Effects of leucine supplemented diet on intestinal absorption in tumor bearing pregnant rats

Background: It is known that amino acid oxidation is increased in tumor-bearing rat muscles and that leucine is an important ketogenic amino acid that provides energy to the skeletal muscle.Methods: To evaluate the effects of a leucine supplemented diet on the intestinal absorption alterations produced by Walker 256, growing pregnant rats were distributed into six groups. Three pregnant groups received a normal protein diet (18% protein):pregnant (N), tumor-bearing (WN), pair-fed rats (Np). Three other pregnant groups were fed a diet supplemented with 3% leucine (15% protein plus 3% leucine):leucine (L), tumor-bearing (WL) and pair-fed with leucine (Lp). Non pregnant rats (C), which received a normal protein diet, were used as a control group. After 20 days, the animals were submitted to intestinal perfusion to measure leucine, methionine and glucose absorption.Results: Tumor-bearing pregnant rats showed impairment in food intake, body weight gain and muscle protein content, which were less accentuated in WL than in WN rats. These metabolic changes led to reduction in both fetal and tumor development. Leucine absorption slightly increased in WN group. In spite of having a significant decrease in leucine and methionine absorption compared to L, the WL group has shown a higher absorption rate of methionine than WN group, probably due to the ingestion of the leucine supplemented diet inducing this amino acid uptake. Glucose absorption was reduced in both tumor-bearing groups.Conclusions: Leucine supplementation during pregnancy in tumor-bearing rats promoted high leucine absorption, increasing the availability of the amino acid for neoplasic cells and, mainly, for fetus and host utilization. This may have contributed to the better preservation of body weight gain, food intake and muscle protein observed in the supplemented rats in relation to the non-supplemented ones.

Purification and biological activity of the thrombin-like substance isolated from Bothrops insularis venom

The venom of Bothrops insidaris snake, known in Brazil as jararaca ilhoa, contains a variety of proteolytic enzymes such as a thrombin-like substance that is responsible for various pharmacological effects. B. insularis venom chromatography profile showed an elution of seven main fractions. The thrombin-like activity was detected in fractions I and 111, the latter being subjected to two other chromatographic procedures, so to say DEAE and Hi Trap Benzamidine. The purity degree of this fraction was confirmed by analytical reverse phase HPLC, which displayed only one main fraction confirmed by SDS-PAGE constituting fraction III. About 5 mu g of fraction III protein potentiated the secretion of insulin induced by 2.8mM of glucose in rats isolated pancreatic beta-cells treated; the increase being around 3-fold higher than its respective control. B. insidaris lectin (BiLec; 10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. BiLec increased perfusion pressure (PP), renal vascular resistence (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, the thrombin-like substance isolated from B. insularis venom induced an increase in insulin secretion, in vitro, and transiently altered vascular, glomerular and tubular parameters in the isolated rat kidney. (c) 2006 Elsevier Ltd. All rights reserved.

Effect of ion reduction of sodium, calcium and potassium on spontaneous contraction of the esophagus of Pomacea lineata SPIX, 1827 (Mollusca-Gastropoda-Prosobranchia)

The invertebrate's musculature still presents many elusive points, especially in molluscs that generally present smooth and cracked fibers with peculiar characteristics. It was found that the molluscs reactions to the ion variation in the bathing are not very clear, mainly in view of the isolated reduction or equivalent of the ions. Suspended in bath, the isolated esophagus of the P. lineata exhibited spontaneous activity. This rhythmic activity was sensitive to the ion variation of the perfusion liquid, evidenced by alterations in the spontaneous contractions. The equivalent reduction of the ion reduced the spontaneous activity, evidenced by the amplitude reduction of the response, besides maintaning an organ contraction, primarily in the reductions below 50%. When the isolated reductions of the Na, Ca or K ion were performed, occurred interference in the spontaneous contractions of the organs, principally in amplitude of the response and maintenance of the contracture in reductions of 50 and 25% of the ion.

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

The antiepileptic activity of JSTX-3 is mediated by N-methyl-D-aspartate receptors in human hippocampal neurons

We analyzed the effect of the acylpolyaminetoxin JSTX-3 on the epileptogenic discharges induced by perfusion of human hippocampal slices with artificial cerebrospinal fluid lacking Mg2+ or N-methyl-D-aspartate. Hippocampi were surgically removed from patients with refractory medial temporal lobe epilepsy, sliced in the surgical room and taken to the laboratory immersed in normal artificial cerebrospinal fluid. Epileptiform activity was induced by perfusion with Mg2+-free artificial cerebrospinal fluid or by iontophoretically applied N-methyl-D-aspartate and intracellular and field recordings of CAI neurons were performed. The ictal-like discharges induced by Mg2+-free artificial cerebrospinal fluid and N-methyl-D-aspartate were blocked by incubation with JSTX-3. This effect was similar to that obtained with the N-methyl-D-aspartate receptor antagonist DL(-)2-amino-5 phosphonovaleric acid. Our findings suggest that in human hippocampal neurons, the antiepileptic effect of JSTX-3 is mediated by its action on N-methyl-D-aspartate receptor.

Renal artery clipping attenuates the progression of adriamycin nephropathy

This study was designed to analyze the impact of diminished renal perfusion pressure due to renal clipping on the rat model of adriamycin nephropathy. Male Wistar rats, divided into four groups (n = 9 per group) were injected with saline as control (C), adriamycin 3 ml/kg (Ad), saline with the left renal artery clipped (Rv), and adriamycin plus clip (AdRv). After 24 weeks mean arterial pressure (MAP), inulin, and p-aminohippurate (PAH) clearances were performed to evaluate renal function. Morphologic analysis included histologic criteria of percentage of glomerulosclerosis and tubulointerstitial lesion index (TILI). The MAP (mm Hg) was similar between Rv (143 +/- 13) and AdRv (154 +/- 20), but higher (P < .05) than C (120 +/- 8) and Ad (124 +/- 11). Inulin clearance (mL/min/100 g) in Ad (0.2 +/- 0.05) was smaller than in C (0.53 +/- 0.17) and Rv (0.4 +/- 0.01) (P < .05), and was at an intermediate level in AdRv (0.33 +/- 0.2). The level of PAH (mL/min/100 g) was normal at 1.76 in C, and diminished more in Ad (0.58) than in Rv (1.06) and AdRv (1.18) (P < .05). Both Ad and the AdRv nonclipped kidneys had the highest degree of glomerulosclerosis (33% and 25%) and TILI (7% and 8%), respectively, compared with C and Rv (both 0%), whereas the clipped kidneys displayed intermediate degrees (9% and 5%) (P < .05 v nonclipped). The data suggest that diminished perfusion pressure of the clipped kidney, by decreasing the intraglomerular pressure, protects the glomerulus from damage and attenuates the evolution of adriamycin nephropathy. Am J Hypertens 1998;11:1124-1128 (C) 1998 American Journal of Hypertension, Ltd.

Hypoxic pulmonary vasoconstriction in reptiles: a comparative study of four species with different lung structures and pulmonary blood pressures

Low O-2 levels in the lungs of birds and mammals cause constriction of the pulmonary vasculature that elevates resistance to pulmonary blood flow and increases pulmonary blood pressure. This hypoxic pulmonary vasoconstriction (HPV) diverts pulmonary blood flow from poorly ventilated and hypoxic areas of the lung to more well-ventilated parts and is considered important for the local matching of ventilation to blood perfusion. In the present study, the effects of acute hypoxia on pulmonary and systemic blood flows and pressures were measured in four species of anesthetized reptiles with diverse lung structures and heart morphologies: varanid lizards (Varanus exanthematicus), caimans (Caiman latirostris), rattlesnakes (Crotalus durissus), and tegu lizards (Tupinambis merianae). As previously shown in turtles, hypoxia causes a reversible constriction of the pulmonary vasculature in varanids and caimans, decreasing pulmonary vascular conductance by 37 and 31%, respectively. These three species possess complex multicameral lungs, and it is likely that HPV would aid to secure ventilation-perfusion homogeneity. There was no HPV in rattlesnakes, which have structurally simple lungs where local ventilation-perfusion inhomogeneities are less likely to occur. However, tegu lizards, which also have simple unicameral lungs, did exhibit HPV, decreasing pulmonary vascular conductance by 32%, albeit at a lower threshold than varanids and caimans (6.2 kPa oxygen in inspired air vs. 8.2 and 13.9 kPa, respectively). Although these observations suggest that HPV is more pronounced in species with complex lungs and functionally divided hearts, it is also clear that other components are involved.

Effects of Antibacterial Agents on Dental Pulps of Monkeys Mechanically Exposed and Contaminated.

The purpose of this study was to compare the effectiveness of antibacterial agents and mineral trioxide aggregate in the healing of bacterial contaminated primate pulps. Study Design: The experiment required four adult male primates (Cebus opella) with 48 teeth prepared with buccal penetrartions into the pulpal tissues. The preparations (Cebus opella) with 48 teeth prepared with buccal penetrations into the exposed to cotton pellets soaked in a bacterial mixture consisting of microorganisms normally found in human pulpal abscesses obtained from the Endodontic Clinic of UNESP. Following bacterial inoculation (30 minute exposure), the pulpal tissue was immediately treated with either sterile saline, Cipro HC Otic solution (12), diluted Buckley formecresol solution (12) or Otosporin otic solution (12) for 5 minutes. After removal of the pellet, hemostasis was obtained and a ZOE base applied to the DFC treated pulps and the non-treated controls (12). After hemostasis, the other exposed pulps were covered with mineral trioxide aggregate (ProRoot). The pulpal bases were all covered with a RMGI (Fuji II LC). The tissue samples were collected at one day, two days, one week and over four weeks (34 days). Results: Following perfusion fixation, the samples were demineralized, sectioned, stained and histologically graded. After histologic analysis, presence of neutrophilic infiltrate and areas of hemorrhage with hyperemia were observed . The depth of the neutrophilic infiltrate depended on the agent or material used. The pupal tissue treated with Otic suspensions demonstrated significantly less inflammation (Kruskal Wallis non parametric analysis, H=9.595 with 1 degree of freedom; P=0.0223) than the formocresol and control groups. The hard tissue bridges formed over the exposure sites were more organized in the MTA treatment groups than in the control and ZOE groups (Kruskal Wallis non parametric analysis, H=18.291 with 1 degree of freedom; P=0.0004). Conclusions: Otic suspensions and MTA are effective in treating bacterial infected pulps and stimulate the production of a hard tissue bridge over the site of the exposure.

Cognitive Deficits in Machado-Joseph Disease Correlate with Hypoperfusion of Visual System Areas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of cardiopulmonary bypass operation on the biodistribution of 99mTc-HMPAO-labelled granulocytes – Evaluation in pigs by planar scintigraphy and section-analyses

Aim of the study was to evaluate the influence of an extra corporal perfusion (cardiopulmonary bypass operation - cpb) on activation and biodistribution of Tc-99m labelled granulocytes in pigs with and without inhibition of the granulocytes by a leukocyte inhibition module (LIM). The cpb is often related to an activation of granulocytes resulting in an inflammatory answer. The biological mechanisms are unsolved yet. First trials of our group showed that LIM may inhibit the activation of neutrophils and therefore antagonize a cpb-caused impairment of cardiac function. This study is the continuation of these experiments with a higher number of animals and the focus on scintigraphic imaging. Animals, material, methods: 39 German landrace pigs were subdivided into three groups: group A (control) median sternotomy without cpb, group B with cpb, group C with LIM in addition to cpb. After labelling with Tc-99m-HMPAO autologues granulocytes were reinjected. Subsequently to cpb, the animals underwent scintigraphic imaging. Quantification was performed with ROI evaluation and with tissue samples (section analysis) examined in a well counter. Results:A high uptake of Tc-99m-HMPAO was found in the liver. The count rates in brain, heart, lung, spleen and kidneys were far below. The amount of Tc-99m-activity in the organ related to the half life corrected administered activity [%] was for the tissue samples (group A/B/C): brain 0.01/0.02/0.03; lung 12.1/8.3/11.5; heart 0.35/0.54/0.42; kidney 1.24/0.87/1.02; spleen 4.0/4.0/4.5, liver 16.8/20.9/19.6. The count rates determined by ROI-evaluation of the scintigraphic images related to the total count rate in the image [%] were (group A/B/C): brain 1.1/0.9/1.0; lung 15.6/10.4/12.2; heart 4.0/3.5/3.4; kidney 4.0/2.9/3.2; spleen 7.6/7.7/9.5, liver 23.1/36.7/31.4. A significant difference in the tracer uptake between the groups could neither be detected by scintigraphic imaging nor evaluation of tissue samples. Conclusion: Scintigraphic imaging as well as section analysis showed a comparable biodistribution of the tracer. Therefore, the initial results of our group were not confirmed with a considerably higher number of animals. Neither cpb nor the use of the LIM influenced distribution of Tc-99m-labelled granulocytes in pigs significantly.

Selective Ablation of the Androgen Receptor in Mouse Sertoli Cells Affects Sertoli Cell Maturation, Barrier Formation and Cytoskeletal Development

The observation that mice with a selective ablation of the androgen receptor (AR) in Sertoli cells (SC) (SCARKO mice) display a complete block in meiosis supports the contention that SC play a pivotal role in the control of germ cell development by androgens. To delineate the physiological and molecular mechanism responsible for this control, we compared tubular development in pubertal SCARKO mice and littermate controls. Particular attention was paid to differences in SC maturation, SC barrier formation and cytoskeletal organization and to the molecular mediators potentially involved. Functional analysis of SC barrier development by hypertonic perfusion and lanthanum permeation techniques and immunohistochemical analysis of junction formation showed that SCARKO mice still attempt to produce a barrier separating basal and adluminal compartment but that barrier formation is delayed and defective. Defective barrier formation was accompanied by disturbances in SC nuclear maturation (immature shape, absence of prominent, tripartite nucleoli) and SC polarization (aberrant positioning of SC nuclei and cytoskeletal elements such as vimentin). Quantitative RT-PCR was used to study the transcript levels of genes potentially related to the described phenomena between day 8 and 35. Differences in the expression of SC genes known to play a role in junction formation could be shown from day 8 for Cldn11, from day 15 for Cldn3 and Espn, from day 20 for Cdh2 and Jam3 and from day 35 for ZO-1. Marked differences were also noted in the transcript levels of several genes that are also related to cell adhesion and cytoskeletal dynamics but that have not yet been studied in SC (Actn3, Ank3, Anxa9, Scin, Emb, Mpzl2). It is concluded that absence of a functional AR in SC impedes the remodeling of testicular tubules expected at the onset of spermatogenesis and interferes with the creation of the specific environment needed for germ cell development.

Cinética do transporte jejunal de glicose em ratos com deficiência de niacina.

The kinetic of jejunal glucose transport was studied using perfused rat jejunum in vivo. Ninety rats were fed a diet deficient in niacin and 90 a control diet. The jejunal loops of 7 groups of animal were infused each group with one of following solutions of glucose: 5, 10, 20, 40, 80, 160 and 300 mM/l. The Vmax and Km values were determined. The results showed that the vitamin-deficient rats absorbed less glucose independently of the amount infused and these animals had lower Vmax (133.7 microM/15 min/15 cm) and Km (192.1 mM/l) than control groups (294.1 microM/15 min/15 cm and 171.8 mM/l, respectively). In conclusion one can assume that niacin deficiency leads to a decreased glucose absorption in the jejunal loops, when tested as in our experimental model.

A influência da perfusão cardíaca nas relações pressão/volume do ventrículo esquerdo.

PURPOSE--To analyze the influence of the myocardial engorgement due to coronary perfusion on the left ventricular diastolic pressure/volume (P/V) relations. METHODS--The study was undertaken in the isovolumic blood-perfused dog heart preparation (n = 7). The P/V relations were determined promoting ventricular volumes variations by steps of 2ml, in two conditions: during coronary perfusion pressure of 100mmHg and after occlusion of the perfusion line. RESULTS--It was verified that the P/V relations obtained without myocardial perfusion shifted to the down right in respect to the perfused heart. A exponential fitting of the P/V relations allows to conclude that there was no difference among the slopes of the curves obtained with and without perfusion. CONCLUSION--Our results indicated that coronary perfusion restrain the capacity of the left ventricle to receive blood, but there is no influence on the passive elastic stiffness of the chamber. This effect was taught to be consequence of the myocardial erectile property.

INFLUENCIA DO ACRESCIMO DE MANITOL A SOLUCAO NUTRIENTE NO DESEMPENHO MECANICO E NO GRAU DE EDEMA MIOCARDICO DE CORACOES ISOLADOS DE RATOS

Purpose - To analyse the influence of mannitol added to Krebs-Henseleit (KH) solution on the myocardium edema and myocardial function. Methods - Isolated rat heart under isovolumetric contractions studied according to Langendorff's technique were perfused with KH solution at constant flow during 90 min. The coronary perfusion pressure, diastolic and systolic pressures were recorded at every 15 min. At the end of the experiment, myocardium water content was measured in hearts perfused with KH solution (group I, n = 9) and in hearts perfused with KH solution plus 8 mM mannitol (group II, n = 8). These results were compared to non-perfused control heart (n = 9). Results - Myocardial water content was statistically higher in group I (80.8 ± 1.3%) compared to group II (78.1 ± 0.7%) and control group (75.5 ± 0.5%). Systolic arterial pressure was statistically higher in group I (86.2 ± 11.5 mmHg) compared to group II (72.7 ± 21.1 mmHg). There was no difference in the diastolic pressure between the two groups. Coronary perfusion pressure (Pp) increased progressively during the experiment in both groups. However, Pp was lower in group II than in group I. Conclusion - Mannitol added to KH solution significantly attenuates the myocardium edema in the isolated perfused rat heart.