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1.
The activation of lymphocytes has been used to study the regulation of mammalian gene expression. Concanavalin A (Con A) added to mouse spleen lymphocytes in serum-free medium leads to an increase in the rate of DNA synthesis as great as 1000 fold, commencing 20 hr after its addition. Prior to 20 hr, the rate of purine synthesis increases 10–100 fold as measured by accumulation of the purine intermediate, formyl glycineamide ribonucleotide (FGAR). Addition of dibutyryl cyclic GMP to the lymphocyte suspensions results in a 10 fold increase in the rate of DNA synthesis in the absence of Con A and enhances both purine synthesis and DNA synthesis in its presence. The activity of phosphoribosyl pyrophosphate synthetase (PRPP synthetase), an enzyme central to purine and pyrimidine biosynthesis, is increased 2–10 fold during the activation. The increase begins to appear 8 hr after Con A addition and requires concomitant protein synthesis. The induced PRPP synthetase activity is stimulated by the presence of cyclic GMP in the enzyme assay. Addition of dibutyryl cyclic AMP to Con A-stimulated lymphocytes inhibits FGAR production, the stimulation of DNA synthesis, and the appearance of cyclic GMP-sensitive PRPP synthetase. These studies suggest that cyclic nucleotides play a significant role in the molecular mechanism of lymphocyte activation, the regulation of purine biosynthesis, and of eucaryotic genetic expression.  相似文献   

2.
B Ullman  L J Gudas  A Cohen  D W Martin 《Cell》1978,14(2):365-375
The inherited absence of either adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with severe immunological impairment. We have developed a cell culture model using a mouse T cell lymphoma to simulate ADA deficiency and to study the relationship between purine salvage enzymes and immune function. 2′-deoxyadenosine triphosphate (deoxyATP) levels have been shown to be greatly elevated in erythrocytes of immunodeficient, ADA-deficient patients, suggesting that deoxyadenosine is the potentially toxic substrate in ADA deficiency. Using a potent ADA inhibitor, we have demonstrated that deoxyadenosine is growth-inhibitory and cytotoxic to S49 cells, and that deoxyATP accumulates in these cells. Cell variants, unable to transport or phosphorylate deoxyadenosine, are much less sensitive to deoxyadenosine, indicating that intracellular phosphorylation of deoxyadenosine is required for the lethal effects.We have partially reversed the cytotoxic effects of deoxyadenosine with deoxycytidine in wild-type cells, but we cannot show any reversal in cell lines lacking deoxycytidine kinase. Adenosine (ado) kinase-deficient cells are extremely resistant to deoxyadenosine in the presence of deoxycytidine. This deoxycytidine reversal of deoxyadenosine toxicity is consistent with an inhibition of ribonucleotide reductase by deoxyATP, and we have shown that incubation of S49 cells with deoxyadenosine markedly reduces intracellular levels of deoxyCTP, deoxyGTP and TTP.Kinetics data in wild-type cells and in cell variants are consistent with the presence of two deoxyadenosine-phosphorylating activities — one associated with ado kinase and another associated with deoxycytidine kinase.The S49 cells appear to be a valid model for the simulation of ADA deficiency in cell culture, and from our results, we can suggest administration of deoxycytidine as a pharmacological regimen to circumvent the clinicopathologic symptoms in ADA deficiency.  相似文献   

3.
Nuclear multiplication stage embryos were punctured in either the anterior, midlateral, or posterior regions. Both embryos and adults were examined for defects, and the defects were correlated with whether there had been any leakage of cytoplasmic material from the egg at the time of puncturing. Embryonic defects were found, correlated to the site of damage, in all three regions. A number of embryos was followed through development and it was found that 15.1% of the embryos which leaked cytoplasm hatched into larvae, compared to 82.3% of those which did not leak any cytoplasm. Morphological defects arising as a result of lateral puncture only were observed in adults. Many sterile adults were obtained from eggs in which the posterior region had been punctured. The results show that nuclear multiplication embryos are well able to tolerate the disturbance of the cortical cytoplasm created by puncture, but only rarely are they able to compensate for the actual loss of material by regulation. The results were similar to those observed after puncturing Drosophila embryos at the cellular blastoderm stage.  相似文献   

4.
Plateau-phase V79 Chinese hamster cells induced to reenter the proliferative cell cycle from G1/G0 by dilution in fresh medium showed an early increase in ornithine decarboxylase (ODC) followed by increases in some polyamines and in DNA synthesis. In contrast, cells accumulated in G1/G0 by growth in extreme hypoxia for 60 h and induced to recycle by reoxygenation did not respond with early increases in either ODC or polyamines.  相似文献   

5.
The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (MB) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite MB possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of MB. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.  相似文献   

6.
The plasma aldosterone radioimmunoassay developed by Ito et al. was found to be non-specific for aldosterone following administration of the spirolactones, spironolactone and canrenoate-K, in rabbits, dogs and humans. The assay interfering principle was identified as a hydroxylated derivative (M-B) of canrenone, which itself is a metabolite common to both spironolactone and canrenoate-K. The metabolite M-B possessed a high cross-reactivity to the 21-hemisuccinate aldosterone antibody relative to other spirolactones. A modified procedure was developed specific for plasma aldosterone in the presence of M-B. Following single doses of spironolactone and canrenoate-K, aldosterone plasma levels were unchanged in humans and in dogs and decreased in rabbits.  相似文献   

7.
Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10−4 M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by α-adrenergic blockade with phenoxybenzamine. Epinephrine (4 · 10−5 M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the β-blocking agent, propranolol. Pure α-adrenergic stimulation with methoxamine (4 · 10−4 M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 · 10−6 M, isoproterenol (a β-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 · 10−5 M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cylcic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 · 10−6 M).These data strongly suggest that cholinergic muscarinic agonists and α-adrenergic agonist stimulate amylase output in rabbit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by α-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this issue to the effects of cholinergic stimuli.  相似文献   

8.
The intravenous administration of αMSH (25 μg/kg) to 11 lambs (3 to 29 days of age) suppressed plasma PRL by 15 minutes. The mean basal concentration was 15.3 ± 2.9 ng/ml and the mean nadir was 4.9 ± 0.8 ng/ml (p<0.01). In chronically catheterized fetuses (128–140 days), intravenous administration of αMSH (25 μg/kg) decreased basal PRL levels (89.6 ± 12.4 ng/ml) significantly at 15–30 minutes to levels of 74.3 ± 11.4 ng/ml (p<.01). The degree of suppression of basal PRL levels was less in fetusus (76.9 ± 4.1%) than that induced in the neonates (40.5 ± 7.1%). In younger fetuses <120 days in whom basal PRL levels are low (3.0 ± 2.1 ng/ml), administration of αMSH was without effect. Plasma GH concentrations were not altered by administration of αMSH. The suppression of PRL secretion by αMSH administration could result from increased release of hypothalamic dopamine or be a direct effect on secretion of prolactin by the pituitary.  相似文献   

9.
The fall in transepithelial electrical resistance which accompanies aldosterone stimulation of short-circuit current (Isc) in toad urinary bladder has been studied further to evaluate the possible causal role of this response in hormonal stimulation of Na+ transport. A steady-state change in tissue conductance was found to depend upon both the simultaneous stimulation of transport by the steroid and the metabolic state of the tissue. Changes in metabolic state alone did not alter resistance. A sustained increase in Na+ transport, dependent on pretreatment with aldosterone and elicited by addition of glucose, could be obtained without a sustained decrease in resistance. Amiloride, an inhibitor of Na+ uptake, produced changes in Isc that were linearly correlated with its effects on tissue conductance. On the basis of the conductance-Isc relationship with amiloride, the Isc response to aldosterone was about two-fold higher than would be predicted from its effects on conductance alone. Despite the apparent lack of a simple quantitative dependence of the change in Isc on the change in conductance when the response is fully developed, the results suggest that conductance changes may mediate the initial or early stage of the response.  相似文献   

10.
Previous work has shown that, as amoebae of the cellular slime mold Polysphondylium pallidum become aggregation competent, they accumulate on their cell surface a carbohydrate-binding protein (lectin) named pallidin. These amoebae also possess cell surface receptors, presumed to contain complex oligosaccharides with a high affinity for the endogenous lectin. If lectin-receptor interactions mediate cell-cell contact, then appropriate concentrations of pallidin inhibitors should block cell cohesion. Two potent macromolecular antagonists of the lectin were employed: the desialylated form of the glycoprotein fetuin and the univalent antibody (Fab) prepared against pallidin. We studied the effects of these inhibitors on rotation-mediated aggregation of P. pallidum amoebae under a variety of assay conditions. Amoebae exposed to hypertonic conditions or to antimetabolites (“Permissive conditions”) were selectively blocked from associating by microgram quantities of the lectin inhibitors, whereas cells in isotonic buffer (“nonpermissive condition”) were only slightly affected. A comparison of the morphology of agglutinates formed under the various conditions allows several explanations for the different susceptibilities to inhibition by antipallidin reagents. Although not conclusive, the work supports a model of cell adhesion in this simple eukaryotic system based at least in part on specific interactions between carbohydrate-binding proteins and receptors on adjoining cells.  相似文献   

11.
L J Gudas  B Ullman  A Cohen  D W Martin 《Cell》1978,14(3):531-538
The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP.The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.  相似文献   

12.
The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [3H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of 32Pi into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.  相似文献   

13.
A murine model of contact sensitization to components of poison oak or ivy urushiol oils was developed. Sensitization was effected by painting such compounds on abdominal skin, and was routinely assessed by challenging on the ears and monitoring increases in ear thickness. Sensitization to 3-heptadecylcatechol (HDC, a component of poison oak urushiol) was studied in detail. Contact sensitivity as indicated by ear swelling reactions was observed from 2 until around 25 days after primary abdominal painting with HDC. In all cases maximal ear swelling occurred 3–4 days after HDC challenge. Sensitivity could also be assessed by monitoring the uptake of radioiodinated deoxyuridine at the ear challenge site, and this correlated with the ear swelling assay in terms of kinetics. The sensitization effect induced by HDC had properties of delayed-type hypersensitivity, being antigen specific, and transferable with sensitized lymph node and spleen cells but not by serum. Also, T cells were required for activity as transfer with spleen cells was abrogated by treatment with anti-Thy-1.2 antibody and complement. In this system HDC and 3-pentadecylcatechol (PDC, a component of poison ivy urushiol oil) were completely cross-reactive both in sensitization and challenge, and both compounds also cross-reacted with native urushiol oil itself. Thus murine sensitization to HDC can be used as a model system for investigating mechanisms for the immunogenicity of such catechols.  相似文献   

14.
The ganglionic potentials elicited by a test dose of acetylcholine (ACh) acting nicotinically consist of an initial depolarization followed by a longer “after-hyperpolarization” (after-HP). The after-HP is abolished selectively and relatively rapidly after treatment with ouabain or with Li-replacement of 90% of Na in Ringer. The hyperpolarizing response to a test dose of dopamine (DA), however, is as or more resistant than the ACh-depolarization to either treatment. These findings provide further strong evidence that the slow inhibitory postsynaptic potential (s-IPSP), for which DA is the transmitter in these ganglia, is not mediated by an activation of an electrogenic NaK pump.  相似文献   

15.
The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10(-12) to 10(-10) M to 10(-13) to 10(-9) M somatostatin, as assessed by the uptake of [3H]thymidine and [3H]leucine, respectively. The inhibitory effect of somatostatin was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 degrees C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.  相似文献   

16.
In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.  相似文献   

17.
A single cutaneous application of components of poison oak or ivy urushiol oils to mice results in contact sensitivity with properties of delayed-type hypersensitivity. The compounds, 3-heptadecylcatechol (HDC, from poison oak urushiol) and 3-pentadecylcatechol (PDC, from poison ivy urushiol) are completely cross-reactive. Covalent bond formation between the o-quinone intermediate of PDC and nucleophilic functionalities such as those found on proteins is known to occur in a regiospecific manner. Amino nucleophiles preferentially attack the 5-position on the catechol ring while thiol nucleophiles attack the 6-position. The present paper describes the immunological properties of the three possible ring monomethylated analogs of PDC. When mice were treated with a single epicutaneous painting with these analogs, only the 5-methyl compound (5-Me-PDC) was found to be an ineffective sensitizer. The 5-Me-PDC analog, however, was capable of inducing cellular proliferation in draining lymph nodes. Furthermore, epicutaneous pretreatment with 5-Me-PDC suppressed the subsequent induction of contact sensitivity to PDC and HDC in an antigen-specific manner. Equivalent treatment with the 6-Me-PDC analog (a good sensitizing agent) resulted in a less consistent and weaker suppressive effect, while the 4-Me-PDC analog did not display any suppressive activity. The suppressive activity could be demonstrated up to 15 days following primary painting with 5-Me-PDC. Lymph node cells obtained from mice 10 days after a single painting with 5-Me-PDC could transfer the suppressive effect. Under certain circumstances 5-Me-PDC could also sensitize, indicating that the analog retains some sensitizing abilities. Hence, blocking the 5-position of the catechol ring results in a major alteration in the type of immune response elicited. Since 5-Me-PDC is specifically blocked in terms of nucleophilic attack by amino groups, it is suggested that attack by amino nucleophiles is of primary importance in the peripheral metabolic processing of these catechols, which in turn determines the outcome of the immune response.  相似文献   

18.
We have developed a rapid and simple technique for the simultaneous isolation of all the major viral proteins from RNA tumor viruses. The basis for this procedure is analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using dansylated virus as internal marker it is possible to follow the migration of unlabeled viral proteins since dansylation does not change the mobility of labeled proteins (8). The method results in approximately 80% recovery of starting protein and is very reproducible. Using radioimmunoassay no alteration of the purified proteins is detectable.  相似文献   

19.
A study was undertaken to determine (1) the effects of endogenous Müllerian inhibiting substance (MIS) on the developing human fetal genital tract; (2) the time in fetal life when MIS is first capable of inhibiting the growth of the embryonic Müllerian ducts; and (3) the reversibility of the effects of MIS on the developing male Müllerian ducts. Human fetal reproductive tracts were transplanted and grown for sustained periods in vivo in athymic nude mice. The genital tracts from 12 male human fetuses, ages 51 to 68 days postovulation, were grafted without their associated gonads into castrated murine hosts and grown for 30 to 70 days. Controls consisted of genital tracts from 8 female human fetuses, ages day 53 to 70 that were grown under identical conditions. Male specimens grew to approximately one-half the size of female specimens and disclosed varying degrees of inhibition of the Müllerian duct system from absence of the Müllerian ducts in older specimens (after Day 63) to poorly segregated segments of stroma as the mildest defect (less than Day 61). It is concluded that (1) MIS secretion by the embryonic testes probably begins before Day 51 of gestation; (2) the effects of MIS are progressive during the so-called critical window; (3) the effects of MIS are permanent; and (4) the mesenchyme is an important target of MIS.  相似文献   

20.
The induction kinetics of human suppressor cells in mixed lymphocyte cultures (MLC) and the influence of prednisolone on the genesis of these suppressor cells is reported. We induced over 1 to 6 days suppressor cells in one-way MLC (MLC-1), the inhibitory activity of which was tested on a secondary MLC (MLC-2), and on responder cells alone, where lymphocytes were obtained from the same lymphocyte donors as for the MLC-1. In four experiments the degree of inhibition (x? ± SE) when suppressor cells were induced for 2, 4, or 6 days was 38.5 ± 11.8, 79.5 ± 7, and 85 ± 6%, respectively, compared to 50.5 ± 9.4, 83.3 ± 7.8, and 85.3 ± 9.8% when 500 ng/ml prednisolone was added to the MLC-1. A similar inhibition pattern was observed when the generated suppressor cells were incubated with responder cells only. The inhibitory activity of these MLC-induced suppressor cells was abrogated by irradiation with 3000 R. Suppressor cells apparently are generated in MLCs between Days 1 and 4; furthermore, their genesis is not affected by usual therapeutic concentrations of prednisolone.  相似文献   

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