Preparation of mitotic chromosomes of leaf-cutting ants from the genera Atta and Acromyrmex

Some modifications were made to the methodology of Imai et al. (Jpn. J. Genet. 63: 159-185, 1988) for cytogenetic analysis of the leaf-cutting ants Atta sexdens piriventris and Acromyrmex heyeri (Hymenoptera, Formicidae), shortening preparation time and improving chromosomal preparations. The brain ganglia of prepupae were dissected in a 0.0025% hypotonic solution of colchicine, placed on a glass slide on a cold plate (4 ± 1oC) for 20 min. The material was fixed directly on the cold slide (with cold fixative I), macerated with a histological needle and fixed again with fixative I, followed by fixatives II and III, all of them cold. The slide was flame-dried right after the use of fixative III, and it was allowed to air-dry at room temperature for 2 h. The resulting metaphases presented less contracted chromosomes, with separated and well defined sister chromatids at a high frequency, when the material was processed in the manner described and stained with 3% Giemsa in phosphate buffer (pH 6.8) for 15 min.

Central lead administration inhibits water intake and sodium appetite in rats

We have demonstrated that acute third ventricle injections of lead acetate (PbAc) exert a powerful antidipsogenic effect and induce a significant increase in renal sodium excretion. In the present study we confirm the antidipsogenic effect of lead and demonstrate that central administration of this metal, in minute amounts, significantly reduces salt intake both during dehydration and after central angiotensinergic stimulation. Adult male Wistar rats had the third ventricle cannulated seven days before the experiments. During this period they had free access to distilled water and hypertonic saline solution (1.5%). After a 24-h period of fluid deprivation, experimental animals received third ventricle injections of PbAc (0.3, N = 8 and 3.0 nmol/rat, N = 14) while controls received sodium acetate (NaAc; 3.0 nmol/rat, N = 10). Rats treated with PbAc at the highest dose showed a significant reduction (P<0.05) both in water and hypertonic saline intake when compared to controls. When the effect of lead administration on angiotensin II-induced water and salt intake was studied, normohydrated animals received third ventricle injections of angiotensin II (9.6 nmol/rat) after pretreatment with 3.0 nmol/rat of PbAc (experimental group, N = 10) or NaAc (controls, N = 8). The group pretreated with PbAc presented a significant reduction (P<0.05) in both water and salt intake compared to controls. Thus, this study confirms the antidipsogenic effect of central lead injections and demonstrates that the presence of lead in the brain exerts a significant inhibition of sodium appetite.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 µM) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 µM), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 µM) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 ± 0.8 µM and k cat = 48.4 ± 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 ± 10, 1,098 ± 91, 38.6 ± 5.2 and 37,340 ± 5,400 µM, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 ± 92.8 and 310,500 ± 38,600 µM, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds.

Effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices

It has been suggested that glucocorticoids released during stress might impair neuronal function by decreasing glucose uptake by hippocampal neurons. Previous work has demonstrated that glucose uptake is reduced in hippocampal and cerebral cortex slices 24 h after exposure to acute stress, while no effect was observed after repeated stress. Here, we report the effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices and on plasma glucose and corticosterone levels. Male adult Wistar rats were exposed to restraint 1 h/day for 50 days in the chronic model. In the acute model there was a single exposure. Immediately or 24 h after stress, the animals were sacrificed and the hippocampus and cerebral cortex were dissected, sliced, and incubated with Krebs buffer, pH 7.4, containing 5 mM glucose and 0.2 µCi D-[U-14C] glucose. CO2 production from glucose was estimated. Trunk blood was also collected, and both corticosterone and glucose were measured. The results showed that corticosterone levels after exposure to acute restraint were increased, but the increase was smaller when the animals were submitted to repeated stress. Blood glucose levels increased after both acute and repeated stress. However, glucose utilization, measured as CO2 production in hippocampal and cerebral cortex slices, was the same in stressed and control groups under conditions of both acute and chronic stress. We conclude that, although stress may induce a decrease in glucose uptake, this effect is not sufficient to affect the energy metabolism of these cells.

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

SDS, C12E8, CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C12E8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C12E8/ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K0.5 = 4.0 µM) and low (K0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C12E8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C12E8 can be purified and retains its activity. The activity is consistent with the form of (alphaß)2 association.

Low pH and calcium effects on net Na+ and K+ fluxes in two catfish species from the Amazon River (Corydoras: Callichthyidae)

The present study analyzes Na+ and K+ disturbances caused by low pH in two catfish species from the Amazon River. Corydoras adolfoi inhabits ion-poor, black-stained, low pH (3.5-4.0) waters, while C. schwartzi is native to ion-rich waters at circumneutral pH. Fish were exposed to pH 3.5 Ca2+-free, and Ca2+-enriched (~500 µmol/l) water to determine the protective effects of calcium. Net Na+ and K+ fluxes were measured in the water collected from the fish experimental chambers. C. adolfoi was unable to control the Na+ efflux at low pH, exhibiting Na+ loss up to -594 ± 84 nmol g-1 h-1 during the first hour. After 3 and 6 h, net Na+ flux increased by 7- and 23-fold, respectively. In C. schwartzi, at pH 3.5, the initial high Na+ loss (-1,063 ± 73 nmol g-1 h-1) was gradually attenuated. A K+ loss occurred in both species, but remained relatively constant throughout exposure. High [Ca2+] affected ion losses in both species. C. adolfoi had 70% loss attenuation, indicating incapacity to control Na+ efflux. In C. schwartzi, elevated [Ca2+] completely prevented the Na+ losses caused by exposure to low pH. Rather different patterns were seen for K+ fluxes, with C. adolfoi showing no K+ disruption when exposed to low pH/high [Ca2+]. Thus, C. adolfoi loses Na+ during acid exposure, but has the ability to control K+ loss, while C. schwartzi controls diffusive Na+ loss but exhibits a slightly higher K+ loss. Ion balance was influenced by [Ca2+] at low pH in C. schwartzi but not in C. adolfoi.

The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein

Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 µg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37ºC. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.

Effect of electrolytic lesion of the dorsal raphe nucleus on water intake and sodium appetite

The present study determined the effect of an electrolytic lesion of the dorsal raphe nucleus (DRN) on water intake and sodium appetite. Male Wistar rats weighing 290-320 g with a lesion of the DRN (L-DRN), performed two days before experiments and confirmed by histology at the end of the experiments, presented increased sensitivity to the dehydration induced by fluid deprivation. The cumulative water intake of L-DRN rats reached 23.3 ± 1.9 ml (a 79% increase, N = 9) while sham-lesioned rats (SL-DRN) did not exceed 13.0 ± 1.0 ml (N = 11, P < 0.0001) after 5 h. The L-DRN rats treated with isoproterenol (300 µg kg-1 ml-1, sc) exhibited an increase in water intake that persisted throughout the experimental period (L-DRN, 15.7 ± 1.47 ml, N = 9 vs SL-DRN, 9.3 ± 1.8 ml, N = 11, P < 0.05). The L-DRN rats also showed an increased spontaneous sodium appetite during the entire period of assessment. The intake of 0.3 M NaCl after 12, 24, 36 and 72 h by the L-DRN rats was always higher than 20.2 ± 4.45 ml (N = 10), while the intake by SL-DRN was always lower than 2.45 ± 0.86 ml (N = 10, P < 0.00001). Sodium- and water-depleted L-DRN rats also exhibited an increased sodium appetite (13.9 ± 2.0 ml, N = 11) compared to SL-DRN (4.6 ± 0.64 ml, N = 11) after 120 min of observation (P < 0.02). The sodium preference of L-DRN rats in both conditions was always higher than that of SL-DRN rats. These results suggest that electrolytic lesion of the DRN overcomes a tonic inhibitory component of sodium appetite.

Characterization of rat heart alkaline phosphatase isoenzymes and modulation of activity

Alkaline phosphatase (ALP) is important in calcification and its expression seems to be associated with the inflammatory process. We investigated the in vitro acute effects of compounds used for the prevention or treatment of cardiovascular diseases on total ALP activity from male Wistar rat heart homogenate. ALP activity was determined by quantifying, at 410 nm, the p-nitrophenol released from p-nitrophenylphosphate (substrate in Tris buffer, pH 10.4). Using specific inhibitors of ALP activity and the reverse transcription-polymerase chain reaction, we showed that the rat heart had high ALP activity (31.73 ± 3.43 nmol p-nitrophenol·mg protein-1·min-1): mainly tissue-nonspecific ALP but also tissue-specific intestinal ALP type II. Both ALP isoenzymes presented myocardial localization (striated pattern) by immunofluorescence. ALP was inhibited a) strongly by 0.5 mM levamisole, 2 mM theophylline and 2 mM aspirin (91, 77 and 84%, respectively) and b) less strongly by 2 mM L-phenylalanine, 100 mL polyphenol-rich beverages and 0.5 mM progesterone (24, 21 to 29 and 11%, respectively). β-estradiol and caffeine (0.5 and 2 mM) had no effect; 0.5 mM simvastatin and 2 mM atenolol activated ALP (32 and 36%, respectively). Propranolol (2 mM) tended to activate ALP activity and corticosterone activated (18%) and inhibited (13%) (0.5 and 2 mM, respectively). We report, for the first time, that the rat heart expresses intestinal ALP type II and has high total ALP activity. ALP activity was inhibited by compounds used in the prevention of cardiovascular pathology. ALP manipulation in vivo may constitute an additional target for intervention in cardiovascular diseases.

PROPRIEDADES EMULSIFICANTES E SOLUBILIDADE DA CASEÍNA BOVINA E DE SEUS HIDROLISADOS TRÍPTICOS: 1. EFEITO DO PH E DO TEMPO DE HIDRÓLISE

Visando a aplicação industrial da caseína e de seus hidrolisados trípticos, foram estudados os efeitos da variação do pH e do tempo de hidrólise sobre suas características de solubilidade e propriedades emulsificantes. Testou-se os valores de pH de 3,0; 4,0; 5,0; 6,0; 7,0 e 8,0 e os tempos de hidrólise: 5, 10 15, 30 e 60min. Foram medidos a solubilidade, a capacidade emulsificante, o índice de atividade emulsificante, a estabilidade da emulsão, e calculdado o tamanho dos glóbulos de gordura. Os resultados obtidos para a caseína nativa indicaram que os melhores valores para estas propriedades funcionais foram encontrados em pH acima de 5,0. A hidrólise tríptica da caseína foi benéfica para sua solubilidade e capacidade emulsificante e prejudicou sua estabilidade, em todos os valores de pH e tempos de hidrólise, exceto no pH 5,0 com 5 min de reação. Por outro lado, este tratamento enzimático contribuiu para melhorar o índice de atividade emulsificante da caseína, entre valores de pH 3,0 e 5,0 e após 10 min de reação.

SENSORY ANALYSIS OF A MODEL SYSTEM USING 5'-IMP AND CYSTEINE AT DIFFERENT pH

Sensory analysis was used to get an overall flavour description of a reaction mixtures containing 5'-IMP and Cysteine. Ribose/cysteine systems were used as reference systems. Results from triangle and aroma profiling show a clear correlation between the terms used and the volatile analysis described in literature for these model systems. For instance reactions at pH 3.0 and 4.5 for 5'-IMP/cysteine systems, which were described as "meaty" and "boiled meat" by panellists, presented, in the literature, the higher number of "meaty" compounds in volatile analysis (1, 7, 8, 20) .

Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Cultivo in vitro de plântulas de orquídea em meios com diferentes concentrações de fertilizante mineral

Existem diversos meios nutritivos utilizados no cultivo in vitro de orquídeas, com diferentes composições e concentrações de sais, resultando em respostas distintas entre eles. O objetivo deste trabalho foi avaliar o crescimento in vitro de plântulas de orquídea (Cattleya walkeriana Gardner) sob diferentes doses de fertilizante NPK, adicionado ao meio de cultivo, como fonte de nutrientes. Foi utilizado o fertilizante Peters® na formulação NPK 10-30-20 + Mg + micronutrientes, nas doses de: 0,0; 1,0; 2,0; 3,0; 4,0; 5,0 e 10,0 g L-1 de meio, acrescido de água de coco (150 mL L-1), sacarose (20 g L-1), carvão ativado (2 g L-1) e pH corrigido para 5,6. Foram utilizados frascos de vidro de 320 mL de capacidade, contendo 35 mL de meio onde foram inoculadas dez plântulas com seis meses de idade, previamente germinadas em meio Knudson C. O experimento foi mantido em sala de crescimento a 27 ± 2 ºC, 16/8 h luz/escuro e irradiância de 48 µmol m-2 s-1. A produção máxima de matéria seca da parte aérea das plantas, aos seis meses de idade, foi estimada em 0,255 g/frasco com uso de 5,22 g L-1 do fertilizante adicionado ao meio de cultura. Considerando a possibilidade de salinidade elevada do meio, para algumas espécies de orquídea, estimou-se a dose de fertilizante (3,55 g L-1) necessária para obtenção de 90% da produção máxima. As raízes apresentaram drástica redução do crescimento (menor número e curtas) em condições de alta concentração de fertilizante.

Conservação de grãos de pólen de mamoneira a baixas temperaturas

A finalidade do armazenamento do grão de pólen é conservar material para futura utilização, proporcionando-lhe condições ótimas, de forma a manter seu poder germinativo, vigor e integridade genética originais. O objetivo deste trabalho foi verificar o poder germinativo dos grãos de pólen de mamoneira (Ricinus communis L.), após armazenamento a baixas temperaturas. Para tanto, grãos de pólen dos cultivares IAC 80 e AL Guarany 2002 foram utilizados e armazenados em quatro temperaturas: -196, -72, -18 e 4 ºC, durante um período de oito semanas. A avaliação da viabilidade deste pólen foi realizada semanalmente, até completar cinco semanas, sendo realizada uma última avaliação após a oitava semana de armazenamento, por meio do teste de germinação in vitro. Meio de cultura, contendo 10 gL-1 de ágar, 100 gL-1 de sacarose, 0,004 gL-1 de ácido bórico e pH 6,0, foi preparado e depositado em placas de lâmina escavada, onde o pólen foi distribuído sobre a superfície do meio. As placas foram incubadas a 20 ºC em BOD. O delineamento experimental foi inteiramente casualizado, em arranjo fatorial 2 x 4 (2 cultivares x 4 temperaturas) para cada tempo de armazenamento, sendo analisados 100 grãos de pólen por repetição, totalizando seis de cada tratamento. Verificou-se diferença significativa entre os tratamentos para a interação cultivar x temperatura na quinta semana de observação (p < 0,05). Demais interações foram altamente significativas em todas as épocas de observação (p < 0,01). Portanto, a utilização de ultrabaixas temperaturas permite a manutenção da viabilidade de grãos de pólen de mamoneira, até a quinta semana de armazenamento.