Strategies of anaerobic sludge granulation in an EGSB reactor.

Anaerobic sludge granulation was evaluated in an expanded granular sludge bed (EGSB) reactor based on the increases in the specific organic loading rate (SOLR). The effect of precursor substances (calcium chloride, sodium chloride, and tannin) on the development of granular sludge was also investigated in batch reactors. The reactors were fed with synthetic sewage and operated in mesophilic conditions. The EGSB was operated with a variable hydraulic retention time (HRT) and the batch reactors, with cycles of 8 h and 16 h. The increase of SOLR from 17.4 ±â€¯7.4 to 104.6 ±â€¯66.7 mgCOD gVSS-1 d-1 in the EGSB resulted in an increase on the average granules diameter from 344.3 to 1583.3 µm. These conditions also favored the reduction rates of chemical oxygen demand (COD) and volatile fatty acids (VFAs) concentration in the reactor. When the upflow velocity suffered an abrupt increase (from 0.06 L h-1 to 0.25 L h-1), the granules size began to decrease and lose their settleability characteristics. Considering this, it is proposed to start the biomass granulation process without effluent recirculation, and, after the granules reach the desired size and settleability capacity, the normal operation of EGSB reactor starts. The results showed that calcium chloride was more efficient for granulation. CaCl2 addition can be performed only during the reactor's start-up, improving granulation and reducing start-up time. Thus, these results have practical implications as granules maintenance is the key to the proper EGSB operation.

The role of angiotensin II on sodium appetite after a low-sodium diet.

The present study aimed to investigate the role of angiotensin II (Ang II) on sodium appetite in rats subjected to a normal or a low-sodium diet (1% or > 0.1% NaCl) for 4 days. During sodium restriction, a reduction in water intake, urinary volume and sodium excretion was observed. After a low-sodium diet, we observed decreased plasma protein concentrations and haematocrit associated with a slight reduction in arterial pressure, without any significant changes in heart rate, natraemia, corticotrophin-releasing hormone mRNA expression in the paraventricular nucleus and corticosterone levels. After providing hypertonic saline, there was an increase in saline intake followed by a small increase in water intake, resulting in an enhanced saline intake ratio and the recovery of arterial pressure. Sodium deprivation increased plasma but not brain Ang I and II concentrations. A low-sodium diet increased kidney renin and liver angiotensinogen mRNA levels but not lung angiotensin-converting enzyme mRNA expression. Moreover, Ang II type 1a receptor mRNA expression was increased in the subfornical organ and the dorsal raphe nucleus and decreased in the medial preoptic nuclei, without changes in the paraventricular nucleus and the nucleus of solitary tract after a low-sodium diet. Blockade of AT(1) receptors or brain Ang II synthesis led to a reduction in sodium intake after a low-sodium diet. Intracerebroventricular injection of Ang II led to a similar increase in sodium and water intake in the control and low-sodium diet groups. In conclusion, the results of the present study suggest that Ang II is involved in the increased sodium appetite after a low-sodium diet.

Effect of the synthetic coumarin, ethyl 2-oxo-2H-chromene-3-carboxylate, on activity of Crotalus durissus ruruima sPLA2 as well as on edema and platelet aggregation induced by this factor.

We show that ethyl 2-oxo-2H-chromene-3-carboxylate (EOCC), a synthetic coumarin, irreversibly inhibits phospholipase A(2) (sPLA2) from Crotalus durissus ruruima venom (sPLA2r) with an IC(50) of 3.1 +/- 0.06 nmol. EOCC strongly decreased the V(max) and K(m), and it virtually abolished the enzyme activity of sPLA2r as well as sPLA2s from other sources. The edema induced by sPLA2r + EOCC was less than that induced by sPLA2r treated with p-bromophenacyl bromide, which was more efficient at neutralizing the platelet aggregation activity of native sPLA2r. Native sPLA2r induced platelet aggregation of 91.54 +/- 9.3%, and sPLA2r + EOCC induced a platelet aggregation of 18.56 +/- 6.5%. EOCC treatment also decreased the myotoxic effect of sPLA2r. Mass spectrometry showed that EOCC formed a stable complex with sPLA2r, which increased the mass of native sPLA2r from 14,299.34 Da to 14,736.22 Da. Moreover, the formation of this complex appeared to be involved in the loss of sPLA2r activity. Our results strongly suggest that EOCC can be used as a pharmacological agent against the sPLA2 in Crotalus durissus sp. venom as well as other sPLA2s.

Behavioural changes induced by angiotensin-converting enzyme inhibition during pregnancy and lactation in adult offspring rats.

1. The use of angiotensin-converting enzyme (ACE) inhibitors during pregnancy is contraindicated because of their association with increased risks of fetopathy, including central nervous systems malformations. In addition, some reports have shown that renin-angiotensin system components are expressed differently during embryonic development and adulthood in the rat. 2. Because angiotensin II and its derivative peptides have been implicated in anxiety and modulation of nociception, the aim of the present study was to investigate whether inhibiting ACE during prenatal and neonatal periods would alter behavioural plasticity in adult male offspring rats. 3. Female Wistar rats were treated with captopril (2 mg/mL water; approximately 200 mg/kg per day) during pregnancy and lactation. At adulthood, the offspring were subjected to the open field, elevated plus maze, social interaction, forced swimming and tail flick tests. 4. Perinatal captopril treatment significantly increased ambulation (33%; P < 0.05) and decreased resting time (37.5%; P < 0.05) in the open field test. Perinatal captopril treatment did not alter any of the behavioural parameters of the elevated plus maze; however, captopril treatment did cause a significant increase in social interaction (75.3%; P < 0.05). In the forced swimming test, there was an increased latency period (102.9%; P < 0.001) and a decreased immobility period (38.7, P < 0.05) in rats treated with perinatal captopril. In the tail flick test, perinatal captopril treatment significantly reduced the latency time (26.3%; P < 0.01). 5. The data show that ACE inhibition during prenatal and neonatal periods affects behavioural responses in adult offspring rats, suggesting that ACE is required for the development of neural systems that are associated with adult anxiety and nociceptive behavioural responses.

Enzymatic and structural characterization of new PLA2 isoform isolated from white venom of Crotalus durissus ruruima.

This work reports the structural and enzymatic characterization of a new sPLA2 from the white venom of Crotalus durissus ruruima, nominated PLA2A. The homogeneity of the PLA2A fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14,299.34Da. Structural investigation, through circular dichroism spectroscopy, revealed that PLA2A has a high content of alpha helix and beta-turn structures, 45.7% and 35.6% respectively. Its amino acid sequence, determined by Edman degradation and "de novo amino acid sequencing", exhibited high identity to PLA2 Cdt F15 from Crotalus durissus terrificus. The enzymatic investigation, conducted using the synthetic substrate 4-nitro-3-(octanoyloxy)-benzoic acid, determined its V(max) (7.56nmoles/min) and K(M) (2.76mM). Moreover, PLA2A showed an allosteric behavior and its enzymatic activity was dependent on Ca(2+). Intrinsic fluorescence measurements suggested that Ca(2+) induced a significant increase of PLA2A fluorescence, whereas its replacement for Mg(2+), Mn(2+), Sn(2+) and Cd(2+) apparently induced no structural modifications. The optimal pH and temperature for the enzymatic activity of PLA2A were 8.4 and 40 degrees C, respectively, and the minimal concentration of p-BPB and crotapotin that significantly inhibited such activity was 0.75mM and 0.4muM, respectively. In addition, PLA2A showed a significant antibacterial effect that was not strictly dependent on the enzymatic activity of such sPLA2.

Oestrogenic influence on brain AT1 receptor signalling on the thirst and sodium appetite in osmotically stimulated and sodium-depleted female rats.

The present work was carried out to investigate the role of angiotensin II type 1 (AT(1)) receptors in nocturnal thirst and sodium appetite induced by classical models of osmotic and sodium depletion challenges in ovariectomized rats chronically treated with oil or oestradiol benzoate (EB, 20 microg per animal, s.c. daily). In both conditions, the animals were given saline or losartan (108 nmol per animal, i.c.v.), a selective AT(1) receptor blocker. Oestrogen therapy significantly reduced the water intake induced by water deprivation, sodium depletion produced by frusemide injected 24 h before, and s.c. acute frusemide plus captopril injection (FUROCAP protocol), with no alteration following s.c. hypertonic saline injection. In contrast, EB therapy decreased the salt intake induced by sodium depletion and FUROCAP protocols, with no alteration following water deprivation and s.c. hypertonic saline injection. Central AT(1) blockade inhibited the dipsogenic response induced by water deprivation, osmotic stimulation, chronic sodium depletion and FUROCAP protocols and inhibited the natriorexigenic response induced by sodium depletion in ovariectomized rats. Oestrogen therapy significantly attenuated the losartan-induced antidipsogenic and antinatriorexigenic actions following sodium depletion and FUROCAP protocols. These results indicate that ovariectomized rats express increased AT(1) receptor signalling related to thirst and sodium appetite responses. Oestrogen therapy and brain AT(1) receptor blockade weakened or markedly decreased the behavioural responses during the nocturnal period, a time at which brain angiotensinergic activity is expected to be more prominent. Finally, we demonstrated through different experimental protocols a clear-cut influence of oestrogenic status on the behavioural AT(1)-induced signalling response.

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 PLA2, 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 microM/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 PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Extratos de Curcuma longa L. e Kalanchoe brasiliensis Camb. no tratamento local do envenenamento por Bothrops alternatus

Este trabalho teve como objetivo a utilização dos extratos aquosos das plantas Curcuma longa e Kalanchoe brasiliensis na terapêutica tópica complementar do envenenamento botrópico experimental em camundongos, visando a antagonização dos efeitos locais (edema, hemorragia e necrose) provocados pelo veneno. O experimento mostrou que os melhores resultados foram obtidos com o extrato de Kalanchoe brasiliensis.

In this contribution, an aqueous extract from rhizomes of Curcuma longa and an aqueous extract from the aerial parts of Kalanchoe brasiliensis were investigated for their properties against the local effects (edema, hemorrhage and necrosis) of Bothrops alternatus venom in mice. The experiment showed that the best results were obtained with Kalanchoe brasiliensis extract.

Dialysable nonspecific inhibitor of lymphocyte proliferation related to E-receptors (CD2).

Several investigators have pointed out that lymphocytic function may be profoundly affected by soluble factors occurring in human serum. It was observed, previously, that the addition of normal human serum dialysate (NHSD), at the beginning of lymphocyte cultures, inhibited the proliferative response to phytohemagglutinin (PHA) and to allogeneic cells. This suppressive effect was removed by previous absorption of NHSD with sheep erythrocytes (E). These data suggested that the dialysable fraction of human serum contains a suppressive factor, apparently related to the human T cell receptor for E (T11 or CD2). In this study we investigated at which phase of the proliferative response does the inhibition induced by NHSD take place. In order to confirm the relationship between this inhibitory factor and E-receptors, the NHSD was subjected to passage through a sepharose affinity column sensitized with an anti-E-receptor serum. This anti-E-receptor serum was obtained by immunizing a sheep with autologous E sensitized with human E-receptors. Time-course experiments showed that NHSD inhibited lymphocyte proliferation induced by PHA even when added to the cultures 18-24 h after mitogenic stimulation. In bidirectional mixed lymphocyte cultures the impairment of the proliferative response was inversely proportional to the time of NHSD addition. NHSD also inhibited the interleukin-2 (IL2) mediated proliferation of lymphoblasts previously exposed to PHA to ensure IL-2-receptors expression. The inhibitory effects of the NHSD were completely removed by absorption with E or by passing NHSD through the affinity column sensitized with the anti-E-receptor serum.(ABSTRACT TRUNCATED AT 250 WORDS)