Anti-tumor properties of blackseed (Nigella sativa L.) extracts

The objective of the present study was to evaluate the in vitro and in vivo anti-cancer effect of Nigella sativa L. seed extracts. The essential oil (IC50 = 0.6%, v/v) and ethyl acetate (IC50 = 0.75%) extracts were more cytotoxic against the P815 cell line than the butanol extract (IC50 = 2%). Similar results were obtained with the Vero cell line. Although all extracts had a comparable cytotoxic effect against the ICO1 cell line, with IC50 values ranging from 0.2 to 0.26% (v/v), tests on the BSR cell line revealed a high cytotoxic effect of the ethyl acetate extract (IC50 = 0.2%) compared to the essential oil (IC50 = 1.2%). These data show that the cytotoxicity of each extract depends on the tumor cell type. In vivo, using the DBA2/P815 (H2d) mouse model, our results clearly showed that the injection of the essential oil into the tumor site significantly inhibited solid tumor development. Indeed, on the 30th day of treatment, the tumor volume of the control animals was 2.5 ± 0.6 cm³, whereas the tumor volumes of the essential oil-treated animals were 0.22 ± 0.1 and 0.16 ± 0.1 cm³ when the animals were injected with 30 µL (28.5 mg)/mouse and 50 µL (47.5 mg)/mouse per 48 h (six times), respectively. Interestingly, the administration of the essential oil into the tumor site inhibited the incidence of liver metastasis development and improved mouse survival.

Lesions of the commissural subnucleus of the nucleus of the solitary tract increase isoproterenol-induced water intake

The nucleus of the solitary tract (NTS) is the primary site of the cardiovascular afferent information about arterial blood pressure and volume. The NTS projects to areas in the central nervous system involved in cardiovascular regulation and hydroelectrolyte balance, such as the anteroventral third ventricle region and the lateral parabrachial nucleus. The aim of the present study was to investigate the effects of electrolytic lesion of the commissural NTS on water and 0.3 M NaCl intake and the cardiovascular responses to subcutaneous injection of isoproterenol. Male Holtzman rats weighing 280 to 320 g were submitted to sham lesion or electrolytic lesion of the commissural NTS (N = 6-15/group). The sham-lesioned rats had the electrode placed along the same coordinates, except that no current was passed. Water intake induced by subcutaneous isoproterenol (30 µg/kg body weight) significantly increased in chronic (15 days) commissural NTS-lesioned rats (to 2.4 ± 0.2 vs sham: 1.9 ± 0.2 mL 100 g body weight-1 60 min-1). Isoproterenol did not induce any sodium intake in sham or in commissural NTS-lesioned rats. The isoproterenol-induced hypotension (sham: -27 ± 4 vs commissural NTS-lesioned rats: -22 ± 4 mmHg/20 min) and tachycardia (sham: 168 ± 10 vs commissural NTS: 144 ± 24 bpm/20 min) were not different between groups. The present results suggest that the commissural NTS is part of an inhibitory neural pathway involved in the control of water intake induced by subcutaneous isoproterenol, and that the overdrinking observed in lesioned rats is not the result of a cardiovascular imbalance in these animals.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Cardiovascular effects of the intracerebroventricular injection of adrenomedullin: roles of the peripheral vasopressin and central cholinergic systems

Our objective was to investigate in conscious Sprague-Dawley (6-8 weeks, 250-300 g) female rats (N = 7 in each group) the effects of intracerebroventricularly (icv) injected adrenomedullin (ADM) on blood pressure and heart rate (HR), and to determine if ADM and calcitonin gene-related peptide (CGRP) receptors, peripheral V1 receptors or the central cholinergic system play roles in these cardiovascular effects. Blood pressure and HR were observed before and for 30 min following drug injections. The following results were obtained: 1) icv ADM (750 ng/10 µL) caused an increase in both blood pressure and HR (DMAP = 11.8 ± 2.3 mmHg and ΔHR = 39.7 ± 4.8 bpm). 2) Pretreatment with a CGRP receptor antagonist (CGRP8-37) and ADM receptor antagonist (ADM22-52) blocked the effect of central ADM on blood pressure and HR. 3) The nicotinic receptor antagonist mecamylamine (25 µg/10 µL, icv) and the muscarinic receptor antagonist atropine (5 µg/10 µL, icv) prevented the stimulating effect of ADM on blood pressure. The effect of ADM on HR was blocked only by atropine (5 µg/10 µL, icv). 4) The V1 receptor antagonist [β-mercapto-β-β-cyclopentamethylenepropionyl¹, O-me-Tyr²,Arg8]-vasopressin (V2255; 10 µg/kg), that was applied intravenously, prevented the effect of ADM on blood pressure and HR. This is the first study reporting the role of specific ADM and CGRP receptors, especially the role of nicotinic and muscarinic central cholinergic receptors and the role of peripheral V1 receptors in the increasing effects of icv ADM on blood pressure and HR.

Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female reproduction

Utilização do Intron Splice Site primer EI-1 na discriminação de leveduras contaminantes do processo de fermentação alcoólica

Neste trabalho, utilizamos o Intron Splice Site primer EI-1 para a análise do perfil de amplificação de diferentes espécies de leveduras consideradas contaminantes no processo de fermentação alcoólica, originadas de uma destilaria no Estado da Paraíba na safra 2004/2005. Foram realizadas as etapas analíticas para discriminação molecular das leveduras a partir da extração do DNA total, amplificação por PCR e análise do perfil genético. Os resultados obtidos indicam que o Intron Splice Site primer EI-1é muito eficaz na discriminação das diferentes espécies de Saccharomyces e não Saccharomyces, evidenciando padrões de bandas específicos para as leveduras analisadas. Este primer, por ser complementar a uma região muito conservada do genoma das leveduras, mostrou-se incapaz de uma discriminação intraespecífica. Isto demonstra a utilidade deste marcador no auxílio à taxonomia de leveduras.