Effect of enzyme supplementation of broiler diets based on corn and soybeans

Digestibility of diets based on corn and soybean meal or soybeans treated by roasting or extrusion, with or without an enzyme supplementation, was measured by true (Sibbald) methods, by analysis of excreta, and by analysis of ileal digesta. Only analysis of ileal digesta was able to consistently measure differences between soybean and enzyme treatments in the digestibility of CP, starch, fat, and ME. The amino acid (AA) digestibility of the diets was measured by analysis of the ileal contents. Whereas enzyme supplementation improved overall CP digestibility by 2.9%, this improvement was not equal for all AA. of the AA most important for broilers fed corn-soybean diets, the digestibilities of Lys, Met, and Arg were not improved or not improved significantly by the enzyme supplementation; however, that of Val was improved by 2.3% and that of Thr was improved by 3.0%. A performance trial demonstrated that enzyme supplementation with equal diet formulation improved BW and the feed conversion ratio by 1.9 and 2.2%, respectively. A second performance trial compared standard diet formulations with formulations using enzyme supplementation and energy levels that were reduced by the amount of improvement provided by the inclusion of enzyme in the first performance trial. No difference was seen between treatments, showing that the improvement of nutrient utilization brought about by enzyme supplementation completely compensated for the reduced energy content. Whereas enzyme supplementation should allow a reduction in CP formulation as well, individual AA were not improved equally by supplementation and should also be balanced.

Liquid-phase blocking sandwich enzyme-linked immunosorbent assay for detection of antibodies against foot-and-mouth disease virus in water buffalo sera

Objective-To develop and apply the liquid-phase blocking sandwich ELISA (BLOCKING-ELISA) for the quantification of antibodies against foot-and-mouth disease virus (FMDV) strains O-1 Campos, A(24) Cruzeiro, and C-3 Indaial.Design-Antibody quantification.Sample Population-158 water buffalo from various premises of São Paulo Stale-Brazil. The sera were collected either from systemically vaccinated or nonvaccinated animals.Procedure-The basic reagents of BLOCKING-ELISA (capture and detector antibodies, virus antigens, and conjugate) were prepared and the reaction was optimized and standardized to quantify water buffalo antibodies against FMDV. An alternative procedure based on mathematical interpolation was adopted to estimate more precisely the antibody 50% competition liters in the BLOCKING-ELISA. These titers were compared with the virus-neutralization test (VNT) titers to determine the correlation between these techniques. The percentages of agreement, cutoff points, and reproducibility also were determined.Results-The antibody liters obtained in the BLOCKING-ELISA had high positive correlation coefficients with VNT, reaching values of 0.90 for O-1 Campos and C-3 Indaial, and 0.82 for the A(24) Cruzeiro (P < 0.0005). The cutoff points obtained by use of the copositivity and conegativity curves allowed determination of high levels of agreement between BLOCKLNG-ELISA and VNT antibody titers against the 3 FMDV strains analyzed.Conclusions-The results characterized by high cor relation coefficients, levels of agreement, and reproducibility indicate that the BLOCKING-ELISA may replace the conventional VNT for detection and quantification of antibodies from water buffalo sera to FMDV.

Turbinatine, a potential key intermediate in the biosynthesis of corynanthean-type indole alkaloids

Extraction of the leaves of Chimarrhis turbinata has led to the isolation of turbinatine (1), a new corynanthean-type indole alkaloid, besides four known indole alkaloids, strictosidine, 5alpha-carboxystrictosidine, vallesiachotamine, and isovallesiachotamine. The structural determination of 1 was based on 1D and 2D spectroscopic data. An evaluation of the DNA-damaging activities of the isolates was performed by means of a bioassay using mutant strains of Saccharomyces cerevisiae, which indicated these compounds were weakly active.

Analysis of pesticides in food and environmental samples by enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays (ELISAs) are the most extensively studied types of immunoassay and their application in pesticide residue monitoring is an area with enormous potential for growth. In comparison with classical analytical methods, ELISA methods offer the possibility of highly sensitive, relatively rapid, and cost-effective measurements. This review introduces the general ELISA formats used, focusing on their use in pesticide analysis. Identifying and studying the effects of interferences in immunoassays is an active area of research and we discuss the matrix effects observed in several studies involving e.g. food, crop and environmental samples. The procedures to eliminate the matrix interferences are briefly discussed. (C) 1998 Elsevier B.V. B.V.

SEROLOGICAL RESPONSE OF CHICKENS TO INFECTION WITH SALMONELLA-GALLINARUM S-PULLORUM DETECTED BY ENZYME-LINKED-IMMUNOSORBENT-ASSAY

The serological response to Salmonella pullorum and S. gallinarum infection in chickens was studied with an indirect enzyme-linked immunosorbent assay (ELISA). In broiler chickens, a more virulent strain of S. pullorum produced a significantly lower serum IgG titer than did a less virulent strain. In laying hens, the serum and egg-yolk IgG titers were very similar. In chickens infected with S. gallinarum, high IgG titers persisted for 30 weeks. In chickens reinfected with this strain, each reinfection was followed by transitory increases in IgG lasting no longer than 2 weeks. Serum samples from Brazil taken from a laying flock with evidence of fowl typhoid showed much higher antibody levels than did those from three uninfected flocks. Using lipopolysaccharide as the detecting antigen, infections caused by these salmonellae could be differentiated from those caused by other groups. Incorporation of the appropriate flagella antigen in the ELISA allowed differentiation between infections caused by S. pullorum and S. enteritidis.

EFFECTS OF DIETARY-PROTEIN, ANGIOTENSIN-I CONVERTING-ENZYME INHIBITION AND MESANGIAL OVERLOAD ON THE PROGRESSION OF ADRIAMYCIN-INDUCED NEPHROPATHY

Adriamycin, a commonly used antineoplastic antibiotic, induces glomerular lesions in rats, resulting in persistent proteinuria and glomerulosclerosis. We studied the effects of dietary protein and of an angiotensin I converting enzyme inhibitor on the progression of this nephropathy and the evolution of the histological lesions, as well as mesangial macromolecule flow. Adriamycin nephropathy was induced by injecting a single iv dose of adriamycin (3 mg/kg body weight) into the tail vein of male Wistar rats (weight, 180-200 g). In Experiment I animals with adriamycin-induced nephropathy were fed diets containing 6% (Low-Protein Diet Group = LPDG), 20% (Normal-Protein Diet Group = NPDG) and 40% (High-protein Diet Group = HPDG) protein and were observed for 30 weeks. In Experiment II the rats with adriamycin nephropathy were divided into 2 groups: ADR, that received adriamycin alone, and ADR-ENA, that received adriamycin plus enalapril, an angiotensin I converting enzyme inhibitor. The animals were sacrificed after a 24-week observation period. Six hours before sacrifice the animals were injected with I-131-ferritin and the amount of I-131-ferritin in the glomeruli was measured. In Experiment III, renal histology was performed 4, 8 and 16 weeks after adriamycin injection. At the end of Experiment I the tubulointerstitial lesion index was 2 for LPDG, 8 for NPDG, and 7.5 for HPDG (P<0.05); the frequency of glomerulosclerosis was 19 +/- 6.1% in LPDG, 42.6 +/- 6% in NPDG, and 54 +/- 9% in HPDG (P<0.05); and proteinuria was 61.1 +/- 25 mg/24 h in LPDG, 218.7 +/- 27.5 mg/24 h in NPDG, and 324.5 +/- 64.8 mg/24 h in HPDG (P<0.05). In Experiment II, at sacrifice, 24-h proteinuria was 189 +/- 16.1 mg in ADR, and 216 +/- 26.1 mg in ADR-ENA (P>0.05); the tubulointerstitial lesion index was 5 for ADR, and 5 for ADR-ENA (P>0.05); the frequency of glomerulosclerosis was 40 +/- 5.2% in ADR and 44 +/- 6% in ADR-ENA (P>0.05); the amount of I-131-ferritin in the mesangium was 214.26 +/- 22.71 cpm/mg protein in ADR and 253.77 +/- 69.72 cpm/mg protein in ADR-ENA (P>0.05). In Experiment III, sequential histological analysis revealed an acute tubulointerstitial cellular infiltrate at week 4, which was decreased at week 8. Tubular casts and dilatation were first seen at week 8 and increased at week 16 when few glomerular lesions were found. The results suggest that the tubulointerstitial lesions may play a role in the development of glomerulosclerosis in adriamycin-induced nephropathy.

Manganese(III) porphyrins: Catalytic activity and intermediate studies in homogeneous systems

We studied how solvent, stirring method, PhIO/MnP molar ratio, presence of water and axial ligand affect the catalytic activities of Mn(TPP)Cl, Mn(MNPP)Cl, Mn(TDCPP)Cl and Mn(TFPP)Cl in the oxidation of cyclohexane by PhIO. A study of the catalytic intermediates in the reaction between Mn(TPP)Cl or Mn(TDCPP)Cl and PhIO was also carried out by UV-Vis and EPR spectroscopies. The reaction of Mn(TPP)Cl with PhIO showed the formation of a mixture of species Mn-IV(OP+ and Mn-V(O)P as intermediates, which were confirmed by the deconvolution of the UV-Vis spectra. Addition of imidazole as cocatalyst favoured the formation of the intermediate species Mn-V(O)P, evidenced by the UV-Vis band at 408 nm. The corresponding EPR spectra gave evidence that in the presence of imidazole, Mn-IV(OP+ species are formed only in very low amounts. For Mn(TDCPP)Cl the dominating intermediate species is Mn-IV(OP+. Addition of imidazole to halogen-substituted MnP systems does not result in increase of the C-ol yields because very stable bis-imidazole-MnP complexes are formed. Anchoring of such MnP on imidazole propyl gel (IPG) results in better catalytic activity because in this case, the catalyst is mono-coordinated to the support and imidazole favours the formation of the intermediate species Mn-V(O)P.

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.

Release of intermediate reactive hydrogen peroxide by macrophage cells activated by natural products

By determining the hydrogen peroxide (H2O2) released in cultures of peritoneal macrophage cells from Swiss mice, we evaluated the action of 27 vegetable compounds (pristimerin, tingenone, jatrophone, palustric acid, lupeol, cladrastin, ocoteine, boldine, tomatine, yohimbine, reserpine, escopoletin, esculine, plumericin, diosgenin, deoxyschizandrin, p-arbutin, mangiferin, and others) using a 2 mg/ml solution of each compound (100 mug/well). Macrophages are cells responsible for the development of the immunological response reaction, liberating more than one hundred compounds into the extracellular environment. Among these are the various cytokines and the intermediate compounds of nitrogen (NO) and oxygen (H2O2). This coordinated sequence of biochemical reactions is known as the oxidative burst. When we compared the results with those obtained with zymosan (an important stimulator of H2O2) we observed that the compounds showing the highest activity were substances 2 (tingenone), 16 (reserpine) and 20. Other substances such as compounds 1, 4, 5, 6, 8, 12, 13, 14, 15, 17, 19, 23, 24, 26, and 27 also showed a certain activity, but with less intensity than the aforementioned ones. Compounds 3, 7, 9, 10, 11, 18, 21, 22 and 25 presented no activity. These results suggest that natural products (mainly tingenone and reserpine and others) with different chemical structures are strong immunological modulators. However, further tests are needed to determine the 'oxidative burst' in future studies.

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.

DO GLIOMA-CELLS EXPRESS CYTOKERATIN

There have been several recent reports of cytokeratin immunoreactivity in glial cells and tumors. We wanted to further test these tissues for cytokeratin immunoreactivity, and to determine whether antibody positivity corresponded to true cytokeratin expression. In the first set of experiments, a series of 10 formalin-fixed, frozen sections of glial tissue were employed; positive immunostaining with a cocktail of monoclonal anti-cytokeratin antibodies was seen only when a pepsin predigestion step was included in the immunostaining procedure. In the second set of experiments, 30 cases of malignant glioma fixed in both methacarn and formalin fixation were employed. Using a panel of three different anti-cytokeratin monoclonal antibodies (35 beta H11, 34 beta E12, CAM5.2), no positive immunostaining was observed in any of the methacarn-fixed tissues; positive immunostaining in the corresponding formalin-fixed tissue was frequently found, but only using the antibodies (35 beta H11, 34 beta E12) requiring enzyme predigestion. In the third set of experiments, immunoblots were performed on cytoskeletal extracts of human gliomas; no bands corresponding to known cytokeratins were observed in any cases. It is concluded that glial tissues and tumors do not, in fact, truly express cytokeratins, despite the fact that it is possible to obtain positive immunostaining of glial tumors and tissues using certain anti-cytokeratin antibodies under certain laboratory conditions.

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.