Cultura de anteras e embriogênese de genótipos selecionados de cevada (Hordeum vulgare L.ssp.vulgare)

A eficiência da técnica de cultura de anteras, em escala comercial, ainda pode ser considerada baixa quando medida em número de plantas duplo-haplóides férteis obtidas para cada antera estabelecida in vitro. Dessa forma, o presente trabalho é pioneiro no estudo detalhado da embriogênese in vitro do micrósporo e do grão de pólen de cevada (Hordeum vulgare L. ssp. vulgare). Com o objetivo de contribuir para o aperfeiçoamento da técnica de cultura de anteras foi analisada a embriogênese, com especial ênfase na etapa da indução, através de análises citológicas e histológicas de anteras cultivadas in vitro. Foram analisadas uma cultivar brasileira de cevada, em comparação com linhagens de duas outras cultivares brasileiras, que foram selecionadas, por seleção divergente para maior ou para menor resposta na indução da rota embriogênica e, respectivamente, para menor ou para maior capacidade de regenerar plântulas verdes. Somente foram estabelecidas em cultivo in vitro as anteras que apresentaram micrósporos e pólens jovens, das linhagens selecionadas da cultivar A-05 (S3A22 e S3A23), e da cultivar BR-2(S3B63 e, apenas na cultura de anteras, S3B61), bem como da cultivar MN-599 (nãoselecionada). Para as análises histológicas, foram fixadas, a cada dois dias, duas anteras, correspondentes a cada fileira da mesma espiga, após o início do cultivo in vitro. As anteras em cultivo e respectivas estruturas multicelulares foram fixadas em FAA 50%, desidratadas em série etílica e incluídas em hidroxietilmetacrilato. Os blocos de resina polimerizada foram secionados longitudinalmente com 3 mm de espessura. Para as análises citológicas foram fixadas, de cada espiga recém-coletada, três espiguetas sendo uma da base, outra do meio e outra do ápice. Após o pré-tratamento à baixa temperatura (5 °C), porém antes do cultivo in vitro, foram fixadas três anteras (amostras utilizadas como controles). A cada três dias, durante o cultivo, três anteras foram fixadas (até 18 dias). As anteras em cultivo e estruturas multicelulares foram fixadas em Farmer e FAA 50%, transferidas após 24 horas para etanol 70%. Na cultura in vitro das anteras houve diferenças entre uma das linhagens da cultivar A-05 em relação a cultivar MN- 599, na produção inicial de estruturas embriogênicas, diferença que desapareceu na produção total. Entretanto, houve diferenças na formação dos xiii embriões: a cv.MN-599 formou embriões bem diferenciados ao passo que a linhagem S3A22 produziu um número aparentemente menor, sendo que os embriões não eram bem diferenciados. A linhagem S3B63 não apresentou embriões até o final da análise histológica. Considerando que a amostra dessa linhagem, mantida em cultura, formou plantas verdes, pode-se propor que a formação de embriões deve ocorrer posteriormente ao desenvolvimento da cv.MN-599. Cabe destacar que houve diferenças significativas entre as cultivares A-05 e BR-2 quanto à regeneração de plântulas verdes. Esses resultados indicam ter havido maior eficiência da seleção em relação à etapa da regeneração. Com relação às categorias classificatórias dos micrósporos e grãos de pólen, constatou-se que desde o início da análise histológica (2o dia de cultivo in vitro) até o final (34o dia), foram observados micrósporos, o mesmo tendo sido observado na análise citológica. Os grãos de pólen multinucleados ocorreram praticamente em todo o período de cultivo in vitro, em ambas análises; não ocorrendo nos controles da citologia (antes do cultivo); os multinucleados foram observados a partir do 3o dia, enquanto que os multicelulares a partir do 4o dia de cultivo. As estruturas multicelulares foram observadas a partir do 8o dia. A quantidade e o tamanho das estruturas multicelulares foram variáveis ao longo da análise histológica, sendo que do 14o ao 20o dia foram encontradas as de maiores dimensões, resultantes da proliferação celular por mitoses sucessivas. A partir do 22o dia (cultivar MN- 599), a ocorrência de estruturas multicelulares no interior dos lóculos da antera diminuiu, predominando o processo de proliferação externo às anteras. Para as linhagens, a partir do 18o dia foram observadas estruturas multicelulares liberadas das anteras. A análise das estruturas multicelulares permitiu classificá-las em quatro categorias: 1. SFD: Sem forma definida; 2. MAC: meristema apical caulinar; 3. MAR: meristema apical radical embrionário adventício; e 4. Embriões. As estruturas amorfas apareceram em maior número, quando comparadas com as outras categorias. Em síntese: as linhagens selecionadas e a cultivar diferiram não apenas no tempo necessário para a formação dos embriões, mas também no desenvolvimento dos mesmos, que foi mais diferenciado na cultivar MN-599, porém sendo observados mais cedo na linhagem S3A22 e S3A23, do que na cultivar MN-599.

Simvastatin improves the healing of infected skin wounds of rats

This study explores the potential of the simvastatin to ameliorate inflammation and infection in open infected skin wounds of rats. Methods: Fourteen Wistar rats weighing 285±12g were used. The study was done in a group whose open infected skin wounds were treated with topical application of sinvastatina microemulsion (SIM, n=7) and a second group with wounds treated with saline 0.9 % (SAL, n=7). A bacteriological exam of the wounds fluid for gram positive and gram negative bacteria, the tecidual expression of TNFá and IL-1â by imunohistochemical technique, and histological analysis by HE stain were performed. Results: The expression of TNFa could be clearly demonstrated in lower degree in skin wounds treated with simvastatin (668.6 ± 74.7 ìm2) than in saline (2120.0 ± 327.1 ìm2). In comparison, wound tissue from SIM group displayed leukocyte infiltration significantly lower than that observed in SAL group (p<0.05). Culture results of the samples taken from wound fluid on fourth post treatment day revealed wound infection in only one rat of group simvastatin (SIM), where Proteus mirabilis, Escherchia coli and Enterobacter sp were isolated. In the rats whose wounds were treated with saline (SAL), polymicrobial infection with more than 100,000 CFU/g was detected in all the wounds. Conclusion: In addition to its antiinflammatory properties, the protective effects of simvastatin in infected open skin wounds is able to reduce infection and probably has antibacterial action. The potential to treat these wounds with statins to ameliorate inflammation and infection is promising

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatatos da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Caracterização parcial e atividades farmacológicas do extrato rico em polissacarídeos sulfatados da angiosperma marinha Halodule wrightii

Sulfated polysaccharides (PS) are biomolecules with a great biotechnological potential. There are few data about PS from high plants. In addition, pharmacological activities of PS from plants have not been carrying out. The aim of this work was extract PS from the angiosperm Halodule wrightii and study their anticoagulant and antioxidant activities. Histological analysis showed the presence of the PS manly in the roots. A polysaccharide-rich extract was obtained from H. wrightii by proteolysis followed by methanol and TCA precipitation. Chemical, infra-red analysis and agarose gel electrophoresis in 1.3 diaminopropane acetate buffer confirmed the presence of sulfated polysaccharides made by glucose, galactose, xylose and sulfate residues in the proportion 1: 0,9: 1: 1. In addition polyacrilamide electrophoresis have shown that extract is mainly compose by 11kDa sulfated polysaccharides. Pharmacological analysis have shown total antioxidant capacity (CAT) that resulted in 15,21 μg for equivalent of ascorbic acid, scavenging activity of the DPPH radical with 41,36 % of scavenging, activity of reducing power with the maximum of 0,290 nm (50 % of vitamin C activity) and scavenging activity superoxide radical (O2-) with a maximum of 32,23 %. Chelating activity of metal less than 4% and scavenging activity of the radical hydroxyl (OH-) less than 2%. Time of activated partial tromboplastin (aPTT) doubling the time of coagulation from 20μg of and protrombin time (PT) was not present. The data indicate that PS from Halodule wrightii could be considered for future applications in medicine, food production or cosmetic industry

Utilização da Berberina na Identificação dos Mastócitos do Molusco Anomalocardia brasiliana

Berberine is an alkaloid used as a fluorochrome in the identification of heparin and DNA. Enerback, 1974, described the technique used until today to study granules rich in heparin of vertebrate mast cells. Santos et al., 2003, studied mast cells of the mollusk Anomalocardia brasiliana using biochemical and histological analysis. This work used the fluorescent dye berberine technique to improve characterization of these cells. Mollusk organs (ctenidium and mantle) were processed with routine histological techniques. Tissue sections were treated with berberine 0,02% in redistilled water acidified to pH 4, by the addition of citric acid for 20 minutes. The visualization was made through fluorescence microscopy with ultraviolet region emission. The mast cell fluorescence had a strong yellow color, where cell nuclei appeared more greenish. This result was very similar to the ones reported before. Mast cells are location at the epithelium surface is the same in both organs, mantle and ctenidium. The fluorescence was easily observed in the granules. Therefore, this technique showed to be good and sensitive to study mast cell of invertebrates

Efeitos do potencial probiótico Enterococcus faecium 32 e de sua associação com o extrato da alga marinha Caulerpa mexicana no tratamento de colite experimental murina

Ulcerative colitis comprising an inflammatory bowel disease, whose most severe consequence is the development of intestinal neoplasia. The drugs currently used to treat the disease trigger a variety of serious adverse effects and are not effective in many cases. Recent studies demonstrated the effectiveness of natural products for the treatment of inflammatory processes. Seaweed extracts and their purified products have shown protective effects in models of inflammation and the association of traditional therapies with probiotics has significantly improved the clinical symptoms of ulcerative colitis. Therefore, the aims of this study include evaluating the potential effects of the use of probiotic strain Enterococcus faecium 32 (Ef32), the methanolic extract of the green seaweed Caulerpa mexicana (M.E.) and their concomitant administration in a murine model of colitis induced by dextran sodium sulfate (DSS). Accordingly, C57BL /6 mice were pretreated orally with Ef32 (109 CFU/ml) for seven days. In the seven days following, the colitis was induced by administration of 3% DSS (w/v) diluted in the animals drinking water. During this period, animals were treated daily with Ef32 and the M.E. (2.0 mg/kg) every other day by intravenous route. The development of colitis was monitored by the disease activity index (DAI), which takes into account the loss of body weight, consistency and presence of blood in stools. After euthanasia, the colon was removed, its length measured and tissue samples were destined for histological analysis and culture for cytokine quantification. The levels of cytokines in the culture supernatant of the colon were measured by ELISA. The treatments with the probiotic Ef32 or the M.E. alone or the combination of these two substances provoked significant improvement as to weight loss and DAI, and prevented the shortening of the colon in response to DSS. The isolated treatments triggered a slight improvement in intestinal mucosal tissue damage. However, their combination was able to completely repair the injury triggered by DSS. The association was also able to reduce the levels of all the cytokines analyzed (IFN-γ, IL-4, IL-6, IL-12, IL-17A and TNF-α). On the other hand, the treatment with Ef32 did not interfere with the levels of TNF-α, whereas treatment with M.E. did not alter the levels of IL-6. Moreover, the treatment with Ef32 not interferes in TNF-α levels, whereas treatment with M.E. did not alter the levels of IL-6. Therefore, the potential probiotic Ef32 and M.E. and especially when these samples were associated proved promising alternatives in the treatment of ulcerative colitis as demonstrated in an experimental model because of its beneficial effects on morphological and clinical parameters, and by reducing the production of proinflammatory cytokines of Th1, Th2 and Th17

Stability and Local Toxicity Evaluation of a Liposomal Prilocaine Formulation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pharmacological and local toxicity studies of a liposomal formulation for the novel local anaesthetic ropivacaine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Analise estrutural do galactano acidico dos ovos de pomacea lineata e avaliacao de suas atividades proliferativa e antiinflamatoria

The acidic galactan (AG) was obtained by extraction and proteolysis by acetone precipitation of the eggs of the mollusc Pomacea lineata. Its structure was elucidated by a combination of chemical analysis, the intrinsic viscosity and NMR spectroscopy 1D and 2D. Biological aspects of AG were evaluated by in vivo testing of healing and peritonitis induced (anti-inflammatory activity) and in vitro assays of cytotoxicity (MTT). This polymer showed a simple structure without the presence of sulfate and uronic acids in its structure. Its intrinsic viscosity and relative were evaluated at 0.44 ± 0.05 and 1.744± 0.07 dl.g-1. Spectroscopy showed that the AG has a constitution composed predominantly of β-D-galactosis, and β-D-glucosamine-NAcetil that comes in a smaller proportion in chain. The character of this acidic polysaccharide is given by the presence of pyruvate in the molecule, forming a cyclic acetal of six states, located in β-D-galactosis. The involvement of AG in the healing process was evaluated and the histological analysis revealed that there was so early in the process of healing, a great stimulation of macrophages with granuloma formation. Suggesting that AG may have promoted the advance of biological events required for tissue healing. In the trial of the GA-induced peritonitis showed dose dependent, demonstrating the anti-inflammatory effect at concentrations above 20 mg/kg, and confirming its inflammatory character and the concentration of 1mg/kg. In vitro tests used in the GA concentration of 1000 μg/mL showed proliferative activity by stimulating the growth of 3T3 cells, corroborating the findings in vivo and demonstrating the absence of cytotoxic activity

Emprego dos ácidos graxos de cadeia curta na colite de derivação fecal : estudo em ratos Wistar

Diversion colitis is a chronic inflammatory process affecting the dysfunctional colon, after a colostomy. It is postulated that nutritional deficiency of the colonic epithelium by the absence of short-chain fatty acids (SCFA) is one of the factors responsible for the appearance of DC and that their employment could reverse the morphological changes of the mucosa. The treatment of choice for fecal diversion colitis (DC) is the reconstruction of the intestinal tract, although they suggested therapeutic options using enemas. This study evaluates the effect of SCFA in atrophy and inflammation in excluded colonic segments before and after the installation DC. Forty Wistar rats were divided into four groups (n = 10 for each group), submitted colostomy with distal colon exclusion. Two control groups (A1 and B1) received rectally administered physiological saline, whereas two experimental groups (A2 and B2) received rectally administered short-chain fatty-acids. The A groups were prophylactically treated (5th to 40th days postoperatively), whereas the B groups were therapeutically treated (after postoperative day 40), for 07 days. Histological sections stained with HE were used for histological analysis of the thickness of the colonic mucosa excluded (t- Student p ≤0.05). Inflammatory reaction of the lamina propria and mucosa were measured with scores previously established (Mann Whitney p ≤ 0.05). There was a significant thickness recovery of the colonic mucosa in group B2 animals (p = 0.0001), which also exhibited a significant reduction in the number of eosinophilic polymorphonuclear cells in the lamina propria (p = 0.0126) and in the intestinal lumen (p = 0.0256). Group A2 did not prevent the mucosal atrophy and significant increases in the numbers of lymphocytes (p=0.0006) and 50 eosinophilic polymorphonuclear cells in the lamina propria of the mucosa (p = 0.0022). Therapeutic use of short-chain fatty-acids significantly reduced eosinophilic polymorphonuclear cell numbers in the intestinal wall and in the colonic lumen; it also reversed the atrophy of the colonic mucosa. Prophylactic use did not impede the development of mucosal atrophy

Histopatologia de fígado, rim e baço de piaractus mesopotamicus, prochilodus lineatus e pseudoplatystoma fasciatum parasitados por myxosporídios, capturados no Rio Aquidauana, Mato Grosso do Sul, Brasil

Este estudo descreveu a histopatologia de rim, baço e fígado de Piaractus mesopotamicus, Prochilodus lineatus e Pseudoplatystoma fasciatum, parasitados por mixosporídios, capturados no Rio Aquidauana, MS. Após necropsia, amostras do fígado, rim cefálico e baço foram colhidas, fixadas em formalina a 10 % tamponada e processadas de acordo com a rotina histológica. Os cortes foram feitos à espessura de 5 μm e corados com hematoxilina-eosina. Foram encontrados Myxobolus porofilus em P. lineatus, M. colossomatis em P. mesopotamicus e Myxobolus spp. nas três espécies de hospedeiros. Cistos de mixosporídios no exame histopatológico foram vistos no fígado e baço de P. mesopotamicus. Mais de 50% das amostras de fígado de P mesopotamicus e P lineatus apresentou hepatodistrofia difusa. Mais de 80 % das amostras de fígado de P. fasciatum apresentou formações hialinas concêntricas e esteatose em 50% das amostras. em 95,23 % das amostras de rins de P. mesopotamicus, foram observadas alterações teciduais, e em mais de 60 % dos casos nefrodistrofia difusa moderada e congestão de sinusóides glomerulares. Alterações teciduais nas amostras de rins de P. lineatus foram observadas em menos de 20 % da amostra. No baço dos peixes ora examinados não foram encontradas lesões dignas de relato.

Efeito de extratos da alga marinha caulerpa mexicana em modelos de inflamação em murinos

The regulation of the inflammatory response is essential to maintain homeostasis. Several studies have been performed to search new drugs that can contribute to avoiding or minimizing an excessive inflammatory process. The aim of this study was to evaluate the effect of extracts of green algae Caulerpa mexican in models of inflammation. In mice, the model of peritonitis induced inflammatory zymosan pretreatment of mice with aqueous and methanol extracts of C. mexican was able to reduce cell migration to the peritoneal cavity. Treatment of mice with extracts of C. mexican also reduced the ear edema induced by xylene and exerted inhibitory action on the migration of leukocytes in inflammation-induced zymosan the air pouch, and timedependent for the extracts tested in the model of ulcerative colitis induced by DSS 3%, the extract methanol, but not the aqueous C. mexican, significantly reduced the clinical symptoms of colitis, as well as the production of proinflammatory cytokines in the culture of mouse colon, in the histological analysis there was a slight reduction of inflammation in the intestinal mucosa. We concluded that the administration of the extracts resulted in the reduction of cell migration to different sites as well as reducing the edema formation induced by chemical irritant. This study demonstrates for the first time the antiinflammatory effect of aqueous and methanolic extracts from green marine algae Caulerpa mexican

MYZOBDELLA PLATENSIS (HIRUNDINIDA: PISCICOLIDAE) IS TRUE PARASITE of BLUE CRABS (CRUSTACEA: PORTUNIDAE)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da composição química, atividade antioxidante, antibacteriana, antinoceptiva, antiinflamatória e toxicidade do extrato metanólico e frações de folhas de Spondias sp. (Anacardiaceae)

Spondias sp. (Anacardiaceae), popularly known as cajá-umbu, is an endemic plant from Northeastern Brazil, where their leaves are widely used in folk medicine to treat inflammatory processes, while their fruits have a great agro industrial potential. This study was designed to evaluate hepatoprotective, antinociceptive, antioxidant, antimicrobial and anti-inflammatory properties, as well as the acute toxicity and repeated dose 28, using a methanolic extract (MES), a fraction rich in flavonoids (FRF) and a precipitate from Spondias sp.leaves. The antioxidant activity of them was valued to evaluate their free radical scavenger capacity by DPPH test, whereas MES and FRF were used to evaluate while the preventive action on carbon tetrachloride (CCl4)-induced hepatotoxicity. Seven groups (n=5) of female Wistar rats were used as follows: control group, CCl4-intoxicated group treated with EMS (500 mg/kg) for 7 days, three CCl4-intoxicated groups treated with FRF (25, 50 and 75 mg/kg) for 7 days and the CCl4-intoxicated group treated with Legalon ® (silimarina; (phytotherapeutic reference) (50 mg/kg; 7 days). MES and FRF showed a protective action against liver injury induced by CCl4, being observed a significant reduction of serum enzyme activity marker of liver damage (alanine transaminase and aspartate transaminase). On the other hand, the lipid peroxidation (SRAT) decrease, as well as the increase of glutathione content and enzyme activity of antioxidant defense system (SOD, CAT, GPx) toward near normal values indicated the ability of EMS to restore the oxidative imbalance induced by CCl4. The histological analysis confirmed the hepatoprotection, compared to degenerative changes in CCl4-treated group. This hepatoprotetor effect was similar to that shown by Legalon®. The in vitro high antioxidant capacity of extract (93.16 ± 1.00%) showed analogous results to those obtained by Carduus marianus BHT (reference standard). This fact explains the obtained results in vivo. Although no antimicrobial activity was detected, EMS and FRF promoted the antinociceptive effect induced in the second phase by the intraplantar formalin test, evidencing the anti-inflammatory action; confirmed by the carrageenan-induced peritonitis model. The evaluation of the mechanical allodynia (CFA a 80%) demonstrated the involvement of the Spondias sp. chemical composition in the anti-inflammatory activity toward the acute processes. The acute exposure and repeated dose during 28 days did not produce significant changes in the parameters that evaluate toxicity. Together the experimental results reveal, that Spondias sp. leaf extracts have a promising potential in pharmaceutical area, and due to its non-toxic condition present efficiency and security

Inhibition of the myotoxic activity of Bothrops jararacussu venom and its two major myotoxins, BthTX-I and BthTX-II, by the aqueous extract of Tabernaemontana catharinensis A. DC. (Apocynaceae)

Partial neutralization of the myotoxic effect of Bothrops jararacussu venom (BV) and two of its myotoxins [bothropstoxin-I (BthTX-I), catalytically inactive, and II (BthTX-II), showing low PLA(2) activity], by the lyophilized aqueous extract of Tabernaemontana catharinensis (AE), was studied in rat isolated soleus muscle preparations (in vitro) and through i.m. injection in the gastrocnemius muscle (in vivo) by determination of creatine kinase (CK) activity and histopathological analysis. Incubation of soleus muscle for 1 h with BV or toxins (20 mug/ml) plus AE (400 mug/ml) added immediately after BV, BthTX-I or BthTX-II reduced CK levels by 53%, 37% and 56%, respectively. The myonecrotic effects of BV (20 mug/ml) upon soleus muscle was reduced 24%, 35% and 36% when AE (400 mug/ml) was added 1 h after BV and CK was evaluated 30 min, 1 and 2 h later, respectively. For BthTX-I these values were 46%, 48% and 47%, while for BthTX-II no inhibitory effect was detected. Histological analysis of soleus muscle after incubation with AE (400 mug/ml, I h) did not reveal any change in muscle fibers, but severe necrosis induced by -BV or toxins (20 mug/ml) was clearly in evidence, and decreased significantly when soleus muscle was protected by AE. This protection was also observed when AE was administered 1 h after BV or BthTX-I, but not after BthTX-II. AE did not inhibit the catalytic PLA(2), activity of BthTX-II or BV and did not change the PAGE pattern of BV, BthTX-I or BthTX-II. In vivo assays were performed in 100-g rats and maximal CK release was attained at a dose of 100 mug of BV, 3 h after injection. AE was not effective when injected 20 s after BV or toxins. However, injecting BV or toxins (100 mug), which were pre-incubated with AE (2 mg) caused an inhibition of 57%, 59% and 51%, respectively, with zero time pre-incubation, but was less effective with I h pre-incubation. This plant represents a potential source of promising myotoxin inhibitors. (C) 2004 Elsevier GmbH. All rights reserved.