Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

Thymidine kinase activity in murine lymphoid organs following glucocorticoid and heparin administration

Thymidine kinase (TK) activity was studied in thymus and spleen of mice after glucocorticoid and heparin administration. Glucocorticoids (cortisone and hydrocortisone) injected intraperitoneally caused a prolonged 80--90% decrease in TK activity of both lymphoid organs. Heparin per se administered in depot-form subcutaneously did not alter the enzyme activity in the lymphoid organs significantly. By contrast, heparin injected 6 hours before glucocorticoid treatment inhibited the decreasing effect of cortisone but not that of hydrocortisone on TK activity in the thymus and fully inhibited the effect of both hormones on the enzyme activity of spleen. In addition the combined use of heparin and cortisone increased the splenic TK activity above the control value on the 2nd day after treatment. The possible mode of action of heparin is discussed.     

Involvement of lysosomes in substrate stabilization of tryptophan-2,3-dioxygenase in rat liver.

Administration of tryptophan or hydrocortisone to rats caused a several-fold increase in tryptophan-2,3-dioxygenase activity in the liver. Highly purified lysosomes were isolated from livers of tryptophan- or hydrocortisone-treated animals as well as the control rats. Immunoblotting of lysosomal proteins with anti-tryptophan-2,3-dioxygenase showed 48 kDa band, corresponding to the subunit molecular weight of the enzyme. The relative amount of the immuno-reactive substance in the lysosomes from hydrocortisone-treated rats was 3 times higher than the control while the value in the lysosomes from tryptophan-treated rats was essentially the same as in the control. These results indicate that administration of tryptophan renders cytosolic tryptophan-2,3-dioxygenase less vulnerable to lysosomal uptake and causes an accumulation of the enzyme in the cytosol.     

Precocious induction of pepsinogen in the stomach of suckling mice by hormones

Effects of hormones on pepsinogen activity in mouse stomach were investigated by enzyme assay and electron microscopy. Administration of hydrocortisone alone to mice on days 5–10 increased the enzyme activity in the stomach to as much as 4.5-fold that of untreated mice and the increase was dose dependent. Thyroxine also evoked precocious differentiation of the stomach. The effects of thyroxine and hydrocortisone were additive. Injections of insulin had little effect when given alone, or in combination with other hormones. Injection of hydrocortisone alone or plus thyroxine also caused morphological differentiation of the chief cells in the stomach mucosa. Administration of thyroxine to mice on days 15–20 induced as much enzyme activity as that induced by hydrocortisone, but neither of these hormones had any effect when injected after day 23.These results suggest that besides hydrocortisone, thyroxine is also involved in differentiation of the stomach in mice for the first 20 days after birth and that the normal increase of pepsinogen activity in the stomach of mice during the late suckling period is brought about by serum glucocorticoids, possibly with thyroxine.     

Hydrocortisone induces an increase of amylase content in individual zymogen granules from rat pancreas

This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses.     

Age-related differential induction of tryptophan pyrrolase by hydrocortisone in the liver of male rats

The activity and the regulatory pattern of tryptophan pyrrolase of the liver of male rats during various phases of the life span were studied with a view to investigate the differential effectiveness of hydrocortisone in relation to growth, development, and senescence of an organism. The level of this enzyme shows no significant change till adulthood but decreases significantly thereafter with increasing age. Adrenalectomy and hydrocortisone treatments decrease and increase, respectively the activity of this enzyme significantly in rats of all the ages. However, the effects of these treatments are highest in the mature rat. Induction of the enzyme by hydrocortisone is actinomycin D-sensitive.     

Inhibition of endogenous RNA polymerase activity from thymus chromatin by transcortin-hydrocortisone complex.

A transortin-hydrocortisone complex has been isolated from human serum by affinity chromatography on oxidized corticosterone coupled to AH-Sepharose 4B. The influence of this complex and of hydrocortisone alone on endogenous RNA polymerase activity from thymus chromatin have been tested. Results show that hydrocortisone alone has no effect on RNA polymerase activity from thymus chromatin. Under the same experimental conditions, The transcortin-hydrocortisone complex induces an important decrease in the incorporation of UMP into RNA. The dose response of thymic RNA polymerase to transcortin-hydrocortisone complex and the effects of alpha-amanitin on this response are also reported.     

Thymus participation in realizing the immunomodulating action of hydrocortisone

It has been shown in experiments on CBA mice that in certain conditions injection of hydrocortisone (10 mg/kg) results in suppression of the delayed type hypersensitivity reaction and prolongation of the skin allograft survival. Preliminary thymectomy abolishes the immunomodulating effect of the drug, being, in the authors' opinion, the evidence for thymus involvement in mediation of the immuno-suppressive effect of hydrocortisone.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.     

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.     

Nuclei of lymphocytes of the T- and B-cell lines during normal embryogenesis and after treatment with hydrocortisone (morphometric and probabilistic-statistical parameters)

Lymphoid cells of the thymus and of the Fabricius bursa have been studied in 18-day-old chick embryos, normal and after injection of hydrocortisone on the 11th day of embryogenesis. By means of optical-structural computer analysis, the complex of morphometric and probability-statistic parameters of the nuclei in the lymphocytes are estimated: area of the nuclei, optical density of chromatin, asymmetry coefficient and variance. Normal T-lymphocytes possess less density of the nuclei, greater optical density of chromatin, greater values of negative asymmetry. The complex of these parameters can be used for identification of visually similar lymphoid cells of T- and B-lines. Under hydrocortisone effect structural changes of the nuclei in the thymus and Fabricius bursa lymphocytes of the chick embryo are uniform: increase in the area of the nuclei, decrease in optical density of chromatin, the asymmetry coefficient becomes positive.     

Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina.

Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone.     

Regulating properties of peptides of the thymus and bursa of Fabricius in immunodepression in birds

The intensity of nucleic acids and protein synthesis in the cells of the thymus and the bursa of Fabricius was studied in chickens against the background of an immunodepression induced by administration of hydrocortisone and cyclophosphamide. It was found out that hydrocortisone causes in chicken a marked lowering of the intensity of inclusion of 3H-thymidine, 3H-uridine and 14C-glycine in thymic cells, and cyclophosphamide--in the cells of the bursa of Fabricius. Under the conditions of selective immunodepression the preparations on the basis of the peptides of the thymus (thymalin) and of the bursa of Fabricius (bursilin) regulate the processes of nucleic acids and protein synthesis chiefly in the cells of organs which produce them.     

Hydrocortisone-induced increase in the number of small intensely fluorescent cells and their histochemically demonstrable catecholamine content in cultures of sympathetic ganglia of the newborn rat

Synopsis It is known that hydrocortisone causes a great increase in the number of small intensely fluorescent (SIF) cells in the sympathetic ganglia when injected into newborn rats. The effect of hydrocortisone on nervous tissuein vitro has not been studied previously.Pieces of newborn rat sympathetic ganglia were cultivated in Rose chambers. Hydrocortisone was dissolved in the medium in concentrations of 1–9 mg/l. Both control and hydrocortisone-containing cultures were examined daily by phase-contrast microscopy, and the catecholamines were demonstrated histochemically by formaldehyde-induced fluorescence after 7 days in culture.All cultures showed outgrowths of axons and supporting cells elements, although these were less extensive in the groups of cultures with hydrocortisone. After a week, SIF cells with a green fluorescence were observed in the control explants. In all cultures with hydrocortisone, a concentration-dependent increase was observed in the fluorescence intensity and the number of the SIF cells in the explant; numerous SIF cells were also seen in the outgrowth. Some SIF cells showed processes and the longest processes were seen in cultures with the highest concentration of hydrocortisone.It is concluded that hydrocortisone causes an increased synthesis of catecholamines in the SIF cellsin vitro, and an increase in their number by affecting either their division or their differentiation from a more immature form, or both. This effect was a direct one and not mediated by any system other than the ganglion itself. Induction of enzyme synthesis by hydrocortisone is proposed as an explanation of the increase in catecholamine concentration.University of Melbourne Senior Research Fellow, September 1971-August 1972Sunshine Foundation and Rowden White Trust Overseas Research Fellow in the University on Melbourne, September 1971-August 1972     

Effect of altered hormonal balance in the mother-fetus system on body weight, the adrenal glands, thymus gland and peripheral blood leukocyte composition in the offspring

Pregnant rats have been injected intramuscularly with hydrocortisone-acetate or desoxycorticosterone-acetate (DOCA) for the III or the II-III trimesters of pregnancy. In the last case the animals are given not only greater doses of the hormones, but during a longer period. By the end of the pregnancy the drug dose decreases. In mother-rats after hydrocortisone injections the adrenals mass increases; after DOCA the body mass increases, and that of the adrenals drops. In the offspring hydrocortisone produces decline of the thymus and adrenals mass, as well as neutrophilic leucocytosis, lympho-, eosino-++- and monocytopenia. Just the opposite, DOCA results in lympho- and monocytosis, in increasing morphofunctional activity of monocytes. Common effects in hormonal action is demonstrated as underdevelopment of the adrenals, in decreasing thymus mass, in development of neutrophilic leucocytosis and eosinopenia. With increasing doses and duration of prenatal injections of corticosteroids in rats the mass of the adrenals and thymus drops even greater, stimulating effect of the hormones on the neutrophilic granulocytopoiesis decreases.     

Comparison of the properties of L-threonine dehydratase isolated from the livers of intact, adrenalectomized or cortisol-treated rats]

L-Threonine dehydratase preparations were isolated from liver of intact, treated with hydrocortisone and adrenalectomized rats. These preparations had different properties in stability, sensitivity to proteases and kinetic patterns. The preparations possessed also serine dehydratase activity, and the ratio threonine: serine activities was modified during the procedure of enzyme purification. It appears that the hormones affect not only the amount of enzyme proteins, but the qualitative properties of these proteins.     

Adenylic acid catabolism in thymocytes of the regenerating thymus in mice

The T-cell non-peptide mitogenic factor isolated from the thymus stimulates thymus regeneration in mice previously treated with hydrocortisone. [8-14C]AMP catabolism in cortisone resistant thymocytes of mice has been investigated. Incorporation of radioactivity into hypoxanthine in cortisone resistant thymocytes is found to increase as compared with the total thymocyte population. Accumulation of labeled AMP catabolites in the form of hypoxanthine grows considerably after in vitro incubation of cortisone-resistant thymocytes with the non-peptide T-cell mitogenic factor. A large proportion of [8-14C]AMP catabolite radioactivity incorporated into cortisone-resistant thymocytes is excreted into the medium as hypoxanthine. It is supposed that hypoxanthine accumulation abrogates limitation of thymocyte DNA synthesis inhibited by relative excess of dGTP.     

DNA ligase changes and cell population in thymus of corticosteroid-treated chick embryo

Two successive forms of DNA ligases normally occur successively in the chicken and chick embryo thymus, a 8.2 S, before hatching and a 6.2 S, after hatching. The disappearance of the 8.2 S and the appearance of the 6.2 S together with its increased activity can be observed earlier under the effect of corticosteroids (dexamethasone (DMSO) and hydrocortisone). The biochemical, histological and cell sorting observations are consistent with the presence of the heavy enzyme in large (7.5 μm) thymocytes and the light enzyme in smaller (5 μm) T-antigen possessing cells. These results are discussed on the basis of the effect of steroids on thymocyte maturation and with regard to cell migration within the lymphoid system.     

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP-ribose) synthetase. It was classified as IgG1/kappa and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA-binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross-reactivity was examined using DNA-binding proteins from 6 different sources, 1F4 reacted with the 120- and 32-kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures of poly(ADP-ribose) synthetase and its degradation products are highly conserved in various animal cells.     

Coexpression of redox partners increases the hydrocortisone (cortisol) production efficiency in CYP11B1 expressing fission yeast Schizosaccharomyces pombe

Cytochromes P450 play a vital role in the steroid biosynthesis pathway of the adrenal gland. An example of an essential P450 cytochrome is the steroid 11beta-hydroxylase CYP11B1, which catalyses the conversion of 11-deoxycorticol to hydrocortisone. However, despite its high biotechnological potential, this enzyme has so far been unsuccessfully employed in present-day biotechnology due to a poor expression yield and inherent protein instability. In this study, CYP11B1 was biotransformed into various strains of the yeast Schizosaccharomyces pombe, all of which also expressed the electron transfer proteins adrenodoxin and/or adrenodoxin reductase - central components of the mitochondrial P450 system - in order to maximise hydrocortisone production efficiency in our proposed model system. Site-directed mutagenesis of CYP11B1 at positions 52 and 78 was performed in order to evaluate the impact of altering the amino acids at these sites. It was found that the presence of an isoleucine at position 78 conferred the highest 11beta-hydroxylation activity of CYP11B1. Coexpression of adrenodoxin and adrenodoxin reductase appeared to further increase the 11beta-hydroxylase activity of the enzyme (3.4 fold). Adrenodoxin mutants which were found to significantly enhance enzyme efficiency in other cytochromes in previous studies were also tested in our system. It was found that, in this case, the wild type adrenodoxin was more efficient. The new fission yeast strain TH75 coexpressing the wild type Adx and AdR displays high hydrocortisone production efficiency at an average of 1mM hydrocortisone over a period of 72h, the highest value published to date for this biotransformation. Finally, our research shows that pTH2 is an ideal plasmid for the coexpression of the mitochondrial electron transfer counterparts, adrenodoxin and adrenodoxin reductase, in Schizosaccharomyces pombe, and so could serve as a convenient tool for future biotechnological applications.