Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

Differential vegetative and reproductive performances among fifteen guinea grass hybrids

The main scope of this work was to detect (Panicum maximum Jacq.) genotype differences as to morphoagronomic and seed quality indices, and to establish character correlations useful for determining vegetative and reproductive trends. Besides the flowering cycle, eight phenological and two seed quality traits were scored in a greenhouse randomized complete block experiment, as follows: plant height (PH), reproductive tiller number/overall tiller number (RTN/OTN), panicle number/reproductive tillers (PN/RT), leaf length (LL), leaf width (LW), panicle length (PL), fresh weight (FW), dry weight (DW), number of seeds/g (NS/G) and seed sample physical purity (SPP). Very-early and early-flowering hybrids consistently showed the highest correlation values among flowering cycle and RTN/OTN (r = -0.59**), PN/RT (r = -0.48**), NS/G (r = -0.88**) and SPP (r = -0.80**) (reproductive parameters) while intermediate and late-flowering hybrids presented the highest values for LL (r = 0.53**), LW (r = 0.60**), PL (r = 0.77**), FW (r = 0.78**) and DW (r = 0.85**) (vegetative traits). The implications of these results for plant breeding and forage management purposes are discussed.

Diversidad zoológica asociada a un silvopastoreo leucaena-guinea con diferentes edades de establecimiento

El objetivo de este trabajo fue evaluar la diversidad zoológica asociada a un silvopastoreo con leucaena-guinea, por medio de la caracterización de la composición y estructura de las aves, insectos y la macrofauna del suelo, en cuatro edades de establecimiento (3, 4, 5 y 6 años de explotación). Con las especies registradas en cada uno de estos grupos zoológicos, se calcularon los índices ecológicos: número de individuos, riqueza, diversidad y abundancia de especies, en diferentes edades del sistema. En todos los grupos, se apreció el aumento significativo en la riqueza de especies y en el índice de diversidad biológica de Shannon, en la medida que se desarrolló el sistema. Se observó incremento en la abundancia de insectos biorreguladores y, en relación con las aves, el horario de muestreo no mostró interacción con los distintos años de siembra. La macrofauna se incrementó, observándose dominancia de anélidos al 6º y 7º año de explotación, caracterizado por Polyferetrina elongata y Oligochaeta elegans. El desarrollo del silvopastoreo leucaena-guinea logra sistemas productivos pecuarios que aumentan la producción de biomasa y de otros componentes biológicos y contribuir para crear un sistema sostenible y compatible con el ambiente.

Chemical constituents from Bakeridesia pickelii Monteiro (Malvaceae) and the relaxant activity of kaempferol-3-O-beta-D-(6"-E-p -coumaroyl) glucopyranoside on guinea-pig ileum

The phytochemical investigation of Bakeridesia pickelii Monteiro led to the isolation of seven compounds: beta-sitosterol, a mixture of sitosteryl-3-O-beta-D-glucopyranoside and stigmasteryl-3-O-beta-D-glucopyranoside, vanillic acid, p-coumaric acid, quercetin 3-O-beta-D-glucopyranoside (isoquercitrin) and kaempferol-3-O-beta-D-(6"-E-p -coumaroyl) glucopyranoside (tiliroside), which was isolated as the major component. Their structures were elucidated on the basis of spectroscopic data such as IR, ¹H and 13C NMR, including two-dimensional techniques. Tiliroside relaxed the guinea-pig ileum pre-contracted with KCl 40 mM (EC50 = 9.5 ± 1.0 x 10-5 M), acetylcholine 10-6 M (EC50 = 2.3 ± 0.9 x 10-5 M) or histamine 10-6 M (EC50 = 4.1 ± 1.0 x 10-5 M) in a concentration-dependent manner.

Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.

Detection of virulence-associated genes of Pasteurella multocida isolated from cases of fowl cholera by multiplex-PCR

The current systems of breeding poultry, based on high population density, increase the risk of spreading pathogens, especially those causing respiratory diseases and those that have more than one host. Fowl Cholera (FC) is one such pathogen, and even though it represents one of several avian diseases that should be considered in the differential diagnosis of notifiable diseases that present with sudden death, the pathogenesis and virulence factors involved in FC are still poorly understood. The objective of this study was to investigate twelve genes related to virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. The protocols developed were capable of detecting all of the proposed genes. The ompH, oma87, sodC, hgbA, hgbB, exBD-tonB and nanB genes were present in 100% of the samples (25/25), the sodA and nanH genes were present in 96% (24/25), ptfA was present in 92% (23/25), and pfhA was present in 60% (15/25). Gene toxA was not identified in any of the samples studied (0/25). Five different genetic profiles were obtained, of which P1 (negative to toxA) was the most common. We concluded that the multiplex-PCR protocols could be useful tools for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and the fact that all samples belonged to the same subspecies of P. multocida, five genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Fowl adenovirus Group I as a causal agent of inclusion body hepatitis/hydropericardium syndrome (IBH/HPS) outbreak in brazilian broiler flocks

Commercial broiler flocks from a farm located in the State of São Paulo, Brazil, presented diarrhea, depression, increased mortality and poor weight gain. Upon post-mortem examination, classical signs of Inclusion Body Hepatitis/Hydropericardium Syndrome (IBH/HPS) were observed, including enlarged pale yellow-colored livers and straw-colored liquid in the pericardial sac. In addition, gross lesions were also observed in the kidneys, pancreas, thymus, intestines and gallbladder. Samples of these organs were analyzed by PCR for the detection of the hexon gene of the Fowl Adenovirus (FAdVs) Group I. The results were positive for both flocks (A and B) assayed by PCR. The macroscopic lesions associated with the detection of FAdV Group I by PCR in several of these affected organs allowed for the identification of IBH/HPS. In fact, this is the first report in Brazil of IBH/HPS in broilers, which identifies FAdVs group I as a causal agent of the disease. These findings may contribute to the worldwide epidemiology of the adenovirus-mediated hepatitis/hydropericardium syndrome.

Effects of piperitenone oxide on the intestinal smooth muscle of the guinea pig

We investigated the effects of piperitenone oxide (PO), a major constituent of the essential oil of Mentha x villosa, on the guinea pig ileum. PO (30 to 740 µg/ml) relaxed basal tonus without significantly altering the resting membrane potential. In addition, PO relaxed preparations precontracted with either 60 mM K+ or 5 mM tetraethylammonium in a concentration-dependent manner. At concentrations from 0.1 to 10 µg/ml PO potentiated acetylcholine-induced contractions, while higher concentrations (>30 µg/ml) blocked this response. These higher PO concentrations also inhibited contractions induced by 60 mM K+. PO also blocked the components of acetylcholine contraction which are not sensitive to nifedipine or to solutions with nominal zero Ca2+ and EGTA. These results show that PO is a relaxant of intestinal smooth muscle and suggest that this activity may be mediated at least in part by an intracellular effect

The effect of L-arginine on guinea-pig and rabbit airway smooth muscle function in vitro

We have investigated the effects of L-arginine, D-arginine and L-lysine on airway smooth muscle responsiveness to spasmogens in vitro. Both L-arginine and D-arginine (100 mM) significantly reduced the contractile potency and maximal contractile response to histamine but not to methacholine or potassium chloride in guinea-pig epithelium-denuded isolated trachea. Similarly, the contractile response to histamine was significantly reduced by L-arginine (100 mM) in rabbit epithelium-denuded isolated bronchus. The amino acid L-lysine (100 mM) failed to significantly alter the contractile potency of histamine in guinea-pig isolated trachea (P>0.05). In guinea-pig isolated trachea precontracted with histamine, both L-arginine and D-arginine produced a concentration-dependent relaxation which was not significantly altered by epithelium removal or by the presence of the nitric oxide synthase inhibitor, NG-nitro L-arginine methyl ester (L-NAME; 50 µM). Thus, at very high concentrations, arginine exhibit a non-competitive antagonism of histamine-induced contraction of isolated airway preparations that was independent of the generation of nitric oxide and was not dependent on charge. These observations confirm previous studies of cutaneous permeability responses and of contractile responses of guinea-pig isolated ileal smooth muscle. Taken together, the data suggest that high concentrations of arginine can exert an anti-histamine effect.

Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM) to a single, non-interacting (nH = 1.1), low capacity (Bmax 1.1 pmol/mg protein) binding site. In competition experiments using several compounds (including ion channel ligands), only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively). PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB) and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits ³H-choline uptake in guinea pig ileum

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of ³H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM) and up to 10 min of treatment, crotoxin decreased ³H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on ³H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits ³H-choline uptake with high affinity (EC25 = 10 ± 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Endogenous opiate analgesia induced by tonic immobility in guinea pigs

A function of the endogenous analgesic system is to prevent recuperative behaviors generated by tissue damage, thus preventing the emission of species-specific defensive behaviors. Activation of intrinsic nociception is fundamental for the maintenance of the behavioral strategy adopted. Tonic immobility (TI) is an inborn defensive behavior characterized by a temporary state of profound and reversible motor inhibition elicited by some forms of physical restraint. We studied the effect of TI behavior on nociception produced by the formalin and hot-plate tests in guinea pigs. The induction of TI produced a significant decrease in the number of flinches (18 ± 6 and 2 ± 1 in phases 1 and 2) and lickings (6 ± 2 and 1 ± 1 in phases 1 and 2) in the formalin test when compared with control (75 ± 13 and 22 ± 6 flinches in phases 1 and 2; 28 ± 7 and 17 ± 7 lickings in phases 1 and 2). In the hot-plate test our results also showed antinociceptive effects of TI, with an increase in the index of analgesia 30 and 45 min after the induction of TI (0.67 ± 0.1 and 0.53 ± 0.13, respectively) when compared with control (-0.10 ± 0.08 at 30 min and -0.09 ± 0.09 at 45 min). These effects were reversed by pretreatment with naloxone (1 mg/kg, ip), suggesting that the hypoalgesia observed after induction of TI behavior, as evaluated by the algesimetric formalin and hot-plate tests, is due to activation of endogenous analgesic mechanisms involving opioid synapses.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10) or 20 µM and Emax of 92% (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6%) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 µM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM) or ODQ (1 µM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM), a VIP receptor antagonist, significantly inhibited (37 ± 7%) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 µM), a CGRP antagonist, only slightly enhanced the action of gentisic acid. Taken together, these results provide functional evidence for the direct activation of voltage and large-conductance Ca+2-activated K+ channels, or indirect modulation of potassium channels induced by VIP receptors and accounts for the predominant relaxation response caused by gentisic acid in the guinea pig trachea.

Short-term evaluation of non-absorbable microgranular hydroxyapatite infiltration in the guinea pig subepidermal abdominal region

Non-absorbable microgranular hydroxyapatite was infiltrated into the subepidermal abdominal region of guinea pigs in order to assess the possibility of using this material to correct deficiencies in orbital volume. Microgranular hydroxyapatite (2.0 ml) was subepidermally infiltrated into the abdominal region of 20 guinea pigs. The animals were divided into four experimental groups of 5 animals each, which were killed 7 (G1), 15 (G2), 30 (G3) and 60 (G4) days after infiltration. The area and the largest and smallest diameters of the nodules formed by infiltration were evaluated at the site of infiltration and histological examination was performed. The mean granuloma area was similar in all groups. Histopathological examination showed that the material remained isolated from surrounding tissues by a pseudocapsule that became denser throughout the experiment. A host reaction started with young fibroblastic tissue that evolved to dense tissue until cartilaginous tissue was formed in G4, progressively advancing towards the center of the granuloma from G1 to G4. Non-absorbable microgranular hydroxyapatite is an inert material that was well tolerated by the animals studied, with maintenance of the infiltrated volume, and may perhaps be useful to fill anophthalmic cavities.

Inotropic effects of extracts of Psidium guajava L. (guava) leaves on the guinea pig atrium

Many pharmacological effects have been ascribed to extracts of Psidium guajava L. (guava) leaves. However, in spite of its widespread use in Brazilian folk medicine and a reasonable number of scientific reports about it, we could not find any study dealing with its action on the mammalian myocardium. In the present study, by measuring isometric force, we observed that the crude extract of P. guajava (water-alcohol extract obtained by macerating dry leaves) depresses the guinea pig atrial contractility in a concentration-dependent fashion (N = 8 hearts, 15 trials). The compound with cardiac activity was concentrated by extraction in a Soxhlet apparatus using 17 M glacial acetic acid after removing the less polar fractions (hexane, chloroform, acetone, ethanol and methanol), suggesting that this compound is a highly polar substance. In the isolated guinea pig left atrium the acetic acid fraction (10-800 mg/l) of P. guajava 1) reversibly decreased myocardial force in a concentration-dependent fashion (EC50 = 0.07g/l, N = 5 hearts, 9 trials, P<0.05), 2) increased the atrial relaxation time measured at 20% of the force amplitude up to 35% (91 ± 15 to 123 ± 30 ms, N = 3 hearts, 6 trials, P<0.05), 3) abolished the positive staircase effect (Bowditch phenomenon) in a concentration-dependent fashion suggesting a decrease of the cellular inward calcium current (N = 4 hearts, 8 trials, P<0.05), and 4) its inotropic effect was abolished by cholinergic receptor blockade with 1.5 mM atropine sulfate, indicating a cholinergic involvement in the mechanism of action of the extract (N = 7 hearts, 15 trials, P<0.05). The acetic acid extract was 20 times more potent than crude extract (EC50 = 1.4 g/l). The results showed that extracts from P. guajava leaves depress myocardial inotropism.