A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones.

Identification of casein kinase 1, casein kinase 2, and cAMP-dependent protein kinase-like activities in Trypanosoma evansi

Trypanosoma evansi contains protein kinases capable of phosphorylating endogenous substrates with apparent molecular masses in the range between 20 and 205 kDa. The major phosphopolypeptide band, pp55, was predominantly localized in the particulate fraction. Anti-alpha and anti-beta tubulin monoclonal antibodies recognized pp55 by Western blot analyses, suggesting that this band corresponds to phosphorylated tubulin. Inhibition experiments in the presence of emodin, heparin, and 2,3-bisphosphoglycerate indicated that the parasite tubulin kinase was a casein kinase 2 (CK2)-like activity. GTP, which can be utilized instead of ATP by CK2, stimulated rather than inactivated the phosphorylation of tubulin in the parasite homogenate and particulate fraction. However, GTP inhibited the cytosolic CK2 responsible for phosphorylating soluble tubulin and other soluble substrates. Casein and two selective peptide substrates, P1 (RRKDLHDDEEDEAMSITA) for casein kinase (CK1) and P2 (RRRADDSDDDDD) for CK2, were recognized as substrates in T. evansi. While the enzymes present in the soluble fraction predominantly phosphorylated P1, P2 was preferentially labeled in the particulate fractions. These results demonstrated the existence of CK1-like and CK2-like activities primarily located in the parasite cytosolic and membranous fractions, respectively. Histone II-A and kemptide (LRRASVA) also behaved as suitable substrates, implying the existence of other Ser/Thr kinases in T. evansi. Cyclic AMP only increased the phosphorylation of histone II-A and kemptide in the cytosol, demonstrating the existence of soluble cAMP-dependent protein kinase-like activities in T. evansi. However, no endogenous substrates for this enzyme were identified in this fraction. Further evidences were obtained by using PKI (6-22), a reported inhibitor of the catalytic subunit of mammalian cAMP-dependent protein kinases, which specifically hindered the cAMP-dependent phosphorylation of histone II-A and kemptide in the parasite soluble fraction. Since the sum of the values obtained in the parasite cytosolic and particulate fractions were always higher than the values observed in the total T. evansi lysate, the kinase activities examined here appeared to be inhibited in the original extract.

Thyroid peroxidase activity is inhibited by amino acids

Normal in vitro thyroid peroxidase (TPO) iodide oxidation activity was completely inhibited by a hydrolyzed TPO preparation (0.15 mg/ml) or hydrolyzed bovine serum albumin (BSA, 0.2 mg/ml). A pancreatic hydrolysate of casein (trypticase peptone, 0.1 mg/ml) and some amino acids (cysteine, tryptophan and methionine, 50 µM each) also inhibited the TPO iodide oxidation reaction completely, whereas casamino acids (0.1 mg/ml), and tyrosine, phenylalanine and histidine (50 µM each) inhibited the TPO reaction by 54% or less. A pancreatic digest of gelatin (0.1 mg/ml) or any other amino acid (50 µM) tested did not significantly decrease TPO activity. The amino acids that impair iodide oxidation also inhibit the TPO albumin iodination activity. The inhibitory amino acids contain side chains with either sulfur atoms (cysteine and methionine) or aromatic rings (tyrosine, tryptophan, histidine and phenylalanine). Among the amino acids tested, only cysteine affected the TPO guaiacol oxidation reaction, producing a transient inhibition at 25 or 50 µM. The iodide oxidation inhibitory activity of cysteine, methionine and tryptophan was reversed by increasing iodide concentrations from 12 to 18 mM, while no such effect was observed when the cofactor (H2O2) concentration was increased. The inhibitory substances might interfere with the enzyme activity by competing with its normal substrates for their binding sites, binding to the free substrates or reducing their oxidized form.

Effect of intraperitoneally administered hydrolyzed whey protein on blood pressure and renal sodium handling in awake spontaneously hypertensive rats

The present study evaluated the acute effect of the intraperitoneal (ip) administration of a whey protein hydrolysate (WPH) on systolic arterial blood pressure (SBP) and renal sodium handling by conscious spontaneously hypertensive rats (SHR). The ip administration of WPH in a volume of 1 ml dose-dependently lowered the SBP in SHR 2 h after administration at doses of 0.5 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 176.6 ± 4.9 mmHg, N = 8, P = 0.001) and 1.0 g/kg (0.15 M NaCl: 188.5 ± 9.3 mmHg vs WPH: 163.8 ± 5.9 mmHg, N = 8, P = 0.0018). Creatinine clearance decreased significantly (P = 0.0084) in the WPH-treated group (326 ± 67 µL min-1 100 g body weight-1) compared to 0.15 M NaCl-treated (890 ± 26 µL min-1 100 g body weight-1) and captopril-treated (903 ± 72 µL min-1 100 g body weight-1) rats. The ip administration of 1.0 g WPH/kg also decreased fractional sodium excretion to 0.021 ± 0.019% compared to 0.126 ± 0.041 and 0.66 ± 0.015% in 0.15 M NaCl and captopril-treated rats, respectively (P = 0.033). Similarly, the fractional potassium excretion in WPH-treated rats (0.25 ± 0.05%) was significantly lower (P = 0.0063) than in control (0.91 ± 0.15%) and captopril-treated rats (1.24 ± 0.30%), respectively. The present study shows a decreased SBP in SHR after the administration of WPH associated with a rise in tubule sodium reabsorption despite an angiotensin I-converting enzyme (ACE)-inhibiting in vitro activity (IC50 = 0.68 mg/mL). The present findings suggest a pathway involving ACE inhibition but measurements of plasma ACE activity and angiotensin II levels are needed to support this suggestion.

Enzyme linked immunosorbent assay: determination of anti-adenovirus antibodies in an infant population

In order to define an accurate assay for anti-adenovirus antibody detection, a recently developed ELISA was compared with IFA and CF. On 58 sera, the ELISA was more sensitive than both CF and IFA, which showed relative sensitivities of 63% and 94%, respectively. It was not possible to determine the exact specificity of the tests because of the lack of a gold standard. Furthermore, the ELISA was used to define the prevalence of adenovirus antibodies in 116 infants between 1 and 24 months old (mean 7.28). The data showed that maternal antibodies waned by the age of 5 to 6 months and that more than 80% of the children had been infected by adenoviruses by the age of 10 months.

Evaluation of enzyme-linked immunosorbent and alternative assays for detection of HIV antibodies using panels of Brasilian sera

Sera from 472 Brazilian subjects, confirmed to be either positive or negative for HIV antibodies and comprising the total clinical spectrum of HIV infection, were utilized in the evaluation of six commercially available enzyme-linked immunosorbent assays (ELISA), as well as of four alternative assays, namely indirect immunofluorescence (IIF), passive hemagglutination (PHA), dot blot and Karpas AIDS cell test. The sensitivities ranged from 100% (Abbott and Roche ELISA) to 84.2% (PHA) and the specificities ranged from 99.3% (IIF) to 80.2% (PHA). The sensitivity and specificity of the PHA and the sensitivity of the Karpas cell test were significantly lower than those of the other tests. Although the IFF and dot blot had good sensitivities and specificities, the six ELISA were more attractive than those tests when other parameters such as ease of reading and duration of assay were considered.

Enzyme-linked immunosorbent assay (ELISA) for measles antibody: a comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copo-sitivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.

Enzyme-linked immunosorbent assay ELISA for the detection of antibodies in the human leptospirosis

An Enzyme-linked immunosorbent assay ELISA was evaluated for the detection of IgA antibodies in the human leptospirosis. The assay proved to be sensitive and specific when compared with the ELISA-IgM, in the examinated serum samples. The results found suggest that IgA antibodies became positive later in leptospirosis, and will can be an evolutive indicator in the development of the disease

Evaluation of the double diffusion, enzyme immunoassay and immunoblotting techniques, for the diagnosis of human hydatid disease in tropical areas

Hydatid disease in tropical areas poses a serious diagnostic problem due to the high frequence of cross-reactivity with other endemic helminthic infections. The enzyme-linked-immunosorbent assay (ELISA) and the double diffusion arc 5 showed respectively a sensitivity of 73% and 57% and a specificity of 84-95% and 100%. However, the specificity of ELISA was greatly increased by using ovine serum and phosphorylcholine in the diluent buffer. The hydatic antigen obtained from ovine cyst fluid showed three main protein bands of 64,58 and 30 KDa using SDS PAGE and immunoblotting. Sera from patients with onchocerciasis, cysticercosis, toxocariasis and Strongyloides infection cross-reacted with the 64 and 58 KDa bands by immunoblotting. However, none of the analyzed sera recognized the 30 KDa band, that seems to be specific in this assay. The immunoblotting showed a sensitivity of 80% and a specificity of 100% when used to recognize the 30 KDa band.

Gelatin particle indirect agglutination test for serodiagnosis of schistosomiasis: comparative study with enzyme-linked immunosorbent assay

A new serological test, the gelatin particle agglutination test (GPAT), was used for the serodiagnosis of schistosomiasis mansoni. This technique showed the sensitivity (90.6%) and specificity (97.8%) close to those of enzyme-linked immunosorbent assay. The GPAT can be easily and rapidly performed without specialized equipment, by using lyophilized antigen-coated gelatin particles. The test also seems to be useful for mass screening of Schistosoma infection in field conditions.

Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for detection of pneumococcal polysaccharide antigens in pleural fluid effusion samples.: Comparison with bacterial culture, counterimmunoelectrophoresis and latex agglutination

A dot-enzyme-linked immunosorbent assay (Dot-ELISA) for pneumococcal antigen detection was standardized in view of the need for a rapid and accurate immunodiagnosis of acute pneumococcal pneumonia. A total of 442 pleural fluid effusion samples (PFES) from children with clinical and laboratory diagnoses of acute bacterial pneumonia, plus 38 control PFES from tuberculosis patients and 20 negative control serum samples from healthy children were evaluated by Dot-ELISA. The samples were previously treated with 0.1 M EDTA pH 7.5 at 90°C for 10 min and dotted on nitrocellulose membrane. Pneumococcal omniserum diluted at 1:200 was employed in this assay for antigen detection. When compared with standard bacterial culture, counterimmunoelectrophoresis and latex agglutination techniques, the Dot-ELISA results showed relative indices of 0.940 to sensitivity, 0.830 to specificity and 0.760 to agreement. Pneumococcal omniserum proved to be an optimal polyvalent antiserum for the detection of pneumococcal antigen by Dot-ELISA. Dot-ELISA proved to be a practical alternative technique for the diagnosis of pneumococcal pneumonia.

Standardization of an enzyme linked immunosorbent assay (ELISA) for detecting circulating toxic venom antigens in patients stung by the scorpion Tityus serrulatus

The sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antigens from toxic components of Tityus serrulatus scorpion venom was determined in patients stung by T. serrulatus before antivenom administration. Thirty-seven patients were classified as mild cases and 19 as moderate or severe cases. The control absorbance in the venom assay was provided by serum samples from 100 individuals of same socioeconomic group and geographical area who had never been stung by scorpions or treated with horse antisera. The negative cutoff value (mean + 2 SD) corresponded to a venom concentration of 4.8 ng/ml. Three out of the 100 normal sera were positive, resulting in a specificity of 97%. The sensitivity of the ELISA when all cases of scorpion sting were included was 39.3%. When mild cases were excluded, the sensitivity increased to 94.7%. This study showed that this ELISA can be used for the detection of circulating venom toxic antigens in patients with systemic manifestations following. T. serrulatus sting but cannot be used for clinical studies in mild cases of envenoming since the test does not discriminate mild cases from control patients.

Diagnosis of human toxoplasmosis by a dot enzyme-linked immunosorbent assay

A Dot enzyme-linked immunosorbent assay (Dot-ELISA) was standardized and evaluated for the serodiagnosis of human toxoplasmosis. Out of 538 serum samples tested by the immunofluorescence test for toxoplasmosis (IFAT-IgG) as reference test, 183 (34%) were positive at cut off 1:16 and 192 (36%) were positive for Dot-ELISA-IgG at cut-off 1:256. For Dot-ELISA, co-positivity was 0.94, co-negativity 0.94 and concordance 0.88 in relation to IFAT-IgG. These results suggest the usefulness of Dot-ELISA (cut-off titer of 1:256) for the serodiagnosis of human toxoplasmosis. The main advantage of this technique is simplicity, positive test can be visually identified (colored precipitate). It does not require a special equipment and it can be used as a qualitative test to screen large numbers of samples or as a quantitative assay to determine end-point titration of individual sera.

Evaluation of the enzyme-linked-immuno-electro-diffusion-assay (ELIEDA) for the diagnosis of Schistosoma mansoni infection with low worm burden

An immunoprecipitation technique, ELIEDA (enzyme-linked-immuno-electro-diffusion assay), was evaluated for the diagnosis of Schistosoma mansoni infection with low worm burden. One hundred of serum samples from patients excreting less than 600 eggs per gram of feces (epg), with unrelated diseases and clinically healthy subjects were studied. In patients with egg counts higher than 200 epg, the sensitivities of IgM and IgG ELIEDA were 1.000 and 0.923, respectively, not differing from other Serologic techniques, such as indirect hemaglutination (IHAT), immunofluorescence (IFT) tests and immuno-electrodiffusion assay (IEDA). However in patients with low egg counts (< 100 epg), the IgG ELIEDA provided better results (0.821) than IgM ELIEDA (0.679), showing sensitivity that did not differ from that of IgG IFT (0.929), but lower than that of IgM IFT (0.964). However, its sensivity was higher than that found with IHAT (0.607) and IEDA (0.536). The specificity of IgG ELIEDA was comparable to that of other techniques. The data indicate that IgG ELIEDA might be useful for the diagnosis of slight S. mansoni infections, and the cellulose acetate membrane strips can be stored for further retrospective studies.

Enzyme linked immunosorbent assay for rubella antibodies: a simple method of antigen production. A preliminary report

A simple method of rubella antigen production by treatment with sodium desoxycholate for use in enzyme immunoassay (IMT-ELISA) is presented. When this assay was compared with a commercial test (Enzygnost-Rubella, Behring), in the study of 108 sera and 118 filter paper blood samples, 96.9% (219/226) overall agreement and correlation coefficient of 0.90 between absorbances were observed. Seven samples showed discordant results, negative by the commercial kit and positive by our test. Four of those 7 samples were available, being 3 positive by HI.