Heat Stability of Serum Lactate Dehydrogenase and its Isoenzymes in Young and Adult Cattle and Sheep

The heat stability of lactate dehydrogenase (LDH) has been investigated in serum from young and adult cattle and sheep. The thermoresistance of the isoenzymes was determined by electrophoresis of serum samples preincubated at different temperatures. Marked differences were found in the percentage distribution of isoenzymes in serum from the two species as well as in the heat stability. LDH in serum from sheep was inactivated at a lower temperature than that in serum from cattle, and inactivation occurred at a lower temperature in young than in adult animals. The enzyme was in both species less tolerant to elevated temperatures than what is reported for human serum. Procedures worked out for a so-called relative heat stability test of LDH in human clinical diagnosis may therefore give misleading results if they were applied uncritically to sera from these animals. The LDH isoenzyme pattern of some main organs in calves and sheep indicates that a serum heat stability test may be useful in the diagnosis of skeletal muscle injuries in sheep. In cattle the tissue isoenzyme distribution is assumed to be too uniform to give information about specific organ lesions either by serum electrophoresis or by a heating technique. In contrast to what has been reported in man, serum levels of α-hydroxybutyrate dehydrogenase (HBD) in cattle and sheep, as earlier reported in swine, are found to be far better correlated to total LDH than to the most thermostable isoenzyme, LDH1.     

Heat stability and pH activity data of alpha-L-fucosidase in human serum vary with enzyme concentration

alpha-L-Fucosidase from serum of humans with either high or low enzyme activity was separately purified. the enzyme from either source had virtually the same heat stability and pH activity profile. It has been widely reported that alpha-L-fucosidase in crude sera from individuals with high and low enzyme activity differed with respect to heat stability and activity at pH 4 relative to activity at pH 5, the pH optimum of the enzyme. We investigated this discrepancy and found that both the heat stability and relative activity at pH 4 of alpha-L-fucosidase from sera with either high or low enzyme activity was dependent upon enzyme concentration. With decreasing enzyme concentration, the enzyme was more heat labile and had less relative activity at pH 4. Consequently, if the data obtained using high and low enzyme activity sera are compared on the basis of equivalent amounts of serum instead of equivalent amounts of enzyme activity, differences between the enzyme from high and low activity serum would be erroneously inferred. Apparently, this is what other investigators have done. Moreover, we found that alpha-L-fucosidase can exist in heat-stable or labile species with sedimentation coefficients of 9.8 S and 4.8 S, respectively. The interconversion and relative proportion of these species is dependent upon enzyme concentration and pH.     

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.     

Molecular forms in human serum of enzymes synthesizing DNA precursors and DNA

Both thymidine kinase (TK) and DNA polymerase (DNAp) are present in measurable amounts in human serum. Even though the use of TK as a clinical marker is rapidly increasing there has been no attempt to characterize the serum TK in a wider extent, i.e.; with respect to Mw or other biochemical parameters. Therefore sera with high TK or DNAp activities derived from patients with cytomegalo-virus (CMV) infection, B₁₂-deficiency and leukaemia were fractionated by gel exclusion chromatography. The TK activity eluted as two peaks, one major TK activity with an apparent molecular weight (Mw) or 730 kD and one minor TK activity corresponding to a Mw of 58 kD. The amount of TK activity at 58 kD varied between 7 and 23% of total activity, depending on the serum fractionated. The DNAp activity in sera from patients with malignant disease and B12 deficiency eluted as a single peak corresponding to a Mw of 240 kD. A DNAp with a different Mw (>1000 kD) was recovered from 1 of 3 investigated immunosuppressed patients with CMV infection. A similar pattern of enzyme forms was observed when sera were separated by glycerol gradient centrifugation.The effect of high salt and various reaction solution components on the enzymes were studied. The only condition found that affected the molecular forms of TK was the state of reduction. Incubation of sera with high concentrations of dithioerythritol (DTE) (400 mM) prior to separation transferred all serum TK to the 58 kD form, it also converted most of the serum DNAp from the 240 kD form to a smaller form (56 kD) without affecting the total recovery of enzymatic activity.The reaction product from both TK forms was exclusively monophosphate and none of the TK forms could efficiently utilize cytidine triphosphate as phosphate donor. The substrate kinetics of the small serum TK fraction was identical with those of an enzyme with similar size purified from proliferating HeLa cells, indicating that both serum TK activities are forms of TK 1, the proliferation associated cellular isozyme.     

Autoantibody to glial fibrillary acidic protein in the sera of cattle with bovine spongiform encephalopathy

It is desirable to make the diagnosis in live cattle with bovine spongiform encephalopathy (BSE), and thus surrogate markers for the disease have been eagerly sought. Serum proteins from BSE cattle were analyzed by 2‐D Western blotting and TOF‐MS. Autoantibodies against proteins in cytoskeletal fractions prepared from normal bovine brains were found in the sera of BSE cattle. The protein recognized was identified to be glial fibrillary acidic protein (GFAP), which is expressed mainly in astrocytes in the brain. The antigen protein, GFAP, was also found in the sera of BSE cattle. The percentages of both positive sera in the autoantibody and GFAP were 44.0% for the BSE cattle, 0% for the healthy cattle, and 5.0% for the clinically suspected BSE‐negative cattle. A significant relationship between the presence of GFAP and the expression of its autoantibody in the serum was recognized in the BSE cattle. These findings suggest a leakage of GFAP into the peripheral blood during neurodegeneration associated with BSE, accompanied by the autoantibody production, and might be useful in understanding the pathogenesis and in developing a serological diagnosis of BSE in live cattle.     

Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.     

Clinico-biochemical use of serum acetylcholine esterase following treatment with synthetic pyrethroids, cypermethrin and fenvalerate, in cattle and buffalo experimentally infested with Boophilus microplus

Following treatment, cypermethrin and fenvalerate, were found to have inhibitory effect on serum acetylcholine esterase (AchE) activity of cattle and buffalo experimentally infested with B. microplus. The pattern of AchE activity in infested-pyrethroid-treated group was found to be significantly different from either healthy or tick-infested control. There was transient increase in the enzyme activity initially, followed by gradual decline and subsequent increase leading to normal level within 7 days of pyrethroid treatment. The enzyme activity was found to be low in buffalo than in cattle and the values remained below normal level up to day 7 in tick-infested group. The reversion of AchE activity to normal level in pyrethroid-treated group indicated that these compounds are prompt and safe ixodicides with least residual effect. The present investigation concludes that estimation of serum AchE might help in the clinico-biochemical diagnosis of tick toxin and pyrethroid toxicity in cattle and buffalo treated with these pyrethroids against tick infestation.     

An association between Schistosoma mansoni worms and an enzymatically-active protease/peptidase in mouse blood

An enzyme found previously in extracts of adult Schistosoma mansoni worms, that hydrolysed the chromogenic substrate N-acetyl-DL-phenylalanine beta-naphthyl-ester, has here been further investigated and characterized. Evidence that the molecule found in the parasite was antigenically and enzymatically homologous with a constituent of normal mouse plasma has been consolidated using a monospecific serum in immunoelectrophoresis and Western immunoblotting. The molecular size of the enzyme was found to be approximately 70 kDa and it was inhibited by a serine protease inhibitor, but not by inhibitors of other classes of protease. The enzymatic activity found in normal mouse serum was also found in normal rat serum, but not in sera from several other mammalian species.     

Human serum biotinidase is a thiol-type enzyme

The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

Human serum lipoamidase

Thirty-two human serum specimens were assayed for lipoamidase (lipoyl-4-aminobenzoate hydrolase) activity. All sera had lipoamidase activities. This substrate was newly synthesized by us and had a satisfying purity as evaluated by HPLC-fluorimetric detection. Product (p-aminobenzoate) liberated was determined directly by the HPLC-fluorimetric method. Liberation of the product was linearly continued for 6 h. The pH optimum of serum lipoamidase was found to be 7.0. The effect of substrate concentration on human serum lipoamidase activity was examined and the reaction was saturated at 0.1 mmol/l. The sera obtained were from individuals aged from 1 to 8 years. The mean value of serum lipoamidase activity was found to be 1.50 U/l (SD 1.037, range 0.04-3.75, n = 32). The difference of sex effects was analyzed and no significant difference was found (males: n = 14, mean 1.48, SD 1.162, range 0.04-3.75; females: n = 18, mean 1.52, SD 0.963, range 0.48-3.51) among this age group. Biotinidase activity was also determined in these 32 serum specimens and the correlation was examined. The mean biotinidase activity was 3.16 U/l (SD 2.567, range 0.35-9.37). The correlation coefficient (r) between lipoamidase activity and biotinidase activity was 0.8931. Although the physiological significance of lipoamidase has not been known, the enzyme might play an important role in recycling of lipoate as biotinidase does.     

Immunoenzyme determination of the total level of ceruloplasmin in the serum from patients with hepatocerebral dystrophy]

An immunoenzymatic method for ceruloplasmin analysis (IEA) based on the use of horseradish peroxidase-labelled monospecific antibodies as markers has been developed. IEA can be used for direct measurements of ceruloplasmin in blood serum, as can be evidenced from the coincidence of calibration plots obtained after the use of potassium-phosphate buffer and ceruloplasmin-free sera. The procedure allows the determination of the total content of ceruloplasmin present in the blood sera of patients with hepatocerebral dystrophies both in the active and inactive forms. The minimum ceruloplasmin concentration detectable by this method is 5 x 10(-9) g/ml. The method was used to determine ceruloplasmin levels in the blood of patients with various grades of hepatocerebral dystrophy. Analysis of blood sera from 6 patients revealed that the ceruloplasmin concentrations determined by IEA were very close, whereas the oxidase activities of this protein differed more than 7-fold. The amount of enzymatically active ceruloplasmin as determined from the oxidase activity made up to 10-68% of the total ceruloplasmin content in the sera, depending of the severity of the pathology.     

Anaphylactogenic, antigeni and avid properties of the antibotulin serum from the blood of various animal species]

It was shown that purified and concentrated by the "Diaferm" method antibotulin sera from horse and cattle blood failed to differ by anaphylactogenic properties; at the same time in sensitization of the organism to protein of one animal species the use of the sera of another species provided a lesser reactogenicity of the preparation. The antigenic activity of the purified and concentrated sera from the blood or horses and cows in testing on rabbits was identical, but in response to cow alpha-globulin the animals responsed by a more intensive production of precipitins. The activity of cow and horse antibotulin serum (determined by the rate and stability of their association with the corresponding toxin) proved to be identical.     

Detection of a tyrosine kinase in human sera and blood cells by pp60src antiserum

Using sera of Rous sarcoma virus-tumor bearing rabbits (TBR-sera) as a tool to detect pp60src kinase in immunoprecipitates, we report here that about 10% of our TBR-sera revealed tyrosine kinase activity in human serum, plasma and in soluble extracts of human blood cells. The activity found in serum represents 5-12% of the total kinase activity in blood. Most of the enzyme activity was detected in lymphocytes and polymorphonuclear cells rather than in platelets and erythrocytes. We also demonstrate that the tyrosine kinase in serum is inhibited by quercetin, a potent inhibitor of the viral pp60src protein kinase.     

Type-specific pneumococcal agglutinating sera in precipitation tests

Diagnostic agglutinating sera to pneumococci of different serotypes have been studied with respect to their capacity for taking part in the reactions of precipitation with capsular pneumococcal polysaccharides. The sera have proved to be highly active and specific in the reactions of double immunodiffusion and immunoelectrophoresis. The sera under study have also been found to react with cattle serum, one of the components of the medium used for the cultivation of pneumococci.     

Chlamydiosis in cattle and in man: an epidemiologic and serologic study

In the Pardubice area, the systematic examinations for Chlamydia-specific antibodies in cattle were started in 1967. In the years 1967-1978 the detectable levels of antibody to Chlamydia were demonstrated in 16.1% of 38,394 sera from cattle and in 18.6% of 5,244 sera from slinking cows. In the year 1979 the percentage of seropositivity in cattle increased significantly to 23.5% of the 4,197 sera examined. Examinations in 1980 revealed that 23.1% of 4,042 sera from cattle, 34.4% of 319 sera from slinking cows and 17.9% of 209 sera from calves were found antibody positive. Over the period from 1974 to 1978 isolates of Chlamydia organisms were obtained from 11.7% of 452 bovine slinks and from 10% of 271 dead or prematurely slaughtered calves. In the years 1979-1981 a total of 775 individuals were subjected to medical and serological examinations for Chlamydia infection using the BIOVETA group antigen. The first group of examinees included 307 farm personnel, attendants to healthy breeds of cattle. Detectable levels of antibody to Chlamydia were recorded in 1.3% of clinically healthy individuals and in 12% of the 75 healthy, randomly selected cows taken care of by this group of personnel. The second group included 280 individuals who were in charge of slinking cows or calves with chlamydial pneumonia, or who were employed in a rendering plant. A total of 7.5% of antibody-positive individuals was found in this group of examinees. Of the 439 calves attended 17.9% had serologic evidence of infection and the 30 dead calves examined were isolate-positive: sera from 176 slinking cows showed positivity in 23.8% of cases. In the third group of 188 persons serving as controls 5.3% had serologic evidence of chlamydial infection. The statistical analysis of deviations from the Gaussian variance normal curve showed neither sex-related nor inter-group differences. At the time of serologic examinations of humans the epidemiologic situation in the given area was extremely favorable. The importance of a close cooperation between the public health and veterinary service personnel (notification of abortions in cattle) and the need of a due attention of gynecologists, dermatologists and urologists are emphasized.     

Serologic distribution of antibodies against adenoviruses in sheep of large-scale farms.

The common soluble antigen of the first subgroup of bovine adenoviruses was used for assaying 793 sheep sera by the agar gel diffusion test. Of the 50 farms included in the study 43 were found infected. The ratio of reacting samples was 73.7% of the sera obtained from infected farms. Virus neutralization tests revealed that a considerable number of sera reacted specifically with all types of ovine adenoviruses, even with serotypes which had never been isolated in Hungary. The results yielded by the agar gel diffusion tests were compared with the results of virus neutralization tests. Of 850 cattle serum samples, agar gel diffusion tests gave positive results in 33.4%. Virus neutralization test was done only with the bovine and adenovirus type 2. No differences could be detected in antibody titres when the prototype strains (No. 19) and the strain isolated from sheep (ORT/111) were compared in parallel titrations. Both ruminant species were found to be infected with hovine adenovirus type 2. Neverthless, inapparent infection with these strains seemed to be less frequent among cattle than in sheep flocks.     

Seroprevalence of Neospora caninum antibodies in cattle and water buffaloes in India

Neospora caninum is now recognized as a major cause of abortion in cattle worldwide, but there is no report of N. caninum infection in cattle in India. Serum samples from 427 dairy cattle and 32 dairy water buffaloes from 7 organized dairy farms located in Punjab, India, were tested for N. caninum antibodies using a commercial monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Antibodies to N. caninum were found in 35 of 427 cattle from 6 of the 7 farms; 9.6% of cows, 5.1% of heifers, and 5.0% of calves were seropositive, suggesting postnatal transmission of N. caninum on the farm. Antibodies to N. caninum were found in 16 of 32 buffaloes tested from 2 dairy farms. In total, 64 cattle and 16 buffalo sera already tested by ELISA were also evaluated by an indirect fluorescent antibody test (IFAT) to verify ELISA results. Of the 64 cattle samples, 29 sera were negative by both tests and of the 35 ELISA-positive sera, 12 had IFAT titers of 1:100 or higher (1 had IFAT titer of 100, 2 had IFAT titer of 200, and 9 had IFAT titers of 400 or higher). Of the 16 buffalo sera positive by ELISA, 1 had an IFAT titer of 1:400. Thus, antibodies to N. caninum were demonstrated in cattle sera by 2 serologic methods. To our knowledge this is the first report of N. caninum infection in cattle and buffaloes in India.     

Study of the immunological properties of Actinomyces rimosus ribonuclease]

The immunological properties of ribonucleases from Act. rimosus were studied in comparison with RNA-ase from the cattle pancreas. The activity of anti-RNA-ase immune sera were determined by the method of specific neutralization of the effect of the above exzymes. The results of the study showed that with the use of homological RNA-ases as the antigens, the maximum level of enzymatic activity neutralization by the immune sera ranged within 70 to 90% and the capacity for induction of specific antibody production expressed in the serum titer was somewhat lower in the actinomycete RNA-ase than in the pancreatic one. When heterological antigens were used, neutralization of the RNA-ase effect by the immune sera was either not detectable or very low which is indicative of the antigenic differences in the actinomycete and pancreatic RNA-ases.     

Antibody to Viruses Affecting Cattle in Commercial Tissue Culture Grade Fetal Calf Serum

Commercial fetal calf serum (FCS) for tissue culture use was tested for neutralizing activity against several viruses which affect cattle. Certain lots of FCS contained no neutralizing activity, whereas other lots contained neutralizing activity to several viruses. It was concluded that the neutralizing activity found in certain lots of sera was due to specific antibody and that its presence could be most easily explained by the contamination of the FCS with serum from postcolostral bovine serum. A nonantibody inhibitor to vesicular stomatitis virus was also found at low levels in most lots of serum. Because those sera which had antibody had antibody to several viruses, it was suggested that the use of the micro-serum neutralization test with a few bovine viruses which are widespread in the bovine population should be satisfactory to detect FCS which was contaminated with postcolostral bovine serum.