Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects

The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

Phytochemical study, antioxidant and antibacterial activities of Stemodia maritima

Stemodinol, a new natural compound, together with known compounds including jaceidin, stemodin, stemodinoside B, isocrenatoside, verbascoside, crenatoside, and isoverbascoside, were isolated from Stemodia maritima Linn. The antioxidant (DPPH method) and antimicrobial activities of stemodin, stemodinoside B, and crenatoside were investigated. Among the components tested, only crenatoside isolated from the roots showed a high antioxidant power. Stemodin and stemodinoside B exhibited antibacterial activities.

Allelopathic potential and systematic evaluation of secondary compounds in extracts from roots of Canavalia ensiformis by capillary electrophoresis

Organic extracts were obtained from roots of Canavalia ensiformis and evaluated for allelopathic potential on the germination of the weed seeds: Mimosa pudica, Cassia tora and Cassia occidentalis showing a strong allelopathic potential. After that, a systematic study of these crude extracts was made using specific protocols developed in capillary electrophoresis (CE) in order to determine some classes of secondary metabolites. Capillary electrophoresis protocols were highly specific, which makes it possible to identify 5 classes of compounds using the same crude extract samples and analyze them fartly. Some of the compounds identified show activity in the inhibition of seeds germination.

Lectin histochemical aspects of the ovarian lamellae epithelium of Genypterus blacodes (Schneider, 1801)

The composition and distribution of the glycoconjugates (GCs) secreted by the epithelium of ovarian lamellae with reference to the reproductive biology of Genypterus blacodes (Schneider, 1801) through lectin hi stochemistry is here discussed. In this species, the epithelial cells that line the ovarian cavity presented sharp morphological variations along the reproductive cycle related to the mucus secretion that accompanies oocyte ma turation. During sp awning season, residues of mannose and N-acetylglucosamine were detected in the glycocalyx of those cells using lectinhistochemistry. N- acetylgalactosamine and fucose were also observed in the same zone. The greatest variations in the lectinhistochemical pattern were found in the apical cytoplasm composition in comparison to the basal zone of the cells. The results of the present study were discussed by comparing their possible functional implications.

Atividade potencialmente alelopática de extratos brutos e hidroalcoólicos de feijão-de-porco (Canavalia ensiformis)

Extratos hidroalcoólicos de parte aérea, raízes e sementes e extratos brutos de sementes de Canavalia ensiformis foram preparados, visando identificar e caracterizar os efeitos potencialmente alelopáticos sobre a germinação de sementes e o alongamento da radícula das plantas daninhas Mimosa pudica, Urena lobata, Senna obtusifolia e Senna occidentalis. Os trabalhos foram desenvolvidos em condições controladas de 25 ºC de temperatura e fotoperíodo de 12 horas, para o bioensaio de germinação, e 24 horas, para o de alongamento da radícula. Os efeitos foram aquilatados tendo por contraste (testemunha) a água destilada. Os resultados variaram em função da espécie receptora, da concentração e da parte da planta utilizada no preparo dos extratos. A inibição da germinação das sementes e do alongamento da radícula foi diretamente proporcional à concentração do extrato, com as mais intensas inibições observadas na concentração de 4%. Independentemente da espécie receptora, as sementes, seguidas das raízes, foram as principais fontes de substâncias químicas com atividades potencialmente alelopáticas no feijão-de-porco. A análise dos diferentes extratos brutos revelou que as substâncias químicas com atividades potencialmente alelopáticas presentes nas sementes do feijão-de-porco têm polaridade compreendida entre o acetato de etila e o metanol. Para o extrato bruto metanólico, concentrações a partir de 0,4% inibiram completamente a germinação das espécies receptoras, enquanto para M. pudica e S. occidentalis concentrações de 0,6 e 0,8% proporcionaram inibições da ordem de 100% para a germinação das sementes dessas espécies. A sensibilidade das espécies aos efeitos potencialmente alelopáticos variou na seguinte ordem decrescente: M. pudica > S. occidentalis > S. obtusifolia > U. lobata.

Development of bean plants in soil contaminated with trifloxysulfuron-sodium after Stizolobium aterrimum and Canavalia ensiformis cultivation

Phytoremediation, the use of plants to decontaminate soils and water resources from organic pollutants such as herbicides, is economically and environmentally a promising technique applied in many areas, including agriculture. The objective of this work was to evaluate the development of bean plants cultivated in the field, in soil with different levels of trifloxysulfuron-sodium contamination, following cultivation of two green manure species, as well as to evaluate the possibility of recontamination of the area by such herbicide with the straw permanence on the soil. The experiment was carried out in Coimbra, MG, Brazil, on a sandy clayey Red - Yellow Argisol from March to November 2003. Four levels of soil contamination with trifloxysulfuron-sodium (0.00; 3.75; 7.50; and 15.00 g ha-1) were used as well as the following five types of cultivation prior to bean sowing in the area after herbicide application: black velvet beans (Stizolobium aterrimum) followed by removal of straw; S. aterrimum, followed by permanence of straw; jack bean (Canavalia ensiformis), followed by removal of straw; C. ensiformis followed by permanence of straw; and without prior cultivation, weed-free (weeded control). The leguminous plants were kept in the area for 65 days, cut close to the soil, and with its aerial part left or not on the surface of the experimental plot, depending on the treatment. Fifteen days after the species were cut, bean was sown in the area. At 45 days after emergence (DAE) of the bean plants, plant height and dry mass of the aerial part were evaluated. Grain productivity was determined during harvest. Height, dry matter of the aerial part and grain productivity of the bean plants, cultivated in an area previously contaminated with trifloxysulfuron-sodium at any of the levels tested, were higher with prior cultivation of S. aterrimum or C. ensiformis. At the lowest level of herbicide contamination, prior cultivation of C. ensiformis was found to be more efficient than that of S. aterrimum in mitigating the harmful effects of trifloxysulfuron-sodium on bean grain production. The permanence of the straw of the green manure species during the bean cycle did not harm the development of the plants or caused culture productivity losses, indicating that straw permanence in the area does not promote recontamination of the area.

Efeito da umidade do solo sobre a capacidade de Canavalia ensiformis e Stizolobium aterrimum em remediar solos contaminados com herbicidas

Objetivou-se, neste trabalho, avaliar os efeitos da umidade do solo sobre a capacidade de Canavalia ensiformis e Stizolobium aterrimum em remediar solos contaminados com os herbicidas tebuthiuron e trifloxysulfuron-sodium. O trabalho foi realizado em duas etapas, sendo na primeira avaliado o crescimento de C. ensiformis e S. aterrimum em solo com diferentes níveis de umidade, contaminados ou não com herbicidas. Os tratamentos foram compostos pela combinação entre os herbicidas tebuthiuron, trifloxysulfuron-sodium e ausência de herbicida, associados a quatro teores de água do solo (0,287, 0,358, 0,431 e 0,575 kg kg-1), dispostos em esquema fatorial 3 x 4, sob delineamento inteiramente casualizado, com três repetições, para cada espécie remediadora. Após o preparo do substrato e enchimento dos vasos, aplicou-se à superfície do solo o herbicida trifloxysulfuron-sodium ou tebuthiuron. Um dia após essa aplicação, procedeu-se à semeadura das espécies remediadoras. Nessa mesma época, utilizaram-se, como testemunha, vasos sem planta remediadora, porém com os mesmos níveis de umidade e com aplicação do herbicida mantido nas mesmas condições daqueles com plantas remediadoras, as quais foram colhidas 60 dias após semeadura. Nessa ocasião, foram avaliadas a altura e a massa seca da parte aérea (MSPA) dessas espécies. Todo o material colhido foi triturado e incorporado ao solo dos seus respectivos vasos. Na segunda etapa, avaliou-se a capacidade remediadora de C. ensiformis e S. aterrimum. Os tratamentos foram compostos pela combinação entre quatro níveis de umidade e cinco tipos de cultivo prévio: cultivo de C. ensiformis e S. aterrimum na presença e ausência do herbicida e um tratamento sem cultivo prévio e com aplicação do herbicida, dispostos em esquema fatorial 4 x 5, sob delineamento inteiramente casualizado, com três repetições, para cada herbicida avaliado. Amostras de 0,5 kg de solo foram retiradas dos vasos (6 L) utilizados na primeira etapa e colocadas em vasos de 0,5 L. Em seguida, cultivaram-se as espécies indicadoras de resíduo dos herbicidas no solo: sorgo (Sorghum bicolor) para o trifloxysulfuron-sodium e soja (Glycine max) para o tebuthiuron. Estas plantas foram colhidas 20 dias após a semeadura, época em que se avaliaram a altura, a MSPA e o grau de intoxicação delas pelos herbicidas. Canavalia ensiformis não sobreviveu até a época de avaliação (60 DAS) em solo contaminado pelo tebuthiuron, independentemente do nível de umidade mantido no solo. S. aterrimum sobreviveu quando cultivada em solo com teor de água entre 0,287 e 0,358 kg kg-1 e, quando comparada a C. ensiformis, foi mais eficiente na descontaminação do solo com residual do tebuthiuron. Nos solos contaminados com trifloxysulfuron-sodium ou com tebuthiuron, com o cultivo prévio das espécies remediadoras, o crescimento do sorgo e da soja foi melhor se comparado ao daquelas plantas cultivadas no solo onde não foi feito o cultivo das espécies remediadoras. De maneira geral, a variação da umidade não interferiu no processo de remediação, sendo os efeitos observados no desenvolvimento das espécies remediadoras. O melhor desenvolvimento de C. ensiformis e S. aterrimum foi observado em solo com teor de água mantido em torno de 0,431 kg kg-1; contudo, nesse teor de água, o tebuthiuron é mais facilmente disponibilizado para a solução do solo.

Biodegradation of glyphosate in rhizospheric soil cultivated with Glycine max, Canavalia ensiformis e Stizolobium aterrimum

Biodegradation of glyphosate was evaluated in rhizospheric soil cultivated with Glycine max (soybean, var. BRS245-RR), Canavalia ensiformis and Stizolobium aterrimum. After these species were cultivated for 60 days, soil samples were collected, placed in flasks and treated with 14C-glyphosate. After 30 days of incubation, the total release rate of C-CO2 was determined along with microbial biomass (MBC), metabolic quotient (qCO2), and degradation percentage of the radio-labeled glyphosate released as 14C-CO2. A higher mass of rhizosphere-associated microorganisms was verified in the soil samples from pots cultivated with soybean, regardless of glyphosate addition. However, in the presence of the herbicide, this characteristic was the most negatively affected. Microorganisms from the C. ensiformis rhizosphere released a lower amount of 14C-CO2, while for those originated from S. aterrimum, the amount released reached 1.3% more than the total carbon derived from the respiratory activity. The rhizospheric soil from S. aterrimum also presented higher glyphosate degradation efficiency per microbial biomass unit. However, considering qCO2, the microbiota of the rhizospheric soil cultivated with soybean was more efficient in herbicide degradation.

Germinação de sementes de Matelea maritima (Jack.) Woods (Asclepiadaceae)

Sementes de M. maritima germinam na temperatura ótima de 25°C com valores máximos na luz, sendo o processo otimizado em temperatura alternada de 25/30°C. Escarificação mecânica promove a germinação. Em sementes nuas, a germinação é promovida e acelerada tanto na luz quanto no escuro. Temperatura de armazenamento e tempo de estocagem alteram a fotossensibilidade das sementes: sementes estocadas à temperatura ambiente por 90 dias perdem o fotoblastismo positivo enquanto que nas estocadas sob baixa temperatura (10°C) este efeito permanece. O teste do tetrazólio demonstra que aos 180 dias de estocagem 46% das sementes permaneceram viáveis. Análises topoquímicas indicam que a principal fonte de reserva destas sementes, localizada nos cotilédones, é proteica.

Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of ß-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (D G') was calculated to be 106.87 kJ . mol-1 at 70 ºC.

Seed characteristics and dispersal of dimorphic fruit segments of Cakile maritima Scopoli (Brassicaceae) population of southern Brazilian coastal dunes

Cakile maritima occurs sporadically along the southern Brazilian coast, where it is restricted to more protected sites at the base of foredunes. Somatic dimorphism in C. maritima is manifested as morphologically distinct upper and lower fruit segments (silicules). The two morphs were tested for differences in size, number of seeds, dispersal ability and natural establishment. In the C. maritima population of southern Brazil, the lower silicule has more seeds than upper silicule, and lower seeds are more likely to abort than the upper ones. Seeds from upper segments were significantly larger than those from lower ones; however, their mass ranges overlap. The mean silicule mass was not significantly different from both segments, but the silicule/seed mass ratio from upper and lower segments was significantly different. Both segments had high ability to float in sea water, more than 50% were still afloat after 70 days. Nevertheless, dispersal occurs mainly to landward due to dominant wind action. Most of the seedlings were restricted to within a one-metre radius of the mother plant, and were principally derived from lower fruit segments.

Leaf optical properties of two liana species Canavalia parviflora Benth. and Gouania virgata Reissk in different light conditions

Lianas are plants that depend on support to reach some appreciable height, and they represent an important structural component of tropical forests. Although they predominate in clearings and gaps, some species survive in the understory. Changes in irradiance between these environments can affect leaf morphology and absorption of photosynthetic active radiation (PAR). We had examined the effects of different light regimes on leaf optical properties, chlorophyll content, specific leaf area, and leaf surface morphology in young seedlings of Canavalia parviflora Benth. (Fabaceae) and Gouania virgata Reissk (Rhamnaceae). The seedlings were distributed on workbenches covered by different layers of neutral shade netting, thus creating three levels of light intensity corresponding to about 40%, 10% and 1.5% of solar irradiance. Plants growing in full sun were used as a control. Both species exhibited an increase in reflectance in full sun and alterations in leaf morphology. Reduction in irradiance induced an increase in absorptance (decrease in reflectance and transmittance) in C. parviflora leaves in the green due to higher chlorophyll content. In G. virgata the spectral leaf changes were less observable. However, the efficiency of absorption was more pronounced in G. virgata than in C. parviflora leaves under 40%, 10% and 1.5% photon flux density (PFD). The greater efficiency of absorption in G. virgata was due to a larger specific leaf area (SLA) under these conditions. The adjustments in leaf optical properties can aid these species in overall carbon gain under limited light conditions.

Accuracy of replication in the polymerase chain reaction. Comparison between Thermotoga maritima DNA polymerase and Thermus aquaticus DNA polymerase

For certain applications of the polymerase chain reaction (PCR), it may be necessary to consider the accuracy of replication. The breakthrough that made PCR user friendly was the commercialization of Thermus aquaticus (Taq) DNA polymerase, an enzyme that would survive the high temperatures needed for DNA denaturation. The development of enzymes with an inherent 3' to 5' exonuclease proofreading activity, lacking in Taq polymerase, would be an improvement when higher fidelity is needed. We used the forward mutation assay to compare the fidelity of Taq polymerase and Thermotoga maritima (ULTMA™) DNA polymerase, an enzyme that does have proofreading activity. We did not find significant differences in the fidelity of either enzyme, even when using optimal buffer conditions, thermal cycling parameters, and number of cycles (0.2% and 0.13% error rates for ULTMA™ and Taq, respectively, after reading about 3,000 bases each). We conclude that for sequencing purposes there is no difference in using a DNA polymerase that contains an inherent 3' to 5' exonuclease activity for DNA amplification. Perhaps the specificity and fidelity of PCR are complex issues influenced by the nature of the target sequence, as well as by each PCR component.

Isolation of a ß-galactoside-binding lectin from cat liver

A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing ß-galactosyl residues, of which the 1-amine-1-deoxy-ß-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4ºC in buffer supplemented with 4 mM ß-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble ß-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.

Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.