Isolation of Campylobacter jejuni from viscera and bile secretion of broiler chickens with diarrhea

Since chickens are important reservoirs of Campylobacter jejuni and their meat is the most frequent route of transmission for human campylobacteriosis, the purpouse of the present study was to investigate the presence of Campylobacter in viscera of chickens with diarrhea, evaluating the frequency of isolation of this microorganism from organs considered to be preferential for its isolation. A total of 107 visceral samples from chickens with diarrhea from different farms in the Ribeirao Preto region, SP, were examined for the presence of Campylobacter jejuni. The material consisted of 73 livers and 34 spleens, plus 29 bile secretion samples. The frequency of Campylobacter jejuni isolation was 54.79% for the liver samples, 35,29% for the spleens and 6,89% for the bile secretion samples. The data suggest that, under the conditions of the present study, the liver may be the organ of choice for the isolation of Campylobacter in the presence of diarrhea and liver involvement in chickens.

Pectinolytic enzyme production by Bacillus species and their potential application on juice extraction

Five Bacillus strains isolated from decaying vegetable material were cultivated on wheat bran and endo-polygalacturonases, exo-polygalacturonase and pectin lyase activities in the crude enzymatic solution obtained were determined. Highest activity was observed for all enzymes when fermentation was carried out at 28 degreesC, the highest activity values were obtained after 120 h of cultivation for exo-PG and after 48 h for endo-PG and PL. The use of the enzymatic solution for treatment of fruits and vegetable mash afforded a high juice extraction and a pulp with good pressing characteristics.

Zinc supplementation does not inhibit basal and metoclopramide-stimulated prolactinemia secretion in healthy men

Dopamine (DA) and zinc (Zn++) share common mechanisms in their inhibition of prolactin (PRL) secretion. Both substances are present in the same brain areas, where Zn++ is released together with DA, suggesting a modulatory effect of Zn++ on dopaminergic receptors. The aim of the present study was to evaluate the effect of Zn supplementation on basal and PRL secretion stimulated by metoclopramide (MCP), a dopaminergic antagonist. Seven healthy men were evaluated in controlled study, where MCP (5 mg) was given intravenously, before and after 3 months of oral Zn++ (25 mg) administration. Our results indicate that chronic Zn++ administration does not change basal or MCP-stimulated plasma PRL secretion suggesting that, in humans, Zn++ does not interfere on PRL secretion mediated through dopaminergic receptors.

Determination of malic acid in real samples by using enzyme immobilized reactors and amperometric detection

The malate dehydrogenase (MDH) and ascorbate oxidase were immobilized independently, onto silanized controlled porous silica and packed in a tygon tube. The reactors were inserted in the flow system, and the malic acid was determined by measurement of NADH produced by enzymatic reaction. The NADH was reoxidized in a wall jet cell that consisted of spectrographic graphite, Ag/AgCl, KCl(sat), and steel needle as work, reference, and counter electrodes, respectively. The current intensities were measured at 390 mV. The malate calibration curve shows a linear range from 5.0 x 10(-6) to 1.0 x 10(-4) molL(-1), the lifetime was 40 analyses, after that a decrease of 20% on the response is observed. Three different citric juices were analyzed and a good correlation between the proposed method and spectrophotometric commercial kit were obtained.

Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers

The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

Purification and characterization of jararassin-I, a thrombin-like enzyme from Bothrops jararaca snake venom

A thrombin-like serine protease, jararassin-I, was isolated from the venom of Bothrops jararaca. The protein was obtained in high yield and purity by a single chromatographic step using the affinity resin Benzamidine-Sepharose CL-6B. SDS-PAGE and dynamic light scattering analyses indicated that the molecular mass of the enzyme was about 30 kD. The enzyme possessed fibrinogenolytic and coagulant activities. The jararassin-I degraded the Bbeta chain of fibrinogen while the Aalpha chain and gammachain were unchanged. Proteases inhibitors, PMSF and benzamidine inhibited the coagulant activity. These results showed jararassin-I is a serine protease similar to coagulating thrombin-like snake venom proteases, but it specifically cleaves Bbeta chain of bovine fibrinogen. Single crystals of enzyme were obtained (0.2 mmx0.2 mmx0.2 mm) and used for X-ray diffraction experiments.

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.

Brazilian individuals with impaired glucose tolerance are characterized by impaired insulin secretion

Background: To better understand the pathogenesis of type 2 diabetes mellitus, insulin secretion and insulin sensitivity (IS) were evaluated in white Brazilians with impaired glucose tolerance (IGT), using the oral glucose tolerance test (OGTT) and the hyperglycemic clamp technique.Methods: Twenty-five IGT subjects were individually matched with normal glucose-tolerant (NGT) subjects for demographic characteristics, At first, they were submitted to the OGTT and plasma glucose and insulin were measured. of the 25 pairs, 20 could participate in the hyperglycemic clamp procedures, at a second visit. All participants had their plasma glucose levels equally increased to 180 mg/dl; this was maintained for three hours by variable glucose infusion. During the procedure, plasma glucose and insulin were measured at established intervals.Results: In the postabsorptive state, the IGT subjects presented higher levels of plasma glucose, blood HbA(1) and serum triglycerides, but similar plasma insulin levels. After the oral glucose load, early and total insulin release, in relation to glucose levels, were respectively, 43 and 67% lower in the IGT individuals, the index of whole-body IS was increased in the IGT individuals (4.36 +/- 1.71 vs 3.61 +/- 1.28 mg(-1).muU(-1) 100.ml(2); p < 0.05). By the hyperglycemic clamp technique first- (82 &PLUSMN; 26 vs 215 &PLUSMN; 88 μU/ml; p < 0.001) and second- (36 +/- 19 vs 73 +/- 44 muU/ml; p < 0.05) phases of insulin secretion was decreased in the IGT individuals, especially the first one. However, the groups did not differ in relation to the IS: IGT = 13.52 &PLUSMN; 7.27 and NGT = 9.96 &PLUSMN; 6.70 mg.ml/kg.μU.min(-1); p > 0.05. Functional relationship of IS (y) on first-phase insulin release (x) showed a smaller (p < 0,05) regression coefficient for the IGT group.Conclusion: Brazilians with IGT well-matched with NGT ones were characterized by impaired first- and second-phase insulin secretion (mainly the former), while defects in IS were not evident.

Use of PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism) in the gene of the enzyme Stearoyl-CoA-Desaturase in Bubalus bubalis

The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis

Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43). To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P. brasiliensis-sensitized subjects. gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10, Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM. gp43-pulsed adherent cells, but not non-adherent cells, mere the main source of IL-10, Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects. Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients. (C) 2001 Academic Press.