The histones of the insect trypanosomatid, Crithidia fasciculata.

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.

Identification of protein complexes containing the c-rel proto-oncogene product in avian hematopoietic cells.

The c-rel proto-oncogene product has been identified as a 75 kDa protein expressed in lymphoid cells transformed by REV-T and Marek's disease virus. A 4.0 kb c-rel transcript is expressed in the bursa, spleen and thymus of chickens with highest levels of expression at 10 days post hatch. Using antiserum specific for the v-rel oncogene product, P75c-rel has been precipitated from [35S]methionine-labeled extracts of bursal, splenic and thymic lymphocytes. Additionally, proteins with the molecular mass of 40 kDa, 115 kDa, and 124 kDa co-immunoprecipitate. These proteins co-migrate with the proteins found associated with pp59v-rel in REV-T transformed lymphoid cells. Antiserum specific for pp40, the most abundant cellular protein associated with pp59v-rel, co-precipitates p75c-rel verifying the existence of p75c-rel/pp40 complexes in normal avian lymphocytes. Antiserum directed against the amino-terminal region of pp59v-rel fails to precipitate native p75c-rel complexes from normal lymphoid cells. In the presence of ionic detergents, antisera directed against the amino, middle and carboxy-regions precipitate equivalent amounts of p75v-rel. These results suggest that the amino-terminal region of p75c-rel is active in binding other proteins or is inaccessible to the antiserum due to the conformation of p75c-rel in the complex. Two p75c-rel complexes exist in the cytosol of normal lymphocytes. The most abundant complex contains 60% of the p75c-rel associated with p115 and p124. The remaining p75c-rel is associated with pp40.

Nonneoplastic and nonhyperplastic thymus in myasthenia gravis. An immunohistochemical study with double immunoenzymatic labeling of basement membrane and cellular components.

Histologically normal thymus (type A) in patients with myasthenia gravis (MG) was immunohistochemically compared with hyperplastic MG thymus (type B) and normal non-MG thymus. In formalin-fixed, paraffin-embedded sections of ten type A, ten type B, and eight non-MG cases, the thymic epithelium and other cellular components were stained in conjunction with the basement membrane by a double immunoenzymatic method. This technique demonstrated a moderate architectural disturbance in type A thymus, with distended perivascular space (PVS), elongated medullary epithelium, and disrupted basement membrane. These changes were more prominent in type B thymus but were minimal to lacking in non-MG thymus. Compared with those in non-MG thymus, the myoid cells in MG thymuses of both types tended to cluster around the Hassall's corpuscles, with a slight decrease in number in type B but not in type A. B-lymphocytes were present in type B, type A, and non-MG thymuses in that order of abundance; the cells were confined to the medullary parenchyma in the non-MG group but were numerous both in the PVS and medulla in the MG groups. T-lymphocytes were increased in the expanded PVS of type A and B MG thymuses. The number of interdigitating reticulum cells was similar in the three groups, but the cellular distribution was more dispersed in MG thymuses of both types. These findings, although previously described in type B thymus, have not been well recognized in type A thymus. They support the view that a common abnormality (presumably chronic thymitis), differing in degree only, underlies MG thymuses regardless of the presence of follicular hyperplasia.

Cholera toxin and its B subunit do not change cytosolic free calcium concentration.

Using the fluorescent Ca2+ probe Quin-2 it has been reported that cholera toxin (CT) and its B subunit (B-CT) increase cytosolic free Ca2+ concentration ([Ca2+]i) in entherocytes, thymocytes and fibroblasts. In this work we show, however, that the fluorescence increases of Quin-2-loaded cells (rat thymocytes, mouse splenocytes, P-388 macrophages and 3T3 fibroblasts) observed upon addition of CT or B-CT are not caused by an increase in [Ca2+]i. The observed effect appears to be accounted for by EDTA-2Na admixtures (present as conservation agent in all CT and B-CT preparations) which 'unquenches' the fluorescence of Quin-2 acid leaked out from the cells into the extracellular medium and produces influorescent complexes with contaminating heavy metal ions. Thus the mitogenic effect of B-CT is not obviously connected with the cytosolic free Ca2+ increase but is probably due to ganglioside-mediated protein phosphorylation.

Hypothalamic modulation of immunoreactive oxytocin in the rat thymus.

Immunoreactive oxytocin was determined in a peptidic extract of rat thymus by means of a highly specific radioimmunoassay combined with high pressure liquid chromatography fractionation. Rat thymus was found to contain 80 +/- 7.5 pg/g wet tissue (congruent to 0.56 pg/mg protein) of oxytocin-like immunoreactivity, which behaved like synthetic oxytocin in the radioimmunoassay and in two different high pressure liquid chromatography columns. Oxytocin concentration was increased by bilateral electrolytic lesion of the paraventricular nucleus of the hypothalamus (PVN), and by high doses of corticosterone (10 mg/kg IM for 7 days) but was not modified by low doses of corticosterone (1 mg/kg IM for 7 days) or by hypophysectomy. The results suggest that rat thymus synthesizes oxytocin and that thymic oxytocin concentration is modulated by the hypothalamus.

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.

Neurohypophyseal hormone receptors in the rat thymus, spleen, and lymphocytes.

Receptor sites for the neurohypophyseal peptides arginine vasopressin (AVP) and oxytocin (OT) have been identified and characterized in some tissues involved in immune function in the rat. Novel radioiodinated ligands for the detection of neurohypophyseal hormone receptors, with a high specific radioactivity and affinity, enabled the selective detection of OT receptors in the thymus and vasopressin (VP) receptors in the spleen. OT receptors were detected in thymic membrane preparations and on thymocytes, which had a ligand selectivity similar to that of uterine OT receptors. AVP receptors of the V1 pressor type were present in a splenic membrane preparation. Specific AVP-binding sites, probably of the V1 type, were also present on splenic lymphocytes. Binding sites for AVP and OT could not be detected on mononuclear cells in peripheral blood of the rat. This study demonstrates that the use of the newly developed radioiodinated AVP and OT receptor ligands, with high specific radioactivity and affinity, enables the selective characterization of receptor sites for the neurohypophyseal hormones, even in the thymus, where previously no binding sites could be detected.

Basic nuclear proteins of the histone-less eukaryote Crypthecodinium cohnii (Pyrrhophyta): two-dimensional electrophoresis and DNA-binding properties.

Unlike typical eukaryotes, the Dinoflagellate Crypthecodinium cohnii does not contain histones but six major basic, low molecular weight nuclear proteins which represent only 10% of the DNA mass and differ from histones in their electrophoretic and DNA-binding properties. These proteins are resolved in two-dimensional electrophoresis (AUT-PAGE x SDS-PAGE). Three proteins with an apparent molecular mass of 16, 16.5 and 17 kDa (p16, p16.5 and p17) are present in addition to the major 14 kDa basic nuclear component (HCc). HCc itself is resolved in three proteins (alpha, beta and gamma). When the proteins are not reduced with 2-mercaptoethanol before 2D-PAGE, the migration of HCc alpha, beta and gamma is modified in a way which suggests the formation of both inter- and intramolecular disulfide bridges and thus, the presence of at least two cysteines. The amino-acid analysis of HCc proteins resolved in 2D gels confirms that they are lysine-rich. HCc alpha, beta and gamma as well as p16, p16.5 and p17 are removed from isolated chromatin with 0.6 M NaCl, indicating that their affinity for DNA in vivo is lower than that of core histones. Furthermore, in vitro, they bind more tightly to single-stranded than to double-stranded DNA.

Nuclear progestin receptors in rat thymic tissue.

The thymus and its associated endothelial cells and lymphocytes act as an important immunological tissue. The endothelial cells of the thymus have been reported to synthesize cytoplasmic progestin receptor in response to estrogen priming. To measure nuclear progestin receptor, female rats were castrated and primed for 3 days with estradiol benzoate (30 micrograms/0.1 ml/d) and immediately before sacrifice injected subcutaneously with 0.2 mg of progesterone. By Scatchard plot analysis we found that specific progestin receptor (KA = 0.89 +/- 0.10 x 10(9) M-1) was present in the KCl-nuclear extract. The concentration of nuclear progestin receptor was found to be in the range of 312.6 +/- 49 fmole/g tissue (n = 9, 1 hour after progesterone injection) while the nuclear receptor was significantly reduced (approximately 44 fmol/g tissue) in the oil treated controls. This level verges on the limits of sensitivity for this assay. For cytoplasmic progestin receptor the concentration was 3.46 +/- 0.20 pmole/g tissue in oil treated controls (n = 14) and 3.36 +/- 0.20 pmole/g tissue in progesterone treated animals (n = 24). The KA of this thymic cytoplasmic progestin receptor was 1.35 +/- 0.06 x 10(9) M-1. By competition assay, the relative binding affinity of nuclear progestin receptor was: R5020 (a potent synthetic progestin) (100%), progesterone (9%), testosterone (0.56%), corticosterone (0.53%), estradiol-17 beta (0%). It is concluded that thymic reticuloepitheleal cells contain nuclear progestin receptor and this finding supports the hypothesis that progesterone, like other sex steroids may play a regulatory role in thymic cell function.

Purification of histone H1 polypeptides by high-performance cation-exchange chromatography.

Calf thymus histone 1 (H1) was cleaved by chemical and enzymatic methods and the resulting polypeptides were fractionated by high-performance cation-exchange. Up to 1 mg of H1 polypeptides were loaded onto a 50 x 5 mm I.D. cation-exchange column and fractionated to greater than 95% purity in less than 30 min. This is the first report on the separation of H1 polypeptides by a strong cation-exchange matrix. In addition, the high-performance cation-exchange chromatography protocol represents a significant decrease in fractionation time when compared to conventional ion-exchange and gel filtration chromatography. The utility of this procedure is shown when the H1 peptides purified by the protocol were used to define antigenic domains of H1 band by procainamide-induced lupus and idiopathic systemic lupus erythematosus. The majority of the sera tested by enzyme-linked immunoassay (ELISA) reacted to the C-terminal peptides of H1 indicating this to be the major antigenic domain of H1.

Reserpine-induced decrease in type I and II corticosteroid receptors in neuronal and lymphoid tissues of adrenalectomized rats.

The effect of the biogenic amine depleting drug, reserpine, on the concentration of type II corticosteroid receptors (i.e., glucocorticoid receptors) in neuronal (hippocampus, frontal cortex, hypothalamus), lymphoid (circulating lymphocytes, spleen, thymus) and pituitary tissues as well as hippocampal type I (i.e., mineralocorticoid) receptors was examined in adrenal-intact and adrenalectomized (ADX) rats. Reserpine (2 mg/kg) or vehicle was administered to adrenal-intact rats for 2 consecutive days. Following the second injection rats were ADX and sacrificed 24 h later. Reserpine significantly decreased type I and II hippocampal receptors as well as type II receptors in frontal cortex, hypothalamus, lymphocytes and spleen. Since the reserpine-induced decreases in receptor content could be due to reserpine-induced elevations in circulating corticosterone levels, reserpine (2 mg/kg) or vehicle was administered to 1-day ADX rats which were then sacrificed 2 days later (i.e., 3 days post ADX). A 1-day ADX control group was also included. The 3-day ADX regimen produced significant or nearly significant increases in type II receptors in hippocampus, frontal cortex, hypothalamus, lymphocytes and spleen in vehicle-treated rats. Reserpine attenuated the ADX-induced upregulation of type II receptors in hippocampus, frontal cortex, lymphocytes and spleen, but had no effect on the ADX-induced upregulation of type II receptors in the hypothalamus. The ADX-induced increase in hippocampal type I receptors was not affected by reserpine treatment. In a final experiment, reserpine (2 mg/kg) or vehicle was administered immediately after ADX and rats were sacrificed 24 h later in order to assess the effect of reserpine on basal (i.e., nonupregulated) corticosteroid receptor levels in the absence of circulating corticosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Instrument for real-time pulse-shape analysis of slit-scan flow cytometry signals.

An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse-shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse-profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 microseconds from the onset of the pulse-profile and particles with a specified morphology of interest were selected for on-line registration of their profiles as digitized pulse-shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.

Estrogen and progesterone receptors in the human thymus.

Thymus tissue samples from 8 prepubertal children of both sexes were analyzed for estrogen and progesterone receptors. With the use of an enzyme immunoassay, estrogen receptors in both the cytosol and nuclear fraction were detected. The mean concentration (+/- SD) of receptors in the cytosol fraction was 2.1 +/- 0.9 and for the sum of receptors in both fractions 2.8 +/- 1.0 fmol/mg of protein. In addition low concentrations of progesterone receptors (mean value +/- SD = 0.9 +/- 0.3 fmol/mg of protein) were detected in all investigated samples. The presence of estrogen receptors in the nuclear fraction and of progesterone receptors suggests that the estrogen receptors may be active in mediating a physiological response and thus supports the view that the human thymus is a target organ for estrogens.

Unusual molecular characteristics of a repeat sequence island within a Giemsa-positive band on the mouse X chromosome.

The mouse genome contains 50 copies of a long complex repeat unit localized as a repeat sequence island to the A3 Giemsa-positive (dark) band on the mouse X chromosome. The repeat units are not tandemly arranged but are juxtaposed and inserted by unrelated sequences of high repetition. The repeat sequence island possesses two notable features that have been suggested as diagnostic features of mammalian Giemsa-positive bands. First, the repeat sequence island encompasses a 1-megabase region devoid of CpG islands; second, it features a high concentration of L1 long interspersed repeat sequences.

Extrahepatic replication of duck hepatitis B virus: more than expected.

Replication of duck hepatitis B virus in extrahepatic tissue such as pancreas, kidney and spleen has been well documented. To assess whether there is more widespread extrahepatic virus replication, we assayed brain, heart, lung, thymus, pancreas, kidney, spleen and intestine of 1- to 16-wk-old ducklings for the presence of duck hepatitis B virus DNA and mRNA by blotting and in situ methods. Replicative intermediates and single-stranded duck hepatitis B virus DNA and RNA transcripts were detected in the brain, lung, heart, intestine, kidney, pancreas and spleen. In situ hybridization showed evidence of viral replication in the lung epithelium, germinal center of spleen, acinar cell of pancreas and tubular epithelium of kidney. These data suggest that extrahepatic duck hepatitis B virus replication is more widespread than previously thought. It is yet to be determined whether widespread extrahepatic replication is unique to duck hepatitis B virus infection or is a common feature of other mammalian hepatitis B-like viruses.

Phosphorylation sites for type N II protein kinase in DNA-topoisomerase I from calf thymus.

1. Calf thymus DNA-topoisomerase I has been isolated, in an improved preparation, nearly to SDS-PAGE homogeneity, as a single major protein (100 kDa). 2. In vitro labeling experiments, which employed the purified enzyme [gamma-32P]ATP and N II protein kinase, also showed that the calf thymus topoisomerase I became phosphorylated. 3. Phosphorylation was accompanied by an increase in topoisomerase I activity. 4. Phosphoaminoacid analysis indicated that only serine residues became phosphorylated. 5. Tryptic peptides mapping, by HV electrophoresis, identified five major [32P]peptides. This number is higher than that reported for topoisomerase I from Novikoff hepatoma cells. 6. Separation of each spot, by reverse phase HPLC, resulted in their elution at fractions 1, 2, 3, 4 and 5 with 9, 11, 16, 27 and 28% acetonitrile, respectively. 7. Isolated phosphopeptides will be subjected to sequencing, to DNA-binding and transcription regulation tests; then, it will be speculated whether type N II protein kinase may contribute to the physiological regulation of DNA topoisomerase I activity from calf thymus, as well.

Evidence for the presence of glycogen in rat thymus.

Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.

Immunohistochemical and histometrical studies of the human thymus with special emphasis on age-related changes in medullary epithelial and dendritic cells.

Age-related changes in medullary epithelial and dendritic cells in the human thymus were examined quantitatively using immunohistochemistry and histometry. The material used was thymic biopsy specimens obtained from 105 patients during cardiac surgery, none of whom had immunological abnormalities. By using keratin and tissue polypeptide antigen markers to identify epithelial cells and S-100 protein and LN-2 markers to identify dendritic cells, the numbers of these cells in the medulla were counted morphometrically. The relative proportions of the cortex, medulla, Hassall's bodies, perivascular space, connective tissue and fatty tissue were evaluated using a computer image-analysis system and the respective relative volumes were calculated. Our results indicate that the number of medullary dendritic cells/mm2 and the relative volume of cortical thymocytes decrease steadily up to the age of 40 years, whereas no major change was found in the number of medullary epithelial cells/mm2.

Organ distribution of proteinase-resistant prion protein in humans and mice with Creutzfeldt-Jakob disease.

We attempted to clarify the organ distribution of human and murine proteinase-resistant prion protein (PrPCJD) in Creutzfeldt-Jakob disease (CJD), and to measure the concentration of PrPCJD, using a semi-quantitative Western blot analysis. Human PrPCJD was restricted to the central nervous system, whereas murine PrPCJD was present in the central nervous system and in the lymphoreticular system at the end stage of CJD. PrPCJD concentration in the central nervous system of mice was almost identical to that of humans. The minimum wet weight of an organ with a positive reaction was 0.3 mg for brain, 1 to 3 mg for spleen, 3 mg for spinal cord, 3 mg for lymph node, 10 mg for thymus and 10 to 30 mg for intestine of the CJD-infected mice. There were no immunoreactions in purified PrPCJD fractions from 300 mg of spleen, lymph node, liver or peripheral nervous systems of humans, nor in 300 mg of liver, lung or kidney of CJD-infected mice. Within the limits of our method, the distribution of murine PrPCJD differed from that of human PrPCJD. Antibodies on the Western blot membrane from murine spleen PrPCJD fractions stained the kuru plaques in the CJD-infected mouse brain. Therefore, PrPCJD in the murine spleen probably shares the epitopes of the antigen in the murine kuru plaques. Although the immunological detection of PrPCJD does have limits of sensitivity, PrPCJD concentrations did correlate with infectivity titres in scrapie-infected or CJD-infected mice.