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1.
Hyperimmune sera against spherules and against arthrospores of Coccidioides immitis were prepared by inoculation of rabbits. The antibody content of these sera was studied by the agar gel diffusion method. It was observed that antispherule pooled sera formed multiple precipitin bands with extracts of spherules and of arthrospores. The antiarthrospore pooled serum, however, failed to precipitate with the spherule extract, and formed a single band in the presence of an arthrospore solution. When the spherule and the arthrospore extracts were tested with a variety of different antisera, it was observed that the spherule preparation formed bands only in combination with anti-purified spherule pooled serum, whereas the arthrospore extract precipitated with anti-purified spherule, antiarthrospore, and anti-Histoplasma capsulatum pooled sera. It was also observed that a spherule culture supernatant solution formed five precipitin bands in combination with anti-spherule pooled sera, formed one band with pooled antiserum from rabbits with coccidioidomycosis, and did not precipitate in the presence of antiarthrospore pooled serum. Coccidioidin, however, formed two bands in the presence of any of these antisera. It was therefore concluded that extracts from the spherule phase of C. immitis differed from solutions obtained from the arthrospore and mycelial phases.  相似文献   

2.
Summary A sonicated spherule preparation was more reactive than a sonicated arthrospore antigen in complement fixation tests with a pooled serum from rabbits with experimental coccidioidomycosis. The reactivity of the sonicated spherule approximated the reactivity of coccidioidin. When the sonicated spherule was separated into its supernatant and sediment fractions, both preparations exhibited serological activity.
Zusammenfassung Ein mit Schallwellen hergestelltes Kügelchen-Präparat war activer in dem Komplement-Tests mit Blutserum von Kaninchen mit einer Coccidioidomycosisinfektion als ein mit Schallwellen hergestelltes Arthrosporantigen. Die Reaktivität der mit Schallwellen hergestellten Kügelchen war der des Coccidioidin ähnlich. Wurden diese Kügelchen in Niederschlag und Lösung getrennt, so hatten beide Präparate serologische Aktivität.


Part of a dissertation submitted to the Graduate School of Duke University in partial fulfillment of requirements for the Ph.D. degree.This work was supported by contract with the Department of the Army, Fort Detrick, Frederick, Maryland.In conducting the research reported herein, the investigators adhered to Guide for Laboratory Animal Facilities and Care established by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, NAS—NRC.  相似文献   

3.
Complement-fixation tests of three different antigen-bovine antibody systems, two antibacterial and one antiviral, were set up with or without normal bovine serum supplement. At the end of the fixation period all mixtures were tested for whole complement activity and for first, second, third and fourth complement components using the conventional, crude reagents R1, R2, R3 and R4. The increased fixation in mixtures containing the bovine serum supplement mainly reflected a greater decrease in second and fourth component activity than in the antigen-bovine antibody mixtures with non-supplemented complement. The decline in first component activity was relatively less. The results of tests for residual third component activity were not consistent.  相似文献   

4.
Kun E  Kirsten E  Hakam A  Bauer PI  Mendeleyev J 《FEBS letters》2008,582(18):2709-2713
The H-bonded complex of ATP with Arg 34 of Zn2+ finger I of poly-ADP-ribose polymerase-1 (PARP-1) determines trans-oligo-ADP-ribosylation from NAD+ to proteins other than PARP-1. This mechanism was tested in lysolecithin fractions of non-malignant and cancer cells separately and after their recombination. Cellular PARP-1 activity was recovered when the centrifugal sediment was recombined with the supernatant fraction containing cellular ADP-ribose oligomer acceptor proteins. Combination of the matrix fraction (Mx) of cancer cells (lacking OXPHOS) with its supernatant had the same PARP-1 activity as the Mx alone. The supernatant of non-malignant cells was replaced by glycolytic enzymes as ADP-ribose acceptor. The hexokinase activity of the supernatant increased when OXPHOS of intact cells was uncoupled by carbonyl cyanide 4-(trifluoro methoxy) phenylhydrazone. trans-ADP-ribosylation was demonstrated by polyacrylamide gel electrophoresis.  相似文献   

5.
Tumor-specific antigens and antisera were prepared for eight of the oncogenic simian adenoviruses. Complement-fixation tests revealed three distinct serological subgroups. This grouping was maintained in studies of virus-infected cells (T antigens) although high titered preparations were obtained for only the major subgroup I. The current grouping is as follows: (I) SV1, SV11, SV25, SV33, SV34, SV38; (II) SV20, SV23; (III) SA 7. Antigens from each subgroup were rapidly inactivated at 56 C, and group II and III antigens were also markedly inactivated at 37 C. One of the tumors (SV1) also contained SV40 T antigen, suggesting origin from a simian adenovirus-SV40 "hybrid."  相似文献   

6.
Binding activity of a putative central neurotransmitter, L-glutamic acid, was examined in the supernatant preparations solubilized from rat retinal membranes by Nonidet P-40. [3H]Glutamate binding activity increased linearly with increasing concentrations of the solubilized proteins up to 15 micrograms. The binding activity reached an equilibrium within 10 min at 2 degrees C, while increasing with incubation time up to 60 min at 30 degrees C. Addition of an excess of nonradioactive glutamate rapidly decreased the activity at 30 degrees C. Scatchard analysis revealed that the solubilized retinal binding activity consisted of a single component with a KD of 0.25 microM and a Bmax of 57.4 pmol/mg protein. The solubilized binding activity exhibited a stereospecificity and a structure selectivity to L-glutamate, and was abolished by quisqualate, L-glutamate diethyl ester, and DL-2-amino-3-phosphonopropionate. None of the other agonists and antagonists for the central excitatory amino acid receptors affected the binding activity. Reduction of incubation temperature from 30 degrees C to 2 degrees C resulted in a drastic attenuation of the binding activity due to decrement of the number of the apparent binding sites. Cation-exchange column chromatography revealed that unidentified radioactive material was in fact formed during the incubation of [3H]glutamate with the retinal preparations at 30 degrees C. These results suggest that retinal [3H]glutamate binding activity may be derived at least in part from the quisqualate-sensitive membranous enzyme with a stereospecific and structure-selective high affinity for the central neurotransmitter.  相似文献   

7.
Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.  相似文献   

8.
Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.  相似文献   

9.
1. Plastid and mitochondrial preparations were obtained by density-gradient centrifugation of homogenates made by gentle disintergration of avocado fruit mesocarp and cauliflower bud tissue. 2. The mitochondrial preparations had respiratory activity but did not incorporate [1-14C]acetate into fatty acids. 3. The plastid preparations incorporated [1--14C]acetate into the range of fatty acids found in the parent tissue. No fatty acid synthetase activity could be detected in the 12000g supernatant of these homogenates. 4. Homogenates produced by rupture of the tissue in an Ato-Mix blender and plastid preparations disintegrated by ultrasonic treatment both had fatty acid synthetase activity which did not sediment at 105000g and which formed mainly [14-C]stearate from [2-14C]malonyl-CoA. 5. It is concluded that the plastids are the principal site of fatty acid biosynthesis in the tissues studied.  相似文献   

10.
Spore differentiation and, in particular, arthrosporogenesis in a clinical strain of T. mentagrophytes was investigated using a variety of methods and by altering environmental conditions. Results are discussed with reference to the in vivo situation. Arthrospores were obtained in the presence of increased CO2 tension but not increased N2 tension. High humidity was necessary for arthrospore formation but maturity (i.e. crops of single spores) was associated with conditions of reduced humidity. Desiccation reduced arthrospore viability. Glucose and peptone based media were suitable for arthrospore formation. Arthrospores were produced at 30 degrees C and 37 degrees C, but 30 degrees C is preferred since chlamydospores were prevalent at 37 degrees C. Conditions for production of arthrospore, microconidial and mycelial suspensions are presented.  相似文献   

11.
cAMP-dependent protein kinase was found in the sediment obtained by centrifuging a homogenate of sea urchin embryos at 10,000g for 20 min, and was solubilized with 1% Triton X-100. This enzyme was eluted at 0.16 M NaCl in a linear concentration gradient on a DEAE-cellulose column, at which cAMP-dependent protein kinase found in the supernatant was also eluted. The enzyme activity was enhanced about 1.5-fold in the presence of 1 μM cAMP, and increased somewhat by adding cGMP or cIMP. The activation by cAMP of protein kinase in the sedimentable fraction was lower than in the supernatant fraction. The properties of the enzyme found in the 10,000g sediment and in the supernatant differ somewhat. The activity of the cAMP-dependent protein kinase in the 10,000g sediment was high in the embryos at the blastula, the swimming blastula, and the mesenchyme blastula stages. On the other hand, the activity was undetectable in unfertilized eggs and in embryos at the morula, the gastrula, and the pluteus stages.  相似文献   

12.
Partition of free dolichol in human urine.   总被引:1,自引:0,他引:1  
We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol.  相似文献   

13.
The activity of an NADPH-dependent PGE2-9-ketoreductase has been demonstrated in rat and human skin. This activity is localized in the high speed supernatant fraction, indicating the presence of an active PGE2-9-ketoreductase associated with the cytoplasmic fraction of the skin. Transformation of PGE2 into PGF2alpha is enhanced by skin specimens from psoriatic plaques and EFA-deficient rats, both characterized by excessive cellular proliferation and increased NADPH production. Incubations of the 105,000 g supernatant fractions from normal and EFA-deficient rats demonstrated that the activity of the PGE2-9-ketoreductase was elevated in high speed preparations from EFA-deficient rats. Results from these studies suggest that the increased activity of PGE2-9-ketoreductase observed in skin from human psoriatic plaques and EFA-deficient rats may be due in part to the increased generation of NADPH by these tissues and in part to alteration of the PGE2-9-ketoreductase by the excessive proliferation of the tissues.  相似文献   

14.
The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig greater than man greater than cat greater than dog greater than rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 times g. The Km for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the Ka for Mn2+ in particulate preparations (0.3-0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8-2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005-10.0 mM Ca2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 times g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4-4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10-4-10-8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012-1.2 mM GTP, 0.3-10.0 mM Mn2+ and 0.005-10.0 mM Ca2+ or 0.0012-1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzyme-lipid suspensions were assayed in the presence of cholinergic agents.  相似文献   

15.
In both supernatant and sediment of thyroid tissue homogenate phospholipase and lysophospholipase activities were demonstrated. In the supernatant, using 1-acyl-2[1-14C]linoleoyl-sn-glycero-3-phosphorocholine in the presence of sodium taurocholate, phospholipase A1 activity with pH optima at 3.6 and 4.8 and phospholipase A2 activity with pH optima at 3.6 and 5.7 were found. The sediment showed mainly phospholipase A2 activity with a pH optimum at pH 6.5. Lysophospholipase activity (optimum pH 7--8), USING 1-[9,10-(3)H]stearyl-sn-glycero-3-phosphorocholine as a substrate was present in both supernatant and sediment. Enzyme assays performed on subcellular fractions suggest the soluble phospholipases to be of lysosomal origin and the solubilized phospholipase A2 activity of homogenate sediment to be of microsomal origin. Incubations with 3H-14C mixed labelled phosphatidylcholine further confirmed the above observations.  相似文献   

16.
The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the 'microsomes' (13000-80000 X g sediment) from pea stem tissue is strongly influenced by concentration of Mg2+ in the homogenization medium. The absence of Mg2+ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at 80000 X g upon addition of 5-10 mM Mg2+ (or Mn2+, Ca2+, Zn2+) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl2 the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg2+. Microsomes prepared with Mg2+ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.  相似文献   

17.
We report that ATP enhances the activity of galactosyltransferase-I, which synthesizes the linkage region between glycosaminoglycan chains and the core proteins of proteoglycans. The enzyme activity in cell-free fractions prepared from cultured human skin fibroblasts was measured by high-performance liquid chromatographic detection of galactosyl-xylosyl-(4-methylumbelliferone) produced from 4-methylumbelliferyl-beta-D-xyloside used as an acceptor. ATP at 2 mM increased the enzyme activity by about 60% in the 110 x g supernatant of the cell homogenate, but not in the supernatant or precipitate fractions obtained by 100,000 x g centrifugation. When both fractions (the 100,000 x g supernatant and precipitate) were mixed, the additional ATP increased the enzyme activity. This increase was canceled by heat treatment or trypsin digestion of the 100,000 x g supernatant. In addition, the 100,000 x g precipitate, which was prepared from the 110 x g supernatant preincubated with ATP, exhibited increased activity, and this increase was abolished by alkaline phosphatase treatment. These results suggest that a protein kinase in the 100,000 x g supernatant activates galactosyltransferase-I activity.  相似文献   

18.
1. The effect of perfusion on the activities of hexokinase and lactate dehydrogenase was studied in the proximal half of the small intestine of fed and starved rats. 2. Perfusion of preparations from starved rats with a medium containing glucose caused a significant increase in hexokinase activity of the particle-free supernatant. The increase in activity was observed as early as 5min after the start of perfusion and persisted for up to 66min of perfusion. No increase in hexokinase activity of the particle-free supernatant was observed when a medium containing mannitol was used. As a further control, preparations from fed rats were perfused under the same conditions. With the medium containing glucose, the hexokinase activity of the particle-free supernatant remained unchanged during the first 15min of perfusion and thereafter fell gradually until, after 66min of perfusion, 73% of the original activity was retained. 3. The activity of lactate dehydrogenase in the particle-free supernatant prepared from the proximal half of the untreated small intestine of starved rats was significantly lower than in corresponding preparations from fed animals. However, it did not change significantly on perfusion with media containing either mannitol or glucose. 4. The distribution of hexokinase activity between total particulate fraction and particle-free supernatant was measured in preparations from starved rats after perfusion for 5–10min. In preparations that had not been perfused the ratio of hexokinase activity in total particulate fraction/particle-free supernatant was significantly higher in starved than in fed animals. After perfusion with a medium containing glucose, the total homogenate activity had not changed significantly, whereas the ratio of hexokinase activity in total particulate fraction/particle-free supernatant decreased significantly and approached the value obtained with fed animals. 5. The results agree with the view that the glucose-dependent increase of hexokinase activity in the soluble cell compartment as observed in vivo and in vitro in the intestinal mucosa of starved rats is brought about by a release of hexokinase activity from a particulate subcellular structure(s).  相似文献   

19.
Glucan synthesis in Pneumocystis carinii.   总被引:5,自引:0,他引:5  
Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphosphoglucose into an insoluble polymer. This enzyme activity was present in both the pellet and the supernatant when the P. carinii preparations were centrifuged. The polymer whose production was catalysed by the supernatant was examined by mass spectrometry and found to be an alpha 1----4 glucan, which is either unbranched or has relatively few branches. Polymer formation was completely inhibited by the addition of alpha amyloglucohydrolase to the supernatant. Polymer formation in the pellet of deoxycholate P. carinii preparations, unlike that in the supernatant, was partially resistant to alpha amyloglucohydrolase. The soluble glucan synthase activity in the supernatant was stable for more than 30 h at room temperature and was approximately 50 times more active on a cell-to-cell basis than the supernatant from deoxycholate preparations of the yeast Saccharomyces cerevisae.  相似文献   

20.
Rat-derived Pneumocystis carinii lysed with sodium deoxycholate catalysed the incorporation of uridine diphospho-glucose into an insoluble polymer. This enzyme activity was present in both the pellet and the supernatant when the P. carinii preparations were centrifuged. The polymer whose production was catalysed by the supernatant was examined by mass spectrometry and found to be an α 1→4 glucan, which is either unbranched or has relatively few branches. Polymer formation was completely inhibited by the addition of α amyloglucohydrolase to the supernatant. Polymer formation in the pellet of deoxycholate P. carinii preparations, unlike that in the supernatant, was partially resistant to α amyloglucohydrolase. The soluble glucan synthase activity in the supernatant was stable for more than 30 h at room temperature and was approximately 50 times more active on a cell-to-cell basis than the supernatant from deoxycholate preparations of the yeast Saccharomyces cerevisae.  相似文献   

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