistance of cells lacking mitochondrial DNA, or other known yeast mitochondrial apoptosis regulators, such as, Aif1p, Cyc3p and Aac1/2/3p. This study provides new insights into a detailed understanding at the molecular level of hLf-induced apoptosis, which may allow the design of new strategies to overcome the emergence of resistance of clinically relevant fungi to conventional antifungals.

Invariant Natural Killer T Cells are Reduced in Hereditary Hemochromatosis Patients.

PURPOSE: Invariant natural killer T (iNKT) cells are CD1d restricted-T cells that react to lipid antigens. iNKT cells were shown to be important in infection, autoimmunity and tumor surveillance. Alterations in the number and function of these cells were described in several pathological conditions including autoimmune and/or liver diseases. CD1d is critical for antigen presentation to iNKT cells, and its expression is increased in liver diseases. The liver is the major organ affected in Hereditary Hemochromatosis (HH), an autosomal recessive disorder caused by excessive iron absorption. Herein, we describe the study of iNKT cells of HH patients. METHODS: Twenty-eight HH patients and 24 control subjects from Santo António Hospital, Porto, were included in this study. Patient's iron biochemical parameters (serum transferrin saturation and ferritin levels) and the liver function marker alanine transaminase (ALT) were determined at the time of study. Peripheral blood iNKT cells were analyzed by flow cytometry using an anti-CD3 antibody and the CD1d tetramer loaded with PBS57. RESULTS: We found a decrease in the percentage and number of circulating iNKT cells from HH patients when compared with control population independently of age. iNKT cell defects were more pronounced in untreated patients, relating with serum ferritin and transferrin saturation levels. No correlation was found with ALT, a marker of active liver dysfunction. CONCLUSIONS: Altogether, our results demonstrate that HH patients have reduced numbers of iNKT cells and that these are influenced by iron overload.

Introgressive hybridization as a promoter of genome reshuffling in natural homoploid fish hybrids (Cyprinidae, Leuciscinae).

Understanding the mechanisms underlying diversification and speciation by introgressive hybridization is currently one of the major challenges in evolutionary biology. Here, the analysis of hybridization between two pairs of Iberian Leuciscinae provided new data on independent hybrid zones involving Achondrostoma oligolepis (AOL) and Pseudochondrostoma duriense (PDU), and confirmed the occurrence of hybrids between AOL and Pseudochondrostoma polylepis (PPO). A multilevel survey combining morphological, genetic and cytogenomic markers on a vast population screening successfully sorted the selected fishes as admixed. Results were similar in both AOL × PDU and AOL × PPO systems. Overall, hybrid morphotypes, cytogenomic data and genetic profiling indicated preferential backcrossing and suggested AOL as a major genomic contributor. Moreover, results implied AOL as more permissive to introgression than PDU or PPO. Although PDU- and PPO-like individuals appeared more resilient to genome modifications, AOL appeared to be more involved and affected by the ongoing hybridization events, as chromosomal translocations were only found in AOL-like individuals. All hybrids analysed evidenced extensive ribosomal DNA (rDNA) polymorphism that was not found in parental species, but usually seen falling within the range of possible parental combinations. Yet, transgressive phenotypes that cannot be explained by normal recombination, including more rDNA clusters than expected or the occurrence of syntenic rDNAs, were also detected. Present results proved rapid genomic evolution providing the genetic novelty for species to persist. In addition, although the ultimate consequences of such apparently extensive and recurrent events remain unknown, modern genome-wide methodologies are of great promise towards answering questions concerning the causes, dynamics and impacts of hybridization.

Chromosomes of Iberian Leuciscinae (Cyprinidae) revisited: Evidence of genome restructuring in homoploid hybrids using dual-color FISH and CGH.

Iberian Leuciscinae have been used in many studies as models to explore topics such as hybridization, allopolyploidy, modes of reproduction, and evolution. This article focuses on the contribution of cytogenomics to foster research in this group of cyprinid fish. Conventional and molecular banding results were reviewed, facilitating comparative analysis between nase and chub taxa inhabiting Portuguese freshwaters. Hybridization is known to occur within both Chondrostoma s.l. and Squalius genera although polyploidy has only been reported in the latter; the reasons behind such differential genome flexibility remain unidentified. FISH tools allowed recognizing additional chromosome markers, confirming NOR polymorphism and distinguishing species and their hybrids. Recombination and genome instability were detected in homoploid and polyploid hybrid genomes supporting active NOR transposition. However, the multiplication of rDNAs in these species does not seem to be associated with Rex3 retroelement, though hybrids were not surveyed. CGH and GISH allowed reaffirming the hybrid origin of S. alburnoides and confirming that the conservative karyotype patterns within Iberian leuciscines are restricted to the macrostructure. Current data also support the usefulness of mapping repetitive DNAs, especially for nonmodel compact genomes with less variable karyotypes and sequence data resources unavailable, like in many cyprinid lineages.

Salinization effects on coastal ecosystems: a terrestrial model ecosystem approach.

In coastal areas, intrusion/irrigation with seawater can threaten biodiversity along with crop yields, and the leaching of salts from areas affected by these processes can increase the salinity of water bodies nearby. The aims of this study were to evaluate the effects of salinization on coastal soil ecosystems due to saline intrusion/irrigation. Terrestrial model ecosystems were used to simulate two soil salinization scenarios: (i) seawater intrusion and irrigation with distilled water and (ii) seawater intrusion and irrigation with saline water. Three sampling periods were established: T0-after acclimation period; T1-salinization effects; and T2-populations' recovery. In each sampling period, the abundance of nematodes, enchytraeids, springtails, mites and earthworms, and plant biomass were measured. Immediate negative effects on enchytraeid abundance were detected, especially at the higher level of saltwater via intrusion+irrigation. Eight weeks after the cessation of saline irrigation, the abundance of enchytraeids fully recovered, and some delayed effects were observed in earthworm abundance and plant biomass, especially at the higher soil conductivity level. The observed low capacity of soil to retain salts suggests that, particularly at high soil conductivities, nearby freshwater bodies can also be endangered. Under saline conditions similar to the ones assayed, survival of some soil communities can be threatened, leading to the loss of biodiversity.This article is part of the theme issue 'Salt in freshwaters: causes, ecological consequences and future prospects'.

Psychosine variants as antigens for natural killer T cells.

Natural killer T (NKT) cells play a central role in the interface between innate and adaptive immunity, and alpha-galactosylceramide was recently shown to be an endogenous antigen for these cells. The source of alpha-galactosylceramide has not yet been determined; however, in vivo degradation of alpha-galactosylceramide involves generation of alpha-psychosine (alpha-galactosylsphingosine). Alpha-psychosine stimulates cytokine release from NKT cells and constitutes an endogenous antigen for these cells. Alpha-psychosine contains a single lipid chain, while most antigens for NKT cells have two lipid chains, and we have investigated if other glycolipids with one lipid chain, derived from know antigens for NKT cells, stimulate cytokine release from NKT cells. Only psychosine variants derived from the most potent NKT cell antigens cause stimulation, and this stimulation occurs in vitro as well as in vivo. Truncated forms of weak antigens for NKT cells are not stimulatory.

Effects of NaCl and seawater induced salinity on survival and reproduction of three soil invertebrate species.

The increase of global mean temperature is raising serious concerns worldwide due to its potential negative effects such as droughts and melting of glaciers and ice caps leading to sea level rise. Expected impacts on soil compartment include floodings, seawater intrusions and use of saltwater for irrigation, with unknown effects on soil ecosystems and their inhabitants. The present study aimed at evaluating the effects of salinisation on soil ecosystems due to sea level rise. The reproduction and mortality of three standard soil invertebrate species (Folsomia candida, Enchytraeus crypticus, Hypoaspis aculeifer) in standard artificial OECD soil spiked with serial dilutions of seawater/gradient of NaCl were evaluated according to standard guidelines. An increased sensitivity was observed in the following order: H. aculeifer≪E. crypticus≈F. candida consistent with the different exposure pathways: springtails and enchytraeids are exposed by ingestion and contact while mites are mainly exposed by ingestion due to a continuous and thick exoskeleton. Although small differences were observed in the calculated effect electrical conductivity values, seawater and NaCl induced the same overall effects (with a difference in the enchytraeid tests where a higher sensitivity was found in relation to NaCl). The adverse effects described in the present study are observed on soils not considered saline. Therefore, the actual limit to define saline soils (4000 µS cm(-1)) does not reflect the existing knowledge when considering soil fauna.

Genetic parameters of multiple ovulation traits in Nellore females.

Variability in superovulatory response is a limiting factor for animal breeding programs using Multiple Ovulation and Embryo Transfer (MOET) nucleus schemes. To evaluate genetic factors affecting superovulory response, 1036 multiple ovulation records from 475 Brazilian Nellore embryo donors (daughters of 139 sires), 2.2-20.5-year olds, were analyzed. Traits used to evaluate superovulatory response included the number of palpable corpora lutea (CL), the total number of recovered structures (RS), and the number of viable embryos (VE). Two data sets were used: data from the first flush only or data from the first three flushes. Genetic parameter estimations were carried out using Restricted Maximum Likelihood (REML) methodology, with single- and multiple-trait animal models. According to the data set used, heritability estimates ranged from 0.47 to 0.57 for CL, from 0.20 to 0.65 for VE, and from 0 to 0.34 for RS, and were higher for the data set that used only the first flushing only. For the first flush, genetic correlations were 0.43 between CL and SF, 0.01 between CL and VE, and 0.73 between SF and VE. Repeatability estimates ranged from 0.47 to 0.51. In conclusion, the use of data from the first flush only might result in better estimates of genetic parameters for MOET traits in Nellore females. Furthermore, moderate to high values for repeatability suggested that selection for a high response to superovulation could be made after the first flush.

PTEN loss in Gleason grade 7 prostate tumors exhibits intratumoral heterogeneity and is associated with unfavorable pathological features

Background: PTEN loss is observed in 20­30% of prostate cancers and is associated with a poor outcome, but clinical details of the impact of this biomarker are unclear for intermediate grade tumors. Methods: We investigated 43 radical prostatectomy-derived grade 7 prostate tumors from the Clinics Hospital of Ribeirão Preto. Tissue microarray (TMA) blocks were constructed and PTEN copy number status was determined for all patients through fluorescence in situ hybridization (FISH). To determine the presence of PTEN protein loss in our study cohort, we performed immunohistochemistry (IHC) in TMA sections. We then developed an automated algorithm in HALO™ to identify regions of PTEN protein loss in whole prostate scanned sections from ten patients with known PTEN deletion status by FISH. Clinical analyses were conducted to determine the associations between PTEN loss and patient outcome. All statistical analyses were conducted in R v3.4.3 with P-values below 0.05 being considered statistically significant. Results: In this study of 43 grade 7 tumors, we found PTEN deletions by FISH in 18.9% of tumors, and PTEN protein loss by IHC in 16.3% of tumors. Both techniques were highly concordant and complementary. Clinical analysis demonstrated that PTEN deletion by FISH was significantly associated with positive margin invasion (P = 0.04) and Gleason score upgrade (P = 0.001). Digital image analysis of ten representative tumors demonstrated distinct intratumoral heterogeneity for PTEN protein loss in four tumors. Conclusions: This study shows that PTEN loss in Gleason grade 7 tumors can be heterogeneous and that a systematic analysis of this biomarker using a combination of FISH, IHC, and digital imaging may identify patients with a greater risk of poor outcome (AU)

The use of papain gel cream and sunflower oil in promoting healing in a wound in dogs: three case reports / Uso de gel creme de papaína na cicatrização de feridas em cães: relato de três casos

Papain is a proteolytic enzyme removed from the leaves of green papaya and/or latex. This enzyme is widely known as a medicinal fruit used extensively in human medicine for the treatment of wounds of various etiologies. However, studies and reports in veterinary medicine are scarce. Another herbal drug widely used in wound healing is Sunflower oil (Helianthus annus). It has inflammatory and antimicrobial properties which stimulate the local neovascularization, promoting tissue granulation, cell migration, fibroblast proliferation, and differentiation. Three dogs were treated with infected necrosis wounds, considered large, extent, and severe, with varied etiology. All cases were treated with papain gel with the exception of one dog, which was given the sunflower oil at the end of the treatment. Papain gel shows effectiveness in the treatment of wounds especially with wound debridement and removal of necrotic tissue. In addition, the healing time was shorter when compared to the treatment with sunflower oil. Finally, the herbal drugs have a low cost and high accessibility. This study contributes to create a new research regarding the use of this drug in animal wound healing.(AU)

A papaína é uma enzima proteolítica retirada do mamão-papaia verde e/ou do látex das folhas do mamoeiro, tendo propriedades medicinais conhecidas na medicina humana para tratamento de feridas de diversas etiologias. Estudos e/ou relatos na medicina veterinária são escassos. Nesse sentido, objetivou-se descrever três casos de cães, que foram atendidos no Hospital Veterinário da Universidade Federal de Lavras, Brasil, todos com feridas infectadas, necrosadas, com etiologia variada e consideradas de grande extensão e gravidade, sendo tratados com gel de papaína. O tratamento demonstrou efetividade na recuperação das feridas, com a formação de grande quantidade de tecido de granulação, em um período menor que os tratamentos convencionais. Por se tratar de um fitoterápico, de baixo custo e alta acessibilidade, o gel de papaína pode ser utilizado no tratamento de feridas de grande extensão em cães e, assim como em humanos, parece ter um efeito benéfico no processo de cicatrização. Pesquisas devem ser conduzidas para elucidar a ação desse produto nos tecidos, bem como as diferentes concentrações a serem administradas.(AU)

Avaliação da depressão miocárdica por meio do feature tracking bidimensional em cadelas com sepse / Assessment of myocardial depression by two-dimensional feature tracking in dogs with sepsis

O objetivo deste estudo prospectivo foi analisar a função do ventrículo esquerdo de cadelas que apresentavam sepse ou não e diagnosticar a depressão miocárdica de forma precoce, por meio do feature tracking imaging bidimensional (FTI-2D).Vinte e nove cadelas diagnosticadas com piometra, patologia usada como modelo experimental para sepse, foram avaliadas. Os animais foram submetidos ao exame ecocardiográfico convencional e ao FTI-2D e divididos em três grupos: grupo I (controle), grupo II (piometra sepse) e grupo III (piometra não sepse). Em relação aos parâmetros da ecocardiografia convencional, não houve diferença estatística entre os grupos avaliados. Quanto ao strain radial, os grupos II e III apresentaram função ventricular inferior no momento do diagnóstico (22,805,80 grupo I; 18,455,96 grupo II; 18,719,45 grupo III; P=0,032) e o grupo III manteve essa redução no retorno. Já no strain rate radial, apenas o grupo III obteve função ventricular inferior no momento do diagnóstico (1,900,57 grupo I; 1,910,70 grupo II, 1,570,53 grupo III; P=0,021). Para o strain longitudinal, observou-se função ventricular mais baixa nos grupos II e III no momento do diagnóstico (11,043,38 grupo I; 8,593,97 grupo II; 8,211,99; P=0,048) e após 10 dias de pós-cirúrgico (11,043,38 grupo I; 9,002,16 grupo II; 8,122,27 grupo III; p=0,048). O FTI-2D foi capaz de detectar alterações precoces da função ventricular esquerda, que não foram observadas no exame ecocardiográfico convencional, sugerindo quadro de depressão miocárdica, até mesmo em cadelas com piometra sem quadro clínico evidente de sepse. Assim, essa modalidade pode ser útil no diagnóstico, pois pode detectar alterações cardíacas precoces, possibilitando uma intervenção terapêutica rápida.(AU)

The aim of this prospective study was to analyze the left ventricular function of dogs who had sepsis or not and diagnose myocardial depression early on through the two-dimensional feature tracking imaging (2D-FTI). Twenty-nine dogs diagnosed with pyometra, a pathology used as an experimental model for sepsis were evaluated. The animals were subjected to conventional echocardiography and 2D-FTI, and divided into three groups: Group I (control), group II (sepsis pyometra) and group III (no sepsis pyometra). Regarding the parameters of conventional echocardiography, there was no statistical difference between the groups. As for the radial Strain, group II and III had lower ventricular function at diagnosis (22.805.80 Group I; 18.455.96 Group II; 18.719.45 Group III; P = 0.032) and group III maintained this reduction in return. Regarding the radial Strain rate, only the III group had lower ventricular function at diagnosis (1.900.57 Group I; 1.910.70 Group II, 1.570.53 Group III; P = 0.021). For the Longitudinal Strain we observed lower ventricular function in groups II and III at diagnosis (11.043.38 Group I, Group II 8.593.97, 8.211.99; P = 0.048) and ten days after surgery (11.043.38 Group I; 9.002.16 Group II; 8.122.27 Group III; P = 0.048). The 2D-FTI was able to detect early changes in left ventricular function, suggesting myocardial depression frame, even in female dogs with pyometra without clear clinical picture of sepsis, which were not seen in conventional echocardiography. Thus, this model can be useful in diagnosis, because it can detect early cardiac changes, enabling a rapid therapeutic intervention.(AU)

Troponina I como biomarcador de lesão cardíaca em cães com sepse / Troponin I as a biomarker of cardiac injury in dogs with sepsis

Avaliou-se a troponina I como biomarcador de lesão cardíaca na sepse, além de outros parâmetros hematológicos, em cadelas com piometra. Os grupos avaliados não diferiram estatisticamente na avaliação da concentração sérica da troponina I cardíaca. A quantidade total de leucócitos (mm3) e a porcentagem de bastonetes foram significativamente maiores no grupo sepse (23.221,74±16.848,80mm3 e 5,91±10,18%) quando comparado ao grupo não sepse (14.492,86±6.828,26mm3 e 1,93±1,64%) e ao grupo controle (10.320,00±3.999,02mm3 e 1,65±2,05%). Houve diferença significativa nas concentrações séricas da proteína C reativa (mg/dL) no grupo sepse (19,57±41,69md/dL) se comparado ao grupo não sepse (10,29±12,02mg/dL) e ao grupo controle (3,60±3,53mg/dL). Na avaliação da concentração sérica do lactato, houve diferença significativa entre cães com piometra e cães saudáveis, porém não houve diferença significativa entre os grupos sepse e não sepse. Os resultados do presente estudo indicam que a troponina I cardíaca não pôde ser considerada um biomarcador precoce para injúria miocárdica nos casos de cadela com piometra, pois os resultados das mensurações foram semelhantes entre os grupos, o que indica que pode não ter ocorrido lesão dos cardiomiócitos nessa fase. Já a proteína C reativa e o lactato são possíveis marcadores para inflamação sistêmica, uma vez que demonstraram concentrações séricas significativamente maiores em cadelas com piometra.(AU)

Troponin I as a biomarker of cardiac injury in sepsis, and other hematological parameters in female dogs with pyometra were evaluated. The groups did not differ in the assessment of serum cardiac troponin I. The total amount of leukocytes (mm3) and percentage of band cells was significantly higher in the sepsis group (23,221.74±16,848.80mm3 and 5.91±10.18%) compared to the non-sepsis group (14,492.86±6,828.26mm3 and 1.93±1.64%) and the control group (10,320.00±3,999.02mm3 and 1.65±2.05% respectively). There were significant differences in serum concentrations of C-reactive protein (mg / dL) in the sepsis group (19.57±41.69md / dL) compared to the non-sepsis group (10.29±12.02mg / dL) and control group (3.60±3.53mg / dL). In the evaluation of serum lactate concentration, there was a significant difference between dogs with pyometra and healthy dogs, but there was no significant difference between the sepsis and non-sepsis groups. The results of this study indicate that troponin I could not be considered an early biomarker of myocardial injury in cases of dogs with pyometra, because the results of the measurements were similar between groups, inferring that there may not have been an cardiomyocytes injury at this stage. C-reactive protein and lactate are potential markers for systemic inflammation, as demonstrated by significantly higher serum concentrations in dogs with pyometra.(AU)

Comparação entre dois protocolos de tratamento de ceratoconjuntivite seca experimentalmente induzida em coelhos / Comparison of two treatment protocols of experimentally induced keratoconjunctivitis sicca in rabbits

O objetivo deste estudo foi avaliar e comparar a eficácia de dois protocolos de tratamento de ceratoconjuntivite seca (CCS) experimentalmente induzida em coelhos: uma formulação oftálmica tópica composta por álcool polivinílico 1,4%, adicionado com acetilcisteína 10% e pilocarpina 1% (AAP), e outro protocolo com o uso do óleo de semente de linhaça (OL) tópico em forma de colírio, durante 12 semanas. Foram utilizados 15 coelhos machos, adultos, da raça Nova Zelândia, alocados aleatoriamente em três grupos: grupo C (controle), grupo AAP (formulação oftálmica) e grupo L (OL tópica). Os animais foram avaliados semanalmente pelo teste lacrimal de Schirmer, teste de fluoresceína e teste de Rosa Bengala; uma vez por mês, pelo exame de citologia esfoliativa ocular; ao final do experimento, pela análise histopatológica da córnea e conjuntiva. Os resultados demonstraram que houve um aumento maior na produção lacrimal quando utilizada a formulação oftálmica, e uma resolução mais rápida das úlceras de córnea, bem como diminuição no número de células desvitalizadas quando utilizado o óleo de semente de linhaça, além de aumento no número de células caliciformes em ambos os grupos de tratamento. A associação desses dois protocolos pode ser no futuro uma alternativa no tratamento da CCS.

The objective of this study was to evaluate and compare the effectiveness of two treatment protocol of experimentally induced keratoconjunctivitis sicca (KCS) in rabbits, a topical ophthalmic formulation composed by 1.4% povinilic alcohol added with 10% acetylcysteine and 1% pilocarpine (AAP) and another protocol with the topical use of the linseed seed oil (LO) in eye drop form f or 12 weeks. Fifteen male New Zealand white rabbits were aleatory allocated in 3 groups: Group C (Control), Group AAP (ophthalmic formulation) and Group L (LO topical). The animals were evaluated weekly using the Schirmer's tear test, fluorescein test and Rose Bengal test monthly for ocular cytology, and at the end of the experiment for histopathological analysis of cornea and conjunctive. The results demonstrated that there was a larger increase in the tear production when the ophthalmic formulation was us ed and a faster rapid resolution of corneal ulcers and decrease in the number of devitalized cells when linseed seed oil was used, besides an increase in the number of caliciform cells in both treatment groups. The association of those two protocols can be a future alternative in the treatment of KCS.

Organ-specific distribution and subcellular localisation of ascorbate peroxidase isoenzymes in potato (Solanum tuberosum L.) plants.

Ascorbate peroxidase (EC 1.11.1.11), a heme-containing homodimeric protein, is a hydrogen peroxide-scavenging enzyme, playing an important role in plants in order to protect them from oxidative stress, thus adverting cellular damage. Several ascorbate peroxidase isoenzymes have been reported but the understanding of their physiological role still depends on a better knowledge of their precise localisation within plant organs. Immunocytochemistry techniques were performed in order to elucidate the peroxisomal and cytosolic ascorbate peroxidase distribution within tissues of leaves and sprouts of potato plants. The peroxisomal isoenzyme was found to have a broad distribution in sprouts, but a differential one in leaves, being restricted to the spongy parenchyma. This differential expression may be associated to the mesophyll asymmetry and the diverse physiological processes that occur in it. The cytosolic isoenzyme was not detected in leaves under the used conditions, probably because it is present in low amounts in these tissues. The results obtained in sprouts were at least curious: cytosolic ascorbate was found to be adjacent to the amyloplasts. Given these results, it is possible to state that apart from their similarity, these two isoenzymes reside in different organelles and seem to take part in different physiological processes as suggested by their organ- and tissue-specific distribution.

New starch-based thermoplastic hydrogels for use as bone cements or drug-delivery carriers.

The development of new biodegradable hydrogels, based on corn starch/cellulose acetate blends, produced by free-radical polymerization with methyl methacrylate monomer (MMA) and/or an acrylic acid monomer (AA), is reported. The polymerization was initiated by a redox system consisting of a benzoyl peroxide and 4-dimethlyaminobenzyl alcohol at low temperature. These hydrogels may constitute an alternative to the materials currently used as bone cements or drug-delivery carriers. Swelling studies were carried out, as a function of pH and temperature, in buffered solutions. The xerogels were further characterized by Fourier transform-infrared spectroscopy. Tensile and compression tests, and dynamic mechanical thermal analysis were used to assess the mechanical performance of the developed materials. The fracture surfaces were observed by scanning electron microscopy. The developed materials are sensitive to the pH, showing a clear reversible transition in a relatively narrow interval of pH, which is just in the range of physiological conditions. These properties make the materials developed in this study very promising for biomedical applications. Fickian-type diffusion is the mechanism predominant in these systems, except for the composition with a higher concentration of AA, that corresponds to the most desirable kinetical behavior for controlled release (case II-transport mechanism). Furthermore, the results obtained in the mechanical tests are in the range of those reported for typical PMMA bone cements, showing that it is possible to develop partially degradable cements with an adequate mechanical behavior.

Rapid deposition of extensin during the elicitation of grapevine callus cultures is specifically catalyzed by a 40-kilodalton peroxidase.

Elicitation or peroxide stimulation of grape (Vitis vinifera L. cv Touriga) vine callus cultures results in the rapid and selective in situ insolubilization of an abundant and ionically bound cell wall protein-denominated GvP1. Surface-enhanced laser desorption/ionization/time of flight-mass spectrometry analysis, the amino acid composition, and the N-terminal sequence of purified GvP1 identified it as an 89.9-kD extensin. Analysis of cell walls following the in situ insolubilization of GvP1 indicates large and specific increases in the major amino acids of GvP1 as compared with the amino acids present in salt-eluted cell walls. We calculate that following deposition, covalently bound GvP1 contributes up to 4% to 5% of the cell wall dry weight. The deposition of GvP1 in situ requires peroxide and endogenous peroxidase activity. Isoelectric focusing of saline eluates of callus revealed only a few basic peroxidases that were all isolated or purified to electrophoretic homogeneity. In vitro and in situ assays of extensin cross-linking activity using GvP1 and peroxidases showed that a 40-kD peroxidase cross-linked GvP1 within minutes, whereas other grapevine peroxidases had no significant activity with GvP1. Internal peptide sequences indicated this extensin peroxidase (EP) is a member of the class III peroxidases. We conclude that we have identified and purified an EP from grapevine callus that is responsible for the catalysis of GvP1 deposition in situ during elicitation. Our results suggest that GvP1 and this EP play an important combined role in grapevine cell wall defense.