Identification of the PR prealbumin proteins in horse serum

The Pr protein, which is one of the major equine acidic prealbumins and which consists of a large number of phenotypes, has been studied with regard to its chemical identity.Serum samples of known Pr phenotype which had been treated with varying amounts of bovine trypsin were subjected to starch gel electrophoresis at pH 4.8. When a certain amount of trypsin was used, the Pr protein was markedly affected, whereas the other acidic prealbumins retained their normal electrophoreitic pattern.Extracts from three different regions of the acidic prealbumin field were tested by the casein precipitating inhibition test (CPI-test). Marked antitrypsin effect appeared against the extract from the Pr zone but not against the extracts from the two other acidic prealbumin zones.When acidic starch gel electrophoresis was combined with the CPI-test, a broad inhibitory zone appeared in the area of the Pr proteins.Pr protein was isolated by the use of agarose gel electrophoresis and sepharose column chromatography. The isolated protein which was tested for purity by gel electrophoresis had a molecular weight of about 60,000.It is concluded that the equine Pr protein corresponds to αi-antitrypsin.     

Properties and isoenzymes of acid phosphatase in erythrocytes and various tissues (kidney, liver, spleen, small intestine mucosa, testis, myocardial and skeletal musculature) of cattle]

The substrate that was split most rapidly by acid phosphatase was p-nitrophenylphosphate. Two peaks of activity were obtained at pH 4.6-4.8 and 5.1-5.4. The enzyme remained stable for a long time when refrigerated. It was inhibited strongly by urea and tartrate, and slightly by fluoride and L-phenylalanine. Mercaptoethanol elicited pronounced activation of the enzyme. Four different forms of isoenzyme, giving rise to 11 phenotypes, were identified. A suitable analytical technique was electrophoresis on polyacrylamide gel with phosphate-citrate buffer. Mean activity was 3.15 +/- 0.41 units per gramme of haemoglobin haemolysate. Some of the isoenzyme preparations showed considerable variation in activity. There was no change in enzyme activity after temporary hypomagnesaemia. Acid phosphatase activity was high in testis, kidney and intestinal mucosa; myocardium, liver and spleen showed moderate activity. Five isoenzymes were demonstrable in a starch column and six in PAA gel.     

Quantitative Comparisons of Acidic Prealbumin (Pr) Phenotypes in Horses

Comparisons of Pr protein amounts in horse sera have been performed using Mancini et al.’s (1965) immunodiffusion technique. Relative values against a chosen standard of 100 % were determined for a total of 435 horses.There was considerable variation between horses, the highest Pr value being 125 % and the lowest 50 % of the standard. In animals of the same Pr phenotype the mean Pr values were significantly higher (P < 0.001) in foals than in mares. In Norwegian Trotter horses the Pr value of Pr NN animals was significantly higher than that of Pr SS phenotypes, whereas the mean Pr values of Pr SS was significantly higher than that of Pr UU Warmblood Trotter horses, the Pr value of Pr SS being 90 % and the Pr-UU 80 % of that of Pr NN.No difference between sexes with regard to Pr values was found.     

Effect of polymorphism in egg white lysozyme on muramidase and antibacterial activities as well as hatchability in the Japanese quail (Coturnix japonica)

Lysozyme is one of the best characterized antimicrobial proteins in egg white. Three phenotypes of egg white lysozyme in Japanese quail, Coturnix japonica, (namely fast; slow; and the combination, FS) were observed by acid polyacrylamide gel electrophoresis. The fast phenotype showed faster mobility on Acid-PAGE than the slow phenotype. Comparison of the coding sequences for lysozyme derived from the slow and fast phenotypes revealed a nonsynonymous SNP at nucleotide position 115 from the translation initiation site, which alters AA sequence of lysozyme. This nonsynonymous SNP converted glutamine (Q) in the slow phenotype to lysine (K) in the fast phenotype at AA residue 21 of mature lysozyme (Q21K). Here, we investigated the effect of these phenotypes on muramidase activity, antibacterial activity, and hatchability. Muramidase activity toward isolated cell walls of Micrococcus lysodeikticus was in the order: fast allozyme > slow allozyme > chicken (Gallus gallus), but no significant difference was found among the 3 (P > 0.05). Antibacterial activity against live Staphylococcus aureus cells was significantly greater for the fast allozyme than the slow allozyme from 20 h after incubation (P < 0.05). For the antibacterial effects against live Escherichia coli cells, the activity of fast was significantly higher than that of slow at 16 h after incubation (P < 0.05). Hatchability was estimated for reciprocal crosses of Japanese quail with the FF (fast) and SS (slow) genotypes. Hatchability was 92.5% in FF male × SS female crosses and 87.2% in SS male × FF female crosses. A Cochran-Mantel-Haenszel test revealed a significant difference between the crosses (P < 0.05) and indicated that the female-derived slow phenotype led to improved rates of hatching. Our results suggest that the nonsynonymous SNP in Japanese quail lysozyme influences the electrophoretic migration, muramidase activity, and antibacterial activity of the protein, in addition to the hatchability of the eggs. These results demonstrate, for the first time, a significant difference in antibacterial activity and hatchability between 2 lysozyme phenotypes in Japanese quail.     

Restriction-endonuclease analysis of Australian isolates of Leptospira interrogans serovar hardjo from cattle with agalactia and abortion

SUMMARY Polyacrylamide gel electrophoresis and agarose gel electrophoresis were used to resolve restriction endonuclease digests of 20 Australian isolates of Leptospira interrogans cultured from urine samples of cattle with agalactia and abortion. The restriction endonuclease profiles of 19 isolates closely matched the profiles of L interrogans serovar hardjo subtype hardjobovis reference strains. The remaining isolate had a different restriction profile from subtype hardjobovis and subtype hardjoprajitno reference strains and was serologically identified as serovar pomona. Silver staining of polyacrylamide gels gave enhanced resolution of restriction fragments compared with the traditional method of ethidium bromide staining of agarose gels.     

Studies on the properties of acid erythrocyte phosphatase in sheep and the isoenzymes of sheep and goat acid erythrocyte phosphatase]

Ovine erythrocytic acid phosphatase showed two peaks of activity at pH 5.0 and 5.7 in acetate buffer with p-nitrophenylphosphate as substrate. The enzyme was only slightly inhibited by fluoride and L-phenylalanine, but high concentrations of urea strongly inhibited it. Activity of the enzyme was greater in goat erythrocytes than in sheep. By means of starch electrophoresis, three isoenzymes belonging to nine types were separated from the ovine enzymes, while three isoenzymes of five types were present in goats. Electrophoresis in polyacrylamide gel was suitable for detecting the rapidly migrating isoenzymes.     

Evaluation of hyaluronidase activity in equine and bovine sera and equine synovial fluid samples by use of enzyme zymography

OBJECTIVE: To investigate the activities of hyaluronidases in equine sera and synovial fluid samples and sera from fetal and adult bovids and evaluate the extent to which the degradation of hyaluronan is influenced by chondrocytes. SAMPLE POPULATION: Commercial and noncommercial samples of equine (n = 6) and bovine (6) sera and 16 synovial fluid samples from horses. PROCEDURE: Hyaluronidase activities in sera and synovial fluid samples were assessed via enzyme zymography (performed at pH 4, 5, 6, or 7). Chondrocytes were isolated from equine cartilage and cultured with or without hyaluronan (1 mg/mL); the degradation of hyaluronan was assessed via agarose gel electrophoresis. RRESULTS: Hyaluronidase activity was detected in equine sera and synovial fluid samples at pH 4, but not at pH 7, and in bovine sera at both pH values. In all samples at pH 4, a major band of activity (molecular weight, approx 60 kd) and some additional higher molecular weight bands were detected; high- and low-molecular-weight activities were detected in bovine sera at pH 7 Hyaluronan in tissue culture medium with or without fetal calf serum was degraded in the presence, but not the absence, of equine chondrocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Hyaluronidase activity was detected in equine sera and synovial fluid at pH 4 and in bovine sera at pH 4 and 7. Primary chondrocytes in monolayer culture can degrade exogenous hyaluronan. Modulating native hyaluronidase activity may offer a new approach to improve the quantity and quality of hyaluronan in articular joints.     

Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

山丹马6-磷酸葡萄糖脱氢酶及葡萄糖磷酸异构酶的多态性研究

用淀粉凝胶电泳及琼脂覆盖技术对山丹马的血液红细胞6磷酸葡萄糖脱氢酶和葡萄糖磷酸异构酶的电泳变异进行了测定。在6磷酸葡萄糖脱氢酶座位发现了3种表现型,即FF,FD和FS,其频率分别为0.958,0.021和0.021。在葡萄糖磷酸异构酶座位发现了两种表现型,即II和FI,其频率分别为0.771和0.229。等位基因频率直接通过表型计算出:PGDF为0.979,PGDD为0.0104,PGDS为0.0104;GPIF为0.1146,GPII为0.8854。亲子关系排除概率在6PGD和GPI座位点分别为0.1009和0.0912。两座位的个体识别概率分别为0.081和0.353。     

Replacement of sodium chloride with dextran or polyethylene glycol for immunoprecipitation of lipopolysaccharide with antibodies in chicken or turkey sera

Dextran or polyethylene glycol could replace sodium chloride in agarose gels for inducing immunoprecipitation of $\text{Pasteurella multocida}$ lipopolysaccharides with antibodies in chicken or turkey sera. Resolution of immunoprecipitates was best when 3% concentrations of either dextran or polyethylene glycol were used. Higher concentrations increased opacity of the gels. Nonspecific precipitation of serum or γ-globulin fractions in gels was caused by the electrophoresis buffer, dextran, and polyethylene glycol. Dialysis of serum or γ-globulin fractions against the electrophoresis buffer and soaking gels in buffers of pH greater than 7.0 that contained 3% polyethylene glycol reduced nonspecific precipitation. Incorporation of dextran or polyethylene glycol into gels enhanced immunoprecipitation in rocket immunoelectrophoresis but resulted in slower mobility of antigen.     

Detection of c-kit mutations in canine mast cell tumors using fluorescent polyacrylamide gel electrophoresis.

Mutations consisting of internal tandem duplications (ITDs) in exons 11 and 12 of the proto-oncogene c-kit are found in 30-50% of malignant canine mast cell tumors (MCTs). Traditionally, identification of such mutations in tumor specimens has been undertaken using standard polymerase chain reaction (PCR) and agarose gel electrophoresis. This procedure is limited to the detection of insertions and deletions larger than 9 base pairs in size. The purpose of this study was to compare the efficiency and accuracy of standard agarose gel electrophoresis with fluorescent polyacrylamide gel electrophoresis (PAGE) for the detection of ITDs in canine MCTs. The results of this study demonstrate that PAGE of labeled PCR products accurately predicts the size of the ITD in each tumor. In addition, other small insertions and deletions were not identified, suggesting that if they occur in canine MCTs, they do so infrequently. Because fluorescent and polyacrylamide formats are automated and have better resolution than agarose gels, fluorescent PAGE provides a more accurate, economical, and higher throughput method for the detection of c-kit mutations in canine MCTs.     

海南水牛血液蛋白多态性研究

采用醋酸纤维薄膜、聚丙烯酰胺凝胶电泳和淀粉凝胶电泳对９７头海南水牛的血红蛋白、血清运铁蛋白和白蛋白多态性进行检测，并计算其基因型和基因频率，发现６头血红蛋白三条变异体和１头一带变异体。     

Thin-layer agarose isoelectric focusing: an improved technique for determining sheep hemoglobin type

An improved technique for rapid screening of sheep flocks for hemoglobin (Hb) type is presented. This technique, isoelectric focusing (IEF) on thin-layer agarose gels is simple, rapid, inexpensive and is suitable for screening large numbers of sheep for Hb type. With this technique, up to 100 sheep blood samples can be prepared, tested and interpreted within 2 h after samples are drawn. The new technique was shown to provide better resolution than polyacrylamide gel electrophoresis (PAGE) and was able to resolve samples in which the Hb had become partially degraded. These same samples could not be resolved by PAGE. The use of a special electroendosmosis-free grade of agarose provided resolution essentially equal to polyacrylamide as a matrix for IEF. The advantages are that the casting of the agarose gels is considerably easier, the focusing of samples is more rapid, staining and destaining times are greatly reduced and hazards from potential neurotoxicity of acrylamide are eliminated. Blood from 138 ewes at the Oregon State University Sheep Center was examined by the new agarose IEF technique to determine and demonstrate its usefulness for screening. No difficulty was encountered with interpretation of any of the samples. Frequencies of the HbA and B alleles were similar to those found in earlier studies when polyacrylamide tube gel electrophoresis was used. The observed frequencies were also similar to those expected with the population in Hardy-Weinberg equilibrium.     

The characterisation of equine interleukin-1

Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.     

Methylation at the CpG doublet in equine adenovirus genome

Viral DNA obtained from the equine adenovirus propagated in equine transitional cell carcinoma (ETCC) cells and in equine fetal dermis cells were compared by cleaving with isoschizomeric restriction enzymes, HpaII and MspI, and then electrophoresed in 1.4 per cent agarose gels. Differences between the HpaII and MspI cleavage patterns were evident in viral DNA obtained only from the equine adenovirus propagated in ETCC cells, suggesting site specific methylation at CpG sequences.     

Studies of Transferrin Polymorphism in Swedish Cattle Using Agarose Gel Electrophoresis

The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with ⁵⁹Fe. Six existing phenotypes based on the alleles Tf^A, Tf^D and Tf^E could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencies between the Swedish Red and White and the Swedish Friesian.     

Characterization of Nitrate-Reducing and Amino Acid–Using Bacteria Prominent in Nitrotoxin-Enriched Equine Cecal Populations

Horses can be at risk for nitropoisoning by consuming plants containing 3-nitro-1-propionic acid or 3-nitro-1-propanol and to a lesser extent by plants containing nitrate. Populations of equine cecal microbes enriched for enhanced rates of 3-nitro-1-propionic acid (NPA) or nitrate metabolism were cultured for NPA- or nitrate-metabolizing bacteria on basal enrichment medium or tryptose soy agar supplemented with either 5-mM NPA or nitrate and under H₂:CO₂ (20:80) as the energy source. After 72 hours, separated colonies picked from plates, or roll tubes were cultured in fresh broth medium for 72 hours and then identified by 16S rRNA gene sequencing. Isolates from the NPA-enriched populations were identified as Streptococcus lutetiensis (five strains), Escherichia coli (two strains), and Sporanaerobacter acetigens (one strain). Strains isolated from nitrate-enriched populations were identified as Escherichia coli (one strain) and Wolinella succinogenes (three strains). None of these strains degraded NPA. Enriched populations of equine cecal microbes, the isolated pure strains and the type strain of Denitrobacterium detoxificans, a competent NPA-metabolizing microbe, were examined using denaturing gradient gel electrophoresis (DGGE). The DGGE analysis indicated that none of the strains in the enriched population of equine cecal microbes were similar to D. detoxificans. However, we report for the first time the isolation of the anaerobic amino acid–using Sporanaerobacter acetigenes from the equine cecum.     

Agarose gel electrophoretic separation of blood serum proteins in cattle.

The agarose gel electrophoresis described by Johansson (1972) was modified so that a buffer of pH 7.9 was used in the gel, whereas the buffer in the electrode vessels had a pH of 8.6.The cattle blood serum protein picture is described in detail. The β₁-globulin zone shows a very distinct picture of the genetically polymorphic bovine transferrins. The region between the α- and β-globulins shows a number of faint and often very distinct bands. A faint background staining over the whole electrophoretogram may partly be caused by a rather strong lipoprotein in the α₁-region, lipids thus having migrated all over the electrophoretogram.The modified method described is well suited as a “screen electrophoresis” for cattle serum and is also useful e.g. in studying bovine transferrin polymorphism.     

福安水牛血液蛋白多态性研究

采用醋酸纤维薄膜、聚丙烯酰胺凝胶电脉及淀粉凝胶电脉对９１头福安水牛的血红蛋白、血清运铁蛋白和白蛋白多态性进行检测，并计算其基因型和基因频率，并发现５头血红蛋白３条带的变异体和１头ＢＸ型白蛋白稀有变异体。     

Sex determination by simultaneous amplification of equine SRY and amelogenin genes

A quick method for sex determination of horses was developed. Simultaneous amplification of the equine sex-determining region of the Y chromosome gene (SRY) and amelogenin gene (AMEL) accomplished the determination of the presence of both the Y chromosome and SRY gene. In agarose gel electrophoresis, a normal stallion showed 1 SRY band and 3 AMEL (AMELX, AMELY, and AMELX/AMELY heteroduplex) bands, and a normal mare showed a single AMELX band. In XY-mares, 3 AMEL bands were detected as in a normal stallion, but no SRY band. The present method enables a quick diagnosis for XY-mare prior to cytogenetic analysis.