首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.  相似文献   

2.
The qualitative profiles of the proteins synthesized by sheep oocytes at various stages of maturation were determined by electrophoretic separation in one dimension on polyacrylamide SDS gels. No change in protein synthetic pattern was observed in ooce changes had taken place in at least 12 separate protein bands. Marked alterations in the synthesis of some proteins were apparent 15 h after LH; formation of proteins in 5 of the original bands was either reduced or not detectable, while new synthesis was evident from the appearance of 7 additional bands. The pattern of proteins produced by oocytes cultured within the follicle corresponded closely with that observed in vivo: changes in synthesis were initiated about 9 h after addition of gonadotrophin and were completed by 15 h. Oocytes cultured outside the follicle in a gonadotrophin-containing medium did not exhibit a change in protein synthesis and at 15 h only those proteins produced during the early stages of maturation were being synthesized.  相似文献   

3.
A five-month-pregnant Suffolk sheep histologically diagnosed as spontaneous scrapie was studied. Western blot analysis was performed with rabbit serum against the sheep scrapie-associated fibrils (SAF). In the proteinase K (pk)-treated parental brain and spleen samples, three major bands (15 K, 18 K, and 23 K) were detected. These major bands were not detected from the placenta. Infectious agents were isolated in mice from the brain samples but not from the placental homogenates. In another case of a three-month-pregnant Corriedale sheep without any clinical sign of, but histologically diagnosed as scrapie, was also studied in a similar approach. In the parental brain samples, three major bands (15 K, 18 K and 23 K) were detected. SAF protein was not detected in the parental spleen and placenta. No bands reactive with the antiserum were detected in any other samples from the fetal brain and spleen in both cases. However, infectious agents were isolated in mice from both brain and placental homogenates. Since the placenta is an important site of natural infection, it is worthwhile to study these tissues for the epidemiological study of scrapie infection.  相似文献   

4.
The serological diagnosis of heartwater based on reactions to the immunodominant Cowdria ruminantium major antigen protein-1 (MAP-1) is impaired by the detection of false-positive reactions. In this study, the prevalence of false-positive reactions on seven heartwater-free farms in Zimbabwe was determined to be 8-94% by immunoblotting against C. ruminantium antigens. The highest prevalence of false-positives on Spring Valley Farm correlated with the presence of Rhipicephalus evertsi evertsi ticks. The other tick species found on these seven farms were Hyalomma truncatum and Hyalomma marginatum rufipes. Rhipicephalus evertsi evertsi ticks collected from Spring Valley Farm and fed on seronegative sheep caused seroconversion in one of two sheep. This sheep developed a mild febrile reaction and C. ruminantium MAP-1 antigen reactive antibodies 3 weeks after the ticks started feeding. Polymerase chain reactions (PCRs), conducted using C. ruminantium-specific primers on ticks collected from the seven farms and on some of the R. e. evertsi ticks that had caused seroconversion in one sheep, were negative. However, some of these ticks gave positive PCRs with DNA primers which amplify a 350 bp DNA fragment of the 16s rRNA gene from all ehrlichial agents indicating the presence of infection with one or more Ehrlichia species. Although attempts to isolate the cross-reacting agent from the sheep were unsuccessful, this study demonstrates that false-positive reactions with the MAP-1 C. ruminantium antigen are associated with agents transmitted by ticks.  相似文献   

5.
Parasitism was studied in white-tailed deer (Odocoileus virginianus) and domestic sheep (Ovis aries) which shared a common range in eastern West Virginia. Of 30 species of internal parasites, 11 were found in deer and 22 in sheep. Five parasites, Sarcocystis sp., Cysticercus tenuicollis, Oesophagostomum venulosum, Cooperia punctata, and Gongylonema pulchrum, occurred in both deer and sheep. An index of similarity of 17.2 suggests that the parasite faunas of these hosts are distinct, and that it is unlikely that white-tailed deer are reservoirs of common parasites of domestic sheep in the southern Appalachian region.  相似文献   

6.
Abstract— The protein patterns from retinae of mice with inherited blindness (C3H/HeJ) were compared with those from normal (DBA/1J) retinae during postnatal maturation. The number of protein bands, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate, increased in the normal retina with development and, by adulthood, 30 bands of protein were observed, with molecular weights ranging from 13,500 to 105,000 daltons. Four bands of retinal protein were suggested to be histones, and one was identified as tubulin. The patterns of protein bands from the immature C3H retinae were similar to those from the DBA retinae but, by adulthood, the pattern from the C3H retina was deficient in three bands of protein, two of which were identified as opsin and a cyclic-nucleotide phosphodiesterase.  相似文献   

7.
Some changes produced in the cell populations of intestinal lymph by infection with the enteric nematode, Trichostrongylus colubriformis, were studied in sheep regularly re-infused with all discharged lymph. Lymphocyte traffic through the intestinal lymphatic duct was reduced until day 35 of primary infection, mainly due to the absence of lymphocytes with smaller cell volumes, but was increased two-fold after day 70 and in immune sheep. Antigen-reactive lymphocytes in blood and lymph were assayed by the uptake of 3H-thymidine in cell culture stimulated by extracts from the larvae of T. colubriformis. Cells from the blood and lymph of immune sheep were highly reactive to worm antigen. A relatively smaller reactivity was present in the blood of worm-free sheep and was abolished during the first 12 days of primary infection. Antigen reactive cells were not detected in intestinal lymph until 12 days after primary infection, and in vitro antigen reactivity in intestinal lymph of immune sheep was increased after challenge with infective larvae. Responses to the mitogens, concanvalin A and phytohaemagglutinin, in cultures of cells from both intestinal lymph and blood were depressed on days 7 and 12 of primary infection. It is proposed that the diminished traffic of lymphocytes in intestinal lymph and the reduced numbers of mitogen and nematode antigen-reactive lymphocytes in both blood and intestinal lymph during the early stages of infection with T. colubriformis is closely related to the slow development of protective immunity to this parasite.  相似文献   

8.
A sheep kappa cDNA probe was isolated, characterized by sequence analysis and shown to have significant sequence identity to other kappa light chains. This probe and a sheep lambda light chain probe were used to estimate the extent of various sheep immunoglobulin light chain gene loci by Southern blot analysis of genomic DNA. The results showed that the sheep has a single hybridizing kappa constant gene and three to five kappa V segment bands. Segregation of three polymorphic bands at the lambda C locus indicated that they were products of separate C segments. Restriction fragment pattern variations were obtained using light chain probes on various sheep breeds, but no pattern or individual band was characteristic for a particular breed.  相似文献   

9.
Haemonchus contortus is a blood-feeding nematode which parasitizes the abomasum of sheep and represents a serious constraint to sheep production. Anthelmintics are currently the most common method of worm control but the worldwide development of multiple-drug resistance and issues of residues in the food chain make alternatives to anthelmintics a priority. Biotechnology-driven solutions to parasitism include vaccines and silencing of genes regulating nematode development. To pursue gene targets that may be suitable for parasite control, a two stage differential-display PCR (dd-PCR) approach was developed to observe differential gene expression between Haemonchus from immune and control sheep. Twenty-four reproducible differentially-expressed bands were identified in 60 pairs of dd-PCR comparisons. The first of these cloned and sequenced corresponded to the H. contortus 60S ribosomal protein L35A. The remaining bands are being cloned and validated and may provide new targets for parasite control.  相似文献   

10.
In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15-25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificities. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

11.
Current methods for the serodiagnosis of sheep fascioliasis show suboptimal sensitivity, specificity, or both. With the aim of developing an improved method, we fractionated native Fasciola hepatica excretory-secretory antigens (ESAs) by size-exclusion FPLC (fast protein liquid chromatography) on a Superdex 75 HR 10/30 column and then tested the serodiagnostic value of the antigens contained in each one of the 4 peaks obtained (peaks I-IV). Serodiagnostic value was assessed using sera from sheep naturally infected with F. hepatica (group A); sera from the individuals of a fluke-free herd (most of which also had other intestinal nematodes, lung nematodes, Moniezia spp., and/or Cysticercus tenuicollis) sera from a fluke-free herd (group B); sera from lambs experimentally infected with 10-40 F. hepatica metacercariae (group C); and sera from uninfected control lambs (group D). Enzyme-linked immunosorbent assay (ELISA) with peak I or II as target antigens (and to a lesser extent with peak III as target) showed reactivity with negative sera, so that it was not possible to establish cutoff values discriminating infected and uninfected animals. In contrast, when peak IV was used as target, a low cutoff value of 0.235 optical density units (mean + 4 SD) discriminated infected and uninfected animals, with 100% sensitivity and 100% specificity. ELISA with peak IV as a target identified infected animals (even animals that had received only 10 metacercariae) within 3-5 wk of infection and subsequently throughout the rest of the 14-wk monitoring period. In Western blotting analysis, again only the antigens contained in peak IV (range 7-40 kDa, under reducing conditions) were specific for diagnosis of infected animals. These results indicate that molecular sieving of F. hepatica ESAs by this procedure is a fast, simple, reproducible way of obtaining antigens useful for serodiagnosis of sheep fascioliasis.  相似文献   

12.
Raymond Miassod  Claude Got 《Planta》1984,162(5):427-433
A polyclonal antiserum was raised against highly purified RNA polymerase II from soybean embryos. Pure RNA polymerase II was fractionated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose sheets, incubated with the immune antiserum and then with iodinated protein A. Autoradiograms showed that the immune antiserum recognized all subunits of RNA polymerase II. Subunits 42, 27 and 16 kdalton were particularly reactive. Application of this transfer technique to protein extracts from soybean embryos or from cultured soybean cells allowed the identification of subunits of RNA polymerase II in the extracts. Analysis of the staining of the bands on the autoradiograms for increasing amounts of pure RNA polymerase II demonstrated that the transfer was quantitative, so that standard curves could be drawn to estimate the unknown amounts of enzyme in the extracts.Abbreviations DEAE diethylaminoethyl - SDS sodium dodecyl sulfate  相似文献   

13.
14.
(1) Membranes from sheep erythrocytes lysed with antibody and human complement were solubilized in Triton X-100 and subjected to isoelectric focusing in polyacrylamide gels containing 1% Triton X-100. Membrane-bound serum proteins were located in the gels by subsequent immunoelectrophoresis against antisera to human serum proteins. Monospecific antisera against C9 and C5 were used to locate the terminal complement complex, which is not dissociated by Triton X-100. The complex focused between pH 5.8 and pH 6.5 and was separated from the bulk of other membrane-bound serum proteins, which focused at pH ranges below than 6.0. (2) In a second step, proteins electrophoretically eluted from the gel sections containing the terminal complement complex were chromatographed on Sepharose 6B equilibrated with 0.05% Triton X-100. Fused rocket immunoelectrophoresis was used to monitor separations. This step separated the terminal complement complex from the remaining contaminating proteins. The complex eluted in a broad peak corresponding to a molecular weight range of 800000-4000000. (3) The terminal complement complex thus obtained migrated with alpha-mobility and yielded a single precipitation arc in crossed immunoelectrophoresis using polyvalent antisera to human serum proteins. A distinct precipitate was obtained with monospecific anti-C9. The presence of C5 and C6, in complex with one another and with C9 was demonstrable by immuno-double-diffusion. No immunoprecipitate was obtained with antisera to sheep erythrocyte membrane proteins. (4) Dodecyl sulfate gel electrophoresis of the complex revealed seven protein bands of 190000, 160000, 115000, 93000, 85000, 68000 and 60000 daltons. Planimetric quantitation of densitometric scans gave a molar ratio of approx. 0.7:0.3:1:1:1:2:1 for these bands, respectively. All bands stained faintly with periodate-Schiff. Two-dimensional dodecyl sulfate gel electrophoresis showed that the first two bands (190000 and 160000 daltons, probably C5b and C5c) represented proteins possessing more than one peptide chain linked by disulfide bonds. The main subunit for both bands was a protein of approximately 68000 daltons. Band 5 (83000 daltons, probably C8alpha) was split into two peptide chains of approximately 68000 and 15000 daltons. The other components were not affected by dithiothreitol treatment. (5) The dodecyl sulfate gel electrophoretograms obtained were very similar to that described by Kolb and Müller-Eberhard (Kolb, W.P. and Müller-Eberhard, H.J. (1975) J. Exp. Med. 141, 724-735) for the terminal complement complex isolated from inulin-activated serum. However, certain minor but consistent deviations were observed. A preliminary correction of the electrophoretograms is presented.  相似文献   

15.
It was found that haptoglobins of camel, cattle, horse, pig, rat, guinea pig and man form upon immunoelectrophoresis precipitation arcs with antibodies against polymeric sheep haptoglobin C, corresponding to alpha 2-globulins. The immunocrossreactivity of haptoglobins of man and various animal species towards antibody to sheep haptoglobin (100, 88.0, 75.2, 72.1, 56.3, 51.0, 41.3 and 28.0% for haptoglobin of sheep, camel, pig, cattle, man, rat, guinea pig and horse, respectively) was determined. The intensity of crossreactions between sheep haptoglobin and the proteins under study towards antibody to haptoglobin C reflects the similarity of their primary structure and, consequently, the immune homology of their molecules. Using quantitative titration, the antigenic valency values for human (6), sheep (5), cattle (4) and horse (3) haptoglobins were determined.  相似文献   

16.
The most complement (C)-sensitive type of erythrocytes (E) occurring in paroxysmal nocturnal hemoglobinuria (type III PNH E) have previously been found to exhibit approximately twofold to fourfold greater lysis than normal human E when exposed to isolated human C5b6, C7, C8, and C9 (reactive lysis), in the absence of a known source of C3- or C5-convertases or fluid-phase C3. In further studies on the mechanism of this phenomenon, we now report that C5b6-dependent binding of 125I-C7 to two samples of PNH E (greater than 95% type III) is equal to that found with normal human E at each of several C5b6 inputs tested. Lysis developed by excess C8 and C9, however, was consistently greater for the PNH E. Thus, the exaggerated sensitivity of type III PNH E to reactive lysis cannot be explained by abnormally high uptake of C5b6 or C7 from the fluid phase. Rather, the data indicate that cell-bound C5b67 sites are converted to effective hemolytic sites with greater efficiency on type III PNH E than on normal human E, assuming that the distribution of cell-bound C7 throughout both cell populations is similar. In related studies we have addressed the proposal by other investigators that C3b putatively bound to PNH E in vivo might account for their increased sensitivity to reactive lysis in vitro, by analogy to prior observations on C3b-potentiated reactive lysis of sheep E. The latter hypothesis was made more appealing by the recent discovery that type III PNH E lack an integral membrane protein, decay-accelerating factor (DAF), which in normal E accelerates the decay of membrane-bound C3 convertases. Against this hypothesis, however, is our present finding that preincubation of PNH E with four different goat or rabbit polyclonal antibodies to human C3 failed to inhibit the subsequent reactive lysis of these cells. Under these same conditions, the C3b-dependent increment in reactive lysis of sheep EAC4b3b was abrogated by pretreatment with similar dilutions of these anti-C3 antibodies, generally in association with agglutination. Furthermore, sheep EAC4b3b displayed increased 125I-C7 binding in proportion to augmented lysis, in contrast to the findings with PNH E. Therefore, deficiency of DAF in type III PNH E does not adequately explain their supranormal sensitivity to reactive lysis unless DAF can modulate the terminal lytic steps by a mechanism distinct from its effect on C3 convertase decay. Alternatively, type III PNH E could have a more general abnormality in which DAF deficiency is one manifestation and increased sensitivity to reactive lysis is another.  相似文献   

17.
Antisera reactive with the C57Bl lymphoma EL4 were induced by hyperimmunization of B6.C-H-2bm-1 (H-2bm-1) mice, a congenic mutant strain of C57Bl/6 (B6). Molecular weight determinations of EL4 surface structures detected with the congenic mutant antibodies were accomplished by electrophoretic analyses (one and two dimensions) on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Analysis of immunoprecipitates in the first dimension under reducing conditions revealed protein bands corresponding to gp 70 and its 33-kDa cleavage fragment, and two-three bands (40-45 kDa) that overlapped with those precipitated from EL4 with AS 8177, an antiserum which detects framework determinants on the T-cell heterodimeric receptor. Further analysis by diagonal electrophoresis confirmed identity of the 40- to 45-kDa proteins precipitated from EL4 with either AS 8177 or H-2bm-1 anti-EL4 sera. Additional diagonal electrophoretic studies showed that the congenic mutant antibodies do not precipitate T-cell receptor molecules from C6VL, an in vitro line derived from the C6XL T-cell lymphoma of C57Bl/ka origin, which was previously shown by some of us to express the heterodimeric T-cell receptor. Together these results demonstrate detection of the heterodimeric T-cell receptor on EL4 with congenic mutant antibodies directed against nonconstant region determinant(s), and indicate a possible linkage between MHC haplotype and the immune response to intraspecies variable regions of the T-cell receptor.  相似文献   

18.
Summary. In the past few years it has been possible by combining enzymatic cleavage of genomic DNA and the Southern blot hybridization technique to explore the endonuclease recognition site polymorphism of the MHC. HLA class I and DR and DQ alpha and beta class II specific probes as well as human C4 and Bf class III probes were used. All these probes were shown to cross-hybridize with DNA from pigs, cattle, sheep and horses. Hybridization of human genomic DNA with a class I probe showed 15–25 bands per genome depending on the enzyme used. Distinct endonucleases generated clusters of restriction fragments (RF) in HLA-informative families which correlated with HLA specificites. While numerous clusters were found associated with HLA-A alleles almost no cluster was related to HLA B or C specificities. Similarly, class II probes provided a large number of clusters. The existence of these clusters suggested that some polymorphic restriction sites are found in strong linkage disequilibrium and that the underlying mechanism might be gene conversion with heteroduplex correction. Since the degree of polymorphism detected by RF appears to be greater than the polymorphism defined by more traditional methods stronger associations between RF and pathological conditions are to be expected. Southern blot analysis was applied to unrelated pigs and sheep, as well as to families. Preliminary studies have also been performed on a few unrelated cattle and horses. Depending on the endonuclease used the HLA class I probe hybridized with around 15 bands in MHC heterozygous pigs and ruminants while up to 20 bands were found in horses. Therefore, a several-fold greater number of potential class I genes exist compared to those actually expressed. With the class II beta probe, cattle and sheep showed around 10 bands whereas 15 were observed in pigs and around 20 in horses. Based on limited results obtained with DQ alpha and beta probes and with the DR alpha probe there appeared to be fewer of these respective genes. Only one C4 gene has been detected in pig and this gene maps within the SLA region. Hybridization with the human C4 probe in cattle, sheep and horses revealed two to four bands which could possibly account for two C4 genes. To date their linkage to the MHC has not been established. The Southern blot hybridization technique represents a powerful tool for future immunogenetic studies. This is even more so in large farm animals where for various reasons it is almost impossible to conduct certain types of investigation that are easily performed in rodents or in man. Although the data are still preliminary, they already extend our knowledge of the MHC in domestic animals far beyond what could have been reasonably anticipated using conventional methods.  相似文献   

19.
Characterization of the baboon erythrocyte C3b-binding protein   总被引:2,自引:0,他引:2  
E from primates demonstrate type 1 CR (CR1) with binding specificities for C3b and C4b. In the present study we characterized the E C3b-binding protein of baboons. We showed that three out of four mouse mAb and one polyclonal antiserum, raised against human E CR1, cross-reacted with baboon E. In addition, one anti-human CR1 mAb (1B4) and a polyclonal anti-human CR1 inhibited the binding of C3b opsonized immune complexes to baboon E. Finally, a mAb to human CR1 (E11) recognized epitopes on E of a variety of nonhuman primates, including baboons. SDS-PAGE analysis of biochemically purified baboon E membrane fractions reactive with E11 demonstrated a 65-kDa protein as a major component. Affinity absorption and elution experiments verified this protein to be E11 reactive as well as a C3b binding protein. E surface radiolabeling, followed by C3i affinity purification, confirmed that this 65-kDa protein is the only C3b-binding protein present on the baboon E membrane. We postulate that the baboon E 65-kDa protein is the equivalent of the human E CR1. In addition, there appear to be antigenic similarities between the baboon E 65-kDa protein and the human E CR1.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号