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61.
Serologic methods for detection of Pasteurella multocida infections in nasal culture negative rabbits 总被引:2,自引:0,他引:2
An agar gel-diffusion test (AGDT) and an enzyme-linked immunosorbent assay (ELISA) were utilized to detect serum antibodies against Pasteurella multocida in naturally infected rabbits derived originally from a Pasteurella-free colony. The antigen used in both assays was purified from a serotype 3 (P-1059) strain of P. multocida. Among 47 serum samples tested 15 (32%) were seropositive; 12 (26%) of which were both AGDT and ELISA-positive, while 3 (6%) were ELISA-positive only. All rabbits examined were normal clinically and negative to repeated nasal cultures, but subsequent cultures at necropsy demonstrated the presence of P. multocida in 11 of the AGDT-positive rabbits and in 14 of the ELISA-positive rabbits. The organism was isolated most frequently from the naso-oropharynx and the tympanic bullae. Serotyping of isolates recovered from the nasopharynx were determined to be serotype 3 or 3,12. Ten seronegative rabbits also were necropsied and none were found harboring P. multocida. These preliminary data indicate that the application of an enzyme-linked immunosorbent assay may prove efficacious in identifying apparently healthy, consistently nasal culture-negative rabbits as subclinical carriers of P. multocida. 相似文献
62.
The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca2+concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca2+ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca2+. In the presence of Ca2+ the two Tms disappeared and a new higherTm appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca2+. Thechromatin reconstituted in the presence of EGTA showed a lowerTm than the chromatin reconstituted in the presence of Ca2+.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca2+. (Received August 12, 1985; Accepted December 7, 1985) 相似文献
63.
Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers 总被引:1,自引:0,他引:1
K Miyata Y Yamamoto M Ueda Y Kawade K Matsumoto I Kubota 《Journal of biochemistry》1986,99(6):1681-1688
A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation. 相似文献
64.
Subaxolemmal cytoskeleton in squid giant axon. II. Morphological identification of microtubule- and microfilament-associated domains of axolemma 总被引:4,自引:2,他引:2
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In the preceding paper (Kobayashi, T., S. Tsukita, S. Tsukita, Y. Yamamoto, and G. Matsumoto, 1986, J. Cell Biol., 102:1710-1725), we demonstrated biochemically that the subaxolemmal cytoskeleton of the squid giant axon was highly specialized and mainly composed of tubulin, actin, axolinin, and a 255-kD protein. In this paper, we analyzed morphologically the molecular organization of the subaxolemmal cytoskeleton in situ. For thin section electron microscopy, the subaxolemmal cytoskeleton was chemically fixed by the intraaxonal perfusion of the fixative containing tannic acid. With this fixation method, the ultrastructural integrity was well preserved. For freeze-etch replica electron microscopy, the intraaxonally perfused axon was opened and rapidly frozen by touching its inner surface against a cooled copper block (4 degrees K), thus permitting the direct stereoscopic observation of the cytoplasmic surface of the axolemma. Using these techniques, it became clear that the major constituents of the subaxolemmal cytoskeleton were microfilaments and microtubules. The microfilaments were observed to be associated with the axolemma through a specialized meshwork of thin strands, forming spot-like clusters just beneath the axolemma. These filaments were decorated with heavy meromyosin showing a characteristic arrowhead appearance. The microtubules were seen to run parallel to the axolemma and embedded in the fine three-dimensional meshwork of thin strands. In vitro observations of the aggregates of axolinin and immunoelectron microscopic analysis showed that this fine meshwork around microtubules mainly consisted of axolinin. Some microtubules grazed along the axolemma and associated laterally with it through slender strands. Therefore, we were led to conclude that the axolemma of the squid giant axon was specialized into two domains (microtubule- and microfilament-associated domains) by its underlying cytoskeletons. 相似文献
65.
Freeze denaturation of enzymes and its prevention with additives 总被引:3,自引:0,他引:3
Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media. 相似文献
66.
M Shimidzu H Shindo U Matsumoto K Mita M Zama 《Archives of biochemistry and biophysics》1985,241(2):692-695
Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5. 相似文献
67.
Subclass-specific antibody-dependent interactions (binding and triggering) between macrophages and supported lipid bilayers have been studied. Percentages of mouse macrophage binding (J774 cell line) to the lipid bilayers were dependent on mouse monoclonal IgG subclasses. The efficiencies were as follows: IgG1 = IgG2a greater than IgG2b greater than IgG3. Furthermore, macrophage triggering (spreading) was more efficient on IgG2a- or IgG1-coated lipid bilayers than on IgG2a, IgG3, or non-specific rabbit IgG. The present experiments show also that phospholipid molecules are able to flip-flop from one side of a supported planar bilayer membrane to the other with a half-life of 10 h-1 day at 25 degrees C. 相似文献
68.
Distinction and similarity in the structure of histones H1 and H5 as indicated by 13C nuclear-magnetic-resonance spectroscopy 总被引:1,自引:0,他引:1
The 13C nuclear magnetic resonance studies have been carried out on histones H1 and H5, by focusing our interest on possible formation of specific salt bridges between acidic and basic amino acid residues in the proteins and also on the structural difference between the two proteins. The 13C chemical shift and pKa values of the carboxyl group of glutamic acid residues in the histones coincided with those of free glutamic acid. Based on this result and another experiment using completely modified lysine residues in the histones, no evidence for a specific interaction between acidic and basic residues has been found. It has also been shown that the pH-effects of aliphatic and aromatic resonances are quite different between H1 and H5, suggesting that the globular domain of H5 is more stable than that of H1. The correlation time (1.5 ns) for the alpha-carbons of H5 estimated from 13C nuclear Overhauser enhancement was twice as long as that of H1 (0.9 ns), indicating that the backbone in the N-terminal and C-terminal domains of H5 is less mobile than that of H1. 相似文献
69.
Catecholamine release from chromaffin cells in response to carbamylcholine and high K+ is transient. Monitoring intracellular free calcium ([Ca2+]i) using quin2 demonstrated a transient rise in [Ca2+]i in response to carbamylcholine. The termination of secretion due to carbamylcholine is probably a consequence of the return of [Ca2+]i to resting levels as the nicotinic receptors desensitise. Depolarisation with 55 mM K+ led to a long-lasting rise in [Ca2+]i which persisted after the secretory response had terminated. The maintained rise in [Ca2+]i appeared to be due to continued opening of verapamil-sensitive Ca2+ channels. These results suggest that inactivation of voltage-dependent Ca2+ channels does not account for the transient nature of the secretory response in chromaffin cells. 相似文献
70.
A mathematical theory is developed for computing the probability that m genes sampled from one population (species) and n genes sampled from another are derived from l genes that existed at the time of population splitting. The expected time of divergence between the two most closely related genes sampled from two different populations and the time of divergence (coalescence) of all genes sampled are studied by using this theory. It is shown that the time of divergence between the two most closely related genes can be used as an approximate estimate of the time of population splitting (T) only when T identical to t/(2N) is small, where t and N are the number of generations and the effective population size, respectively. The variance of Nei and Li's estimate (d) of the number of net nucleotide differences between two populations is also studied. It is shown that the standard error (Sd) of d is larger than the mean when T is small (T much less than 1). In this case, Sd is reduced considerably by increasing sample size. When T is large (T greater than 1), however, a large proportion of the variance of d is caused by stochastic factors, and increase in the sample size does not help to reduce Sd. To reduce the stochastic variance of d, one must use data from many independent unlinked gene loci. 相似文献