(Table 4) Mesozoolpankton abundance and biomass and relative percentage of key zooplankton groups along the Bulgarian coast, Black Sea

Results of studies during Project of an international expedition onboard R/V Vladimir Parshin in September-October 2005 are presented. Intensive development of Bacillariophyceae and Dynophyceae was recorded in coastal waters of Bulgaria, Turkey, and in the Danube River delta during period of investigations. Increase in algae population was accompanied by rising of chlorophyll a concentration up to 2.0-5.5 µg/l. In the deep water region it did not exceed 0.5 µg/l. Phytoplankton growth rate in the surface water layer varied from 0.1 to 1.0 1/day. This parameter and NO2+NO3 concentration, as well as the silicon concentration were correlative, as was described by the Michaelis-Menten equation. Phytoplankton growth was affected by basic nutrients. Zooplankton grazing varied from 0.10 to 0.69 1/day and average values in different regions varied by 1.5 times. Microalgae size range is one of major factors of grazing regulation. Rate of phytoplankton consumption was decreasing with increasing the largest diatom Pseudosolenia calcar-avis impact on total biomass of nano- and microphytoplankton.

Biogenic silica in the coastal plume of the Mississippi River

The surface distributions of dissolved silicic acid, chlorophyll and diatom abundance were measured in the plume of the Mississippi River and adjacent waters during spring (late April and early May 1993) and summer (July 1992). In spring, the time of maximum river flow, there was an intense diatom bloom with a mean diatom abundance of 1.5 x 10**7 cells/l, more than an order of magnitude higher than in summer. Mixing curves of silicic acid concentration ([Si(OH)4]) versus salinity indicate that biological uptake within the river plume removed >99% of the Si(OH)4 supplied by the river in spring and 80 to 95% in summer. In spring [Si(OH)4] was occasionally depleted to <0.2 µM-among the lowest values ever reported from the ocean-with extensive depletion to >=0.5 µM over the shelf. In summer [Si(OH)4] was less severely depleted; the lowest measured was 0.93 µM and all others were >=2.4 µM. 30Si kinetic experiments were performed during both spring and summer to measure the degree to which the rate of Si uptake by the natural diatom assemblages was limited in situ by substrate availability. In spring the dependence of the specific uptake rate (V) on extracellular [Si(OH)4] conformed much more closely to the Michaelis-Menten saturation function than has been observed in past studies. Strong dependence of V on [Si(OH)4] was observed throughout the most Si(OH)4-depleted (<0.5 µM) region, where V was limited to 12 to 45% of the diatom assemblages' maximum uptake rate (Vmax). Half-saturation concentrations for Si uptake (Ks) averaged 0.85 uM (range = 0.48 to 1.71; n = 7) in spring, with the lowest values equal to the lowest previously reported for natural diatom assemblages. There was only 1 station in summer where V was limited by [Si(OH)4], and at that station Ks was 5.3 µM-quite high in comparison with previous studies. At stations where V was limited by [Si(OH)4], in both spring and summer, Chaetoceros spp. were numerically dominant; where there was no Si limitation other diatoms, usually Skeletonema costatum, dominated. The data thus indicate strong Si limitation in spring, with diatom assemblages well adapted to low [Si(OH)4], but little or no Si limitation in summer. Historical data suggest that coastal Si(OH)4 depletion and Si limitation may be recent phenomena in the northern Gulf of Mexico, resulting from increasing [NO3-] and decreasing [Si(OH)4] in the Mississippi River during the past 30 to 50 yr.

Functional and physiological consequences of genetic variation at phosphoglucose isomerase: Heat shock protein expression is related to enzyme genotype in a montane beetle

Allele frequency variation at the phosphoglucose isomerase (PGI) locus in Californian populations of the beetle Chrysomela aeneicollis suggests that PGI may be undergoing natural selection. We quantified (i) apparent Michaelis-Menten constant (Km) of fructose 6-phosphate at different temperatures and (ii) thermal stability for three common PGI genotypes (1–1, 1–4, and 4–4). We also measured air temperature (Ta) and beetle body temperature (Tb) in three montane drainages in the Sierra Nevada, California. Finally, we measured 70-kDa heat shock protein (Hsp70) expression in field-collected and laboratory-acclimated beetles. We found that PGI allele 1 predominated in the northernmost drainage, Rock Creek (RC), which was also significantly cooler than the southernmost drainage, Big Pine Creek (BPC), where PGI allele 4 predominated. Allele frequencies and air temperatures were intermediate in the middle drainage, Bishop Creek (BC). Differences among genotypes in Km (1–1 > 1–4 > 4–4) and thermal stability (4–4 > 1–4 > 1–1) followed a pattern consistent with temperature adaptation. In nature, Tb was closely related to Ta. Hsp70 expression in adult beetles decreased with elevation and differed among drainages (BPC > BC > RC). After laboratory acclimation (8 days, 20°C day, 4°C night) and heat shock (4 h, 28–36°C), Hsp70 expression was greater for RC than BPC beetles. In RC, field-collected beetles homozygous for PGI 1–1 had higher Hsp70 levels than heterozygotes or a 4–4 homozygote. These results reveal functional and physiological differences among PGI genotypes, which suggest that montane populations of this beetle are locally adapted to temperature.

The Role of Iron-Deficiency Stress Responses in Stimulating Heavy-Metal Transport in Plants1

Plant accumulation of Fe and other metals can be enhanced under Fe deficiency. We investigated the influence of Fe status on heavy-metal and divalent-cation uptake in roots of pea (Pisum sativum L. cv Sparkle) seedlings using Cd2+ uptake as a model system. Radiotracer techniques were used to quantify unidirectional 109Cd influx into roots of Fe-deficient and Fe-sufficient pea seedlings. The concentration-dependent kinetics for 109Cd influx were graphically complex and nonsaturating but could be resolved into a linear component and a saturable component exhibiting Michaelis-Menten kinetics. We demonstrated that the linear component was apoplastically bound Cd2+ remaining in the root cell wall after desorption, whereas the saturable component was transporter-mediated Cd2+ influx across the root-cell plasma membrane. The Cd2+ transport system in roots of both Fe-deficient and Fe-sufficient seedlings exhibited similar Michaelis constant values, 1.5 and 0.6 μm, respectively, for saturable Cd2+ influx, whereas the maximum initial velocity for Cd2+ uptake in Fe-deficient seedlings was nearly 7-fold higher than that in Fe-grown seedlings. Investigations into the mechanistic basis for this response demonstrated that Fe-deficiency-induced stimulation of the plasma membrane H+-ATPase did not play a role in the enhanced Cd2+ uptake. Expression studies with the Fe2+ transporter cloned from Arabidopsis, IRT1, indicated that Fe deficiency induced the expression of this transporter, which might facilitate the transport of heavy-metal divalent cations such as Cd2+ and Zn2+, in addition to Fe2+.

Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM.

The nonclassical major histocompatibility complex class II molecule HLA-DM (DM) has recently been shown to play a central role in the class II-associated antigen presentation pathway: DM releases invariant chain-derived CLIP peptides (class II-associated invariant chain protein peptide) from HLA-DR (DR) molecules and thereby facilitates loading with antigenic peptides. Some observations have led to the suggestion that DM acts in a catalytic manner, but so far direct proof is missing. Here, we investigated in vitro the kinetics of exchange of endogenously bound CLIP for various peptides on DR1 and DR2a molecules: we found that in the presence of DM the peptide loading process follows Michaelis-Menten kinetics with turnover numbers of 3-12 DR molecules per minute per DM molecule, and with KM values of 500-1000 nM. In addition, surface plasmon resonance measurements showed that DM interacts efficiently with DR-CLIP complexes but only weakly with DR-peptide complexes isolated from DM-positive cells. Taken together, our data provide evidence that DM functions as an enzyme-like catalyst of peptide exchange and favors the generation of long-lived DR-peptide complexes that are no longer substrates for DM.

Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels.

Caracterização in vitro do metabolismo e da absorção intestinal da casearina X

As principais propriedades farmacológicas da Casearia sylvestris, uma espécie de árvore cujas folhas são utilizadas na medicina popular, já foram descritas na literatura. Recentemente foi demonstrada a potente atividade citotóxica in vitro da casearina X (CAS X), o diterpeno clerodânico majoritário isolado das folhas de C. sylvestris, contra linhagens de células tumorais humanas. Apesar dos resultados promissores, sua potente atividade citotóxica in vitro não pode ser extrapolada para uma potente atividade in vivo, a menos que possua boa biodisponibilidade e duração desejável do seu efeito. Tendo em vista que o avanço nas pesquisas de produtos naturais requer a avaliação pré-clínica de propriedades farmacocinéticas, no presente trabalho foi realizada a caracterização in vitro do metabolismo e da absorção intestinal da CAS X, com o objetivo de prever sua biodisponibilidade in vivo. Para os estudos de metabolismo in vitro, foi utilizado o modelo microssomal hepático de ratos e de humanos. Foi desenvolvido um método analítico para a quantificação da CAS X em microssomas, empregando a precipitação de proteínas com acetonitrila no preparo das amostras e a cromatografia líquida de alta eficiência para as análises. O método foi validado de acordo com os guias oficiais da Agência Nacional de Vigilância Sanitária e da European Medicine Agency (EMA). A CAS X demonstrou ser substrato para as reações de hidrólise mediada pelas carboxilesterases (CES) e apresentou um perfil cinético de Michaelis-Menten. Foram estimados os parâmetros de Vmax e KM, demonstrando que o clearance intrínseco em microssomas hepático de humanos foi 1,7 vezes maior que o de ratos. O clearance hepático foi estimado por extrapolação in vitro-in vivo, resultando em mais de 90% do fluxo sanguíneo hepático em ambas as espécies. Um estudo qualitativo para a pesquisa de metabólitos foi feito utilizando espectrometria de massas, pelo qual foi possível sugerir a formação da casearina X dialdeído como produto de metabolismo. Nos estudos de absorção intestinal in vitro foi utilizado o modelo de monocamadas de células Caco-2. Um método analítico por cromatografia líquida acoplada a espectrometria de massas foi desenvolvido e validado de acordo com o EMA, para as etapas de quantificação da CAS X no sistema de células. Os parâmetros cinéticos de permeabilidade aparente absortiva e secretória da CAS X foram estimados em um sistema celular, no qual a atividade hidrolítica da CES foi inibida. Assim, a CAS X foi capaz de permear a monocamada de células Caco-2, provavelmente por transporte ativo, sem a ocorrência de efluxo, mas com significativa retenção do composto dentro das células. Em conjunto, os ensaios in vitro realizados demonstraram a susceptibilidade da CAS X ao metabolismo de primeira passagem, como substrato para as CES específicas expressas no fígado e intestino.

A numerical approach to modeling the catalytic voltammetry of surface-confined redox enzymes

A finite difference method for simulating voltammograms of electrochemically driven enzyme catalysis is presented. The method enables any enzyme mechanism to be simulated. The finite difference equations can be represented as a matrix equation containing a nonlinear sparse matrix. This equation has been solved using the software package Mathematica. Our focus is on the use of cyclic voltammetry since this is the most commonly employed electrochemical method used to elucidate mechanisms. The use of cyclic voltammetry to obtain data from systems obeying Michaelis-Menten kinetics is discussed, and we then verify our observations on the Michaelis-Menten system using the finite difference simulation. Finally, we demonstrate how the method can be used to obtain mechanistic information on a real redox enzyme system, the complex bacterial molybdoenzyme xanthine dehydrogenase.

In vitro characterization of lamotrigine N2-glucuronidation and the lamotrigine-valproic acid interaction

Studies were performed to investigate the UDP-glucuronosyltransferase enzyme( s) responsible for the human liver microsomal N2-glucuronidation of the anticonvulsant drug lamotrigine ( LTG) and the mechanistic basis for the LTG-valproic acid ( VPA) interaction in vivo. LTG N2-glucuronidation by microsomes from five livers exhibited atypical kinetics, best described by a model comprising the expressions for the Hill ( 1869 +/- 1286 mu M, n = 0.65 +/- 0.16) and Michaelis-Menten ( Km 2234 +/- 774 mu M) equations. The UGT1A4 inhibitor hecogenin abolished the Michaelis-Menten component, without affecting the Hill component. LTG N2-glucuronidation by recombinant UGT1A4 exhibited Michaelis-Menten kinetics, with a K-m of 1558 mu M. Although recombinant UGT2B7 exhibited only low activity toward LTG, inhibition by zidovudine and fluconazole and activation by bovine serum albumin ( BSA) ( 2%) strongly suggested that this enzyme was responsible for the Hill component of microsomal LTG N2-glucuronidation. VPA ( 10 mM) abolished the Hill component of microsomal LTG N2-glucuronidation, without affecting the Michaelis-Menten component or UGT1A4-catalyzed LTG metabolism. K-i values for inhibition of the Hill component of LTG N2-glucuronidation by VPA were 2465 +/- 370 mu M and 387 +/- 12 mu M in the absence and presence, respectively, of BSA ( 2%). Consistent with published data for the effect of fluconazole on zidovudine glucuronidation by human liver microsomal UGT2B7, the Ki value generated in the presence of BSA predicted the magnitude of the LTG-VPA interaction reported in vivo. These data indicate that UGT2B7 and UGT1A4 are responsible for the Hill and Michaelis-Menten components, respectively, of microsomal LTG N2-glucuronidation, and the LTG-VPA interaction in vivo arises from inhibition of UGT2B7.

Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

The Effects of Brominated Flame Retardants on Thyroid Hormone Homeostasis in Human Placenta Tissues and Cell Culture

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.

In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.

In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation.

In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure.

This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.

Conversão da lactose e síntese de galacto-oligossacarídeos: uma abordagem experimental e teórica

O presente trabalho avaliou, na etapa experimental, um processo simultâneo de catálise e fermentação láctica visando obter um iogurte com potenciais características nutracêuticas e, na sua etapa teórica, estabeleceu uma interlocução entre a vivência experimentalista e a teoria da cinética enzimática, no que se refere à conversão da lactose e à síntese de galactooligossacarídeos (GOS). Na abordagem experimental, para um substrato específico, avaliouse biocatálise conduzida simultaneamente à fermentação, defasando a adição da enzima em relação ao início do processo fermentativo. A fermentação foi realizada a partir de cultura láctica liofilizada comercial contendo dois micro-organismos probióticos, Bifidobacterium animalis e Lactobacillus acidophilus, associados aos micro-organismos característicos do iogurte, Lactobacillus bulgaricus e Streptococcus thermophilus. Foi utilizado um preparado enzimático contendo -galactosidases obtidas de duas origens distintas: Kluyveromyces lactis e Aspergillus niger. Foram avaliados os efeitos da concentração da enzima e do tempo de adição da enzima em um planejamento experimental 2 2 . As respostas foram às concentrações, ao final do processo, de lactose, de GOS, de glicose e de galactose e a hidrólise dos galactooligossacarídeos ao longo do tempo. No que se refere à abordagem teórica, o presente trabalho considerou modelos matemáticos de hidrólise de dissacarídeos e conversão da lactose, em que a inibição foi representada a partir do incremento da concentração dos produtos da reação. No que se refere à conversão da lactose e síntese de GOS, o presente trabalho buscou estabelecer um modelo matemático em que a inibição ocorreu por efeito do incremento das concentrações de glicose e de galactose, comparando-o com os modelos conhecidos na literatura. Verificou-se que o desempenho do modelo obtido no presente trabalho foi robusto em relação às premissas estabelecidas. Na comparação com resultados experimentais de conversão enzimática, o modelo mostrou-se capaz de minimizar o erro e de ajustar-se aos dados experimentais.

Estudo do crescimento, eficiência de biofiltração e cinética de absorção de nutrientes (N-NH, N-NO e P-PO4³) da macroalga Gracilaria cervicornis (Turner) J. Agardh

The objective of this study was to examine the growth of Gracilaria cervicornis cultured in a shrimp (Litopenaeus vannamei) pond and to determine the absorption efficiency and the kinetics parameters (Vmax, Ks e Vmax:Ks) of this macroalgae for the nutrients N-NO3-, N-NH4+ and P-PO4-3, aiming at its use as bioremediatory of eutrophicated environments. For this study, two experiments (field and laboratory) were developed. In the field study, the seaweed was examined in relation to the growth and the biomass. In the laboratory experiment, the absorption efficiency of G. cervicornis was measured through the monitoring of the concentration of the three nutrients (N-NO3-, N-NH4+ e P-PO4-3) during 5 hours and the kinetic parameters were determined through the formula of Michaelis-Menten. The results obtained in this study demonstrated that G. cervicornis benefited from the available nutrients in the pond, increasing 52.4% of its biomass value after 30 days of culture. It was evidenced that the variability of the biomass could be explained through the salinity, availability of light (transparency and solid particle in suspension) and concentration of N-NO3- in the environment. In the laboratory experiment, the highest absorption efficiency was found in the treatments with low concentration (5 µmol.L-1), being evidenced a reduction of up to 85,3%, 97,5% and 81,2% of N-NH4+, N-NO3- and P-PO43-, respectively. Regarding the kinetic parameters, G. cervicornis presented better ability in absorbing N-NH4+ in high concentrations (Vmax = 158,5 µmol g-1 dry wt h-1) and P-PO43- in low concentrations (Ks = 5 µmol.L-1 e Vmax:Ks = 10,3). The results of this study show that G. cervicornis could be cultivated in shrimp ponds, presents a good capacity of absorption for the tested nutrients and is a promising candidate for biorremediation in shrimp pond effluent

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Kinetic Analysis of Gill (Na+,K+)-ATPase Activity in Selected Ontogenetic Stages of the Amazon River Shrimp, (Decapoda, Palaemonidae): Interactions at ATP- and Cation-Binding Sites

We investigated modulation by ATP, Mg2+, Na+, K+ and NH4 (+) and inhibition by ouabain of (Na+,K+)-ATPase activity in microsomal homogenates of whole zoeae I and decapodid III (formerly zoea IX) and whole-body and gill homogenates of juvenile and adult Amazon River shrimps, . (Na+,K+)-ATPase-specific activity was increased twofold in decapodid III compared to zoea I, juveniles and adults, suggesting an important role in this ontogenetic stage. The apparent affinity for ATP ( (M) = 0.09 +/- A 0.01 mmol L-1) of the decapodid III (Na+,K+)-ATPase, about twofold greater than the other stages, further highlights this relevance. Modulation of (Na+,K+)-ATPase activity by K+ also revealed a threefold greater affinity for K+ ( (0.5) = 0.91 +/- A 0.04 mmol L-1) in decapodid III than in other stages; NH4 (+) had no modulatory effect. The affinity for Na+ ( (0.5) = 13.2 +/- A 0.6 mmol L-1) of zoea I (Na+,K+)-ATPase was fourfold less than other stages. Modulation by Na+, Mg2+ and NH4 (+) obeyed cooperative kinetics, while K+ modulation exhibited Michaelis-Menten behavior. Rates of maximal Mg2+ stimulation of ouabain-insensitive ATPase activity differed in each ontogenetic stage, suggesting that Mg2+-stimulated ATPases other than (Na+,K+)-ATPase are present. Ouabain inhibition suggests that, among the various ATPase activities present in the different stages, Na+-ATPase may be involved in the ontogeny of osmoregulation in larval The NH4 (+)-stimulated, ouabain-insensitive ATPase activity seen in zoea I and decapodid III may reflect a stage-specific means of ammonia excretion since functional gills are absent in the early larval stages.