Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor.

We have previously raised an anti-c-erb A peptide antibody (designated 4B II) which immunoprecipitated in vitro transcription/translation products of c-erb A alpha 1 and beta. 4B II could recognize nuclear T3 receptor (NT3R) without distinction between difference in species and tissues. Using 4B II, we studied immunohistochemical localization of NT3R proteins in various tissues of the rat. Cryostat sections (4-6 microns) of selected rat tissues were incubated with 4B II at 4C overnight, followed by fluorescein-isothyocianate-conjugated anti-rabbit immunoglobulin G for 60 min at 25 C. The cellular localization of fluorescence in all tissues examined was exclusively nuclear. Under the same conditions, control sections stained with antiserum which had previously absorbed with c-erb A peptide or inactive serum showed no specific staining. In the brain the large nuclei, supposed to be neuronal, were strongly stained in the cerebral cortex and the granular layer of the cerebellum. In the kidney, cells in the glomerulus, the distal, but not the proximal, tubules, and the collecting ducts exhibited nuclear staining. Nuclear fluorescence was observed homogeneously in the heart and liver, but the intensity was much weaker in the latter. Less intense fluorescence was seen in the testis and spleen, although specific immunostaining was clearly observed in the nuclei of spermatocytes, Leydig cells, and the heads of the sperms in the testis, and many lymphocytes in the spleen. Nuclei of follicular cells of the thyroid exhibited very strong fluorescence, suggesting existence of plenty of NT3R proteins. The anterior pituitary showed strong immunostaining in most nuclei, and clear nuclear fluorescence was also detected in the intermediate lobe of the pituitary. The present study showed that NT3R distributes selectively in certain types of cells in many tissues and that the content of NT3R proteins seems to correlate with the concentration of c-erb A mRNA alpha 1 and beta among many organs.

Presence of 3-hydroxyanthranilic acid in rat tissues and evidence for its production from anthranilic acid in the brain.

As assessed by HPLC with electrochemical detection, 3-hydroxyanthranilic acid (3-HANA) was found to be present in the rat brain and peripheral organs. The highest concentrations were measured in the kidney (86 fmol/mg of tissue) and spleen (56 fmol/mg of tissue), whereas the adrenal gland, liver, heart, and several forebrain areas (hippocampus, striatum, parietal cortex, thalamus, amygdala/pyriform cortex, and frontal cortex) contained less 3-HANA (between 15 and 22 fmol/mg of tissue). Slightly lower concentrations of 3-HANA were found in the brainstem and the cerebellum. The metabolic disposition of 3-HANA was examined in tissue slices which were incubated in Krebs-Ringer buffer at 37 degrees C in vitro. Incubation for up to 2 h did not affect 3-HANA concentration in brain tissue. However, inhibition of 3-HANA degradation by the specific 3-hydroxyanthranilic acid oxygenase blocker 4-chloro-3-hydroxyanthranilic acid (4-Cl-3-HANA; 10 microM) resulted in a rapid (within 2.5 min) doubling of 3-HANA levels in slices from cerebral cortex. No further increases were observed after incubations of up to 120 min. Exposure of cortical slices to 3-HANA's putative bioprecursors, 3-hydroxykynurenine (3-HK) and anthranilic acid (ANA), in the absence of 4-Cl-3-HANA resulted in rapid, transient increases in 3-HANA production. Maximal 3-HANA synthesis from ANA exceeded the maximal effect of 3-HK by approximately 11-fold.2+ In the presence of 4-Cl-3-HANA, 1 mM ANA produced 9.0 +/- 0.3 and 89.0 +/- 9.3 (5 min) or 51.6 +/- 7.9 and 187.5 +/- 11.2 (120 min) fmol of newly synthesized 3-HANA/mg of brain tissue, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoprotein storage in Gaucher disease: lectin histochemistry and biochemical studies.

Lectin histochemical studies were performed on formalin-fixed, frozen, and paraffin-embedded tissue sections from 19 patients with glucosylceramide lipidosis (i.e., Gaucher disease). Eleven different lectins were used to identify the specific carbohydrate residues in the undegraded stored compounds in the cytoplasm of Gaucher cells. In all cases studied, Gaucher cells stained with Concanavalia ensiformis agglutinin, Datura stramonium agglutinin, Lens culinaris, Ricinus communis agglutinin-I, and wheat germ agglutinin. These results demonstrated common carbohydrate residues in the undegraded material stored within Gaucher cells and indicated the presence of fucosylated N-linked complex oligosaccharides, and glycans containing N-acetyllactosamine repeating sequences, as well as nonreducing terminal beta-galactosyl and sialyl residues. In order to confirm these findings using biochemical methods, livers and spleens from Gaucher patients and controls, and from a patient with Niemann-Pick disease type C (included for comparison) were digested with Pronase and the resulting glycopeptides separated by gel filtration into fractions with high and low molecular weight. In the high-molecular-weight fractions from livers of Gaucher patients, the levels of sugars corresponding to N-linked glycans, as measured by gas-liquid chromatography, were elevated over those in controls. In the high-molecular-weight fractions from spleens, the levels of the same sugars were elevated in both Gaucher and Niemann-Pick type C patients. Digestion of the glycopeptides with endo-beta-galactosidase, which specifically cleaves polylactosaminoglycans, showed the presence of material containing N-acetyllactosamine repeating units in Gaucher liver glycopeptide fractions, but not in control and Niemann-Pick type C derived glycopeptide fractions. Our histochemical and biochemical studies demonstrated that in addition to glucosylceramide, affected tissues of patients with Gaucher disease accumulate glycoproteins. This accumulation could not have been predicted on the basis of the primary enzymatic defect.

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.

Identification of novel gangliosides containing lactosaminyl-GM1 structure from rat spleen.

Two novel monosialogangliosides were isolated from rat spleen. The structures of the gangliosides (shown below, where NeuNGc is N-glycolylneuraminic acid) were characterized by compositional analysis, methylation analysis, hydrolyses with exoglycosidases, direct probe fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectrometry. A novel structure common to both gangliosides was N-acetyllactosaminyl-GM1 LacN-GM1; where GM1 is II3NeuAc-GgOse4Cer), and this is the first paper to report the occurrence of a new group of gangliosides. [formula: see text] Furthermore, in a monosialoganglioside fraction of rat spleen, the occurrence of a ganglioside having two lactosamine units (Gal alpha 1-3(Gal beta 1-4GlcNAc beta 1-3)2-GM1(NeuAc) (alpha Gal-(LacN)2-GM1] was suggested. These gangliosides have a unique structure, which includes the ganglio series ganglioside core and the extended modification characteristic of the lacto series.

Immunohistochemical localization of cathepsin D in ocular tissues.

Cathepsin D has been believed to play an important role in the catabolism of protein in various tissues. In retinal pigment epithelium, cathepsin D degrades rod outer segments and rhodopsin into glycopeptides. To our knowledge, no reports have described the immunohistochemical localization of cathepsin D in whole ocular tissues. We investigated the reaction of bovine, rat, and human eyes with a polyclonal antibody to cathepsin D from bovine spleen. Cathepsin D immunoreactivity was observed in the cytoplasm of the following cells: epithelium and endothelium of the cornea; keratocytes; pigmented and nonpigmented epithelium of the ciliary body; epithelium and cortex of the lens; epithelium and sphincter and dilator muscles of the iris; Müller cells; ganglion cells and pigment epithelium of the retina; and endothelium of various vessels. Positively stained ocular tissues were believed to have a high activity of protein catabolism. Since cathepsin D was closely associated with phagosomes in retinal pigment epithelium, we concluded that cathepsin D probably contributes to the physiologic degradation of rod outer segments.

Isolation of a specific granulopoiesis inhibitor from calf spleen and identification as glutathione.

A small peptide was isolated from calf spleen, specifically inhibiting murine granulopoiesis in vitro. Purification involved ultrafiltration, anion-exchange chromatography with a FPLC system and reversed-phase chromatography on HPLC. Determination of the amino acid composition, following acid hydrolysis and phenyl isothiocyanate and dabsyl chloride derivatization, revealed the amino acids glutamic acid, cysteine and glycine. Although the N-terminal amino group was not blocked, peptide sequencing with common techniques was not possible. Comparison of the isolated peptide with the well-known tripeptide glutathione by HPLC and fast atom bombardment (FAB)/tandem mass spectrometry showed the identity of both substances. Moreover, glutathione was found to be a specific granulopoiesis inhibitor in vitro at 10-100 nM, a so far unknown property of this well-known peptide.

Allele specific oligonucleotide probes for mouse I-A region and gene: I-A beta.

MHC Class II gene expression is being intensively studied in vitro. In vivo class II gene expression that occurs during G.V.H.R. and allograft reaction has not been studied as well as the gene expression in vitro because such studies require unambiguous detection of graft or host specific MHC class II genes. Although this is possible by using allele specific oligonucleotide probes for individual class II genes, such probes for class II genes in mice have not been described before. We compared the nucleotide sequences of I-A beta genes from various haplotypes and selected a region of minimal homology in the 2nd exon of this gene. We synthesized two oligonucleotide probes corresponding to a 20 nucleotide stretch in the hypervariable region of the I-A beta 1 genes of H-2k and H-2d haplotype mice. These probes specifically detect I-A beta mRNA from mice of appropriate haplotypes. These allele-specific probes should help study the in vivo expression of graft or host specific I-A genes in G.V.H.R. or allograft reaction.

Production of yeast killer toxin in experimentally infected animals.

The ability of a killer yeast (Pichia anomala, UCSC 25F) to produce toxin in vivo was demonstrated, for the first time, in tissues of normal and immunosuppressed experimentally infected mice by means of a fluorescent antibody technique and a killer toxin specific monoclonal antibody. The possible significance of the findings is discussed.

Distribution of mouse mammary tumor virus-related sequences does not correlate with the taxonomic position of their hosts.

Sequences (MRS) distantly related to mouse mammary tumor virus (MMTV) were found in genomes of a wide range of mammalian species using blot hybridization. The number of MRS copies and the degree of their homology with the hybridization probe varied and did not correlate with the taxonomic position of the species. Nevertheless, within a genus the set of MRS was species specific and reflected the taxonomic relation between the species. MRS were also found in avian genomes and the degree of their homology did not correlate with the taxonomic position of the species either. The origin and distribution of MRS is discussed on the basis of the authors' and published data.

Experimental selenium poisoning in Nubian goats.

The toxicity of sodium selenite was studied in 28 Nubian goats, 20 of which died or were killed in extremis 2 h to 21 d after dosing. Single or repeated daily oral doses of 160, 80, 40, 20 and 5 mg sodium selenite/kg were toxic to goats while daily doses of selenite ranging from 0.25 to 1 mg/kg/d for 225 d were not toxic to this species of animals. The main signs of poisoning were uneasiness, inappetence, dyspnea, salivation, diarhea, paresis of the hind limbs, arching of the back, and recumbency. The main lesions were hemorrhages in the rumen, reticulum, osmasum and abomasum, hemorrhagic or catarrhal abomasitis and enteritis, fatty change and necrosis of the centrilobular hepatocytes and of the cells of the renal convoluted tubules, splenic hemosiderosis, pulmonary congestion, haemorrhage, edema and emphysema, accumulation of lymphocytes in the vital organs, and straw-colored fluid in the serous cavities.

Sexual and age variations of organ iron content in Shaver chickens.

1. Haematological values and iron content in liver, spleen, kidneys and intestine were determined in Shaver chickens of both sexes at 4, 8, 13 and 18 weeks and in females at 24 weeks (the beginning of the laying period). 2. The haematocrit decreased significantly in laying compared with non-laying females and the haemoglobin concentration was similar to that in the prelaying state. Plasma iron in laying females increased to four times the basal value at 13 weeks. 3. Females of 13 and 18 weeks (prelaying state) stored more iron than males at the same age. A simultaneous liver and spleen mobilisation of stored iron and increased intestinal iron accumulation took place in the laying process. The haematological variables examined were minimally altered. 4. The iron contents of both heart and kidneys were influenced by age and followed a linear trend, except that in the heart of females where a quadratic response was observed.

Gaucher's disease type I. Report of a case with prominent deposition of ceroid in splenic endothelial cells and intestinal smooth muscle fibers.

A case of type I Gaucher's disease in a 39-year-old male is reported. Autopsy showed marked enlargement of the spleen (3,070 g) and infiltration of typical Gaucher's cells in the spleen, liver, bone, marrow, gastrointestinal tract, lymph nodes and adrenal glands. The diagnosis of Gaucher's disease was ascertained by the very low beta-glucosidase activity of cultured subcutaneous fibroblasts and the high content of glucocerebroside in the spleen tissue. A peculiar finding in this case was prominent deposition of brown pigment in endothelial cells of the spleen and smooth muscles fibers of the gastrointestinal tract, urinary bladder and prostate. Histochemical examination revealed that the granules in endothelial cells and smooth muscle fibers were ceroid. Such deposition of ceroid has never been reported previously in Gaucher's disease. Ceroid deposition in generalized smooth muscle fibers is known as brown bowel syndrome, and is highly suggestive of severe vitamin E deficiency. Although other symptoms of vitamin E deficiency were not noticed in this case, some malnutritional condition might play a role in prominent deposition of ceroid in lysosomes, possibly together with deficient activity of a lysosomal enzyme.

Human splenic galaptin: physicochemical characterization.

Mammalian spleens were previously reported to contain beta-galactoside-binding lectins [Allen, H. J., Cywinski, M., Palmberg, R., & DiCioccio, R. (1987) Arch. Biochem. Biophys. 256, 523-533]. The aim of the present investigation was to determine the relationship of human splenic galaptin to other beta-galactoside-binding lectins identified in other human and animal tissues. Galaptin of subunit molecular mass 14.5 kDa was the only lectin of this type found in human spleen as assessed by SDS-PAGE, RP-HPLC, and Western blot analyses. Three polypeptides of pI 4.60, 4.80, and 4.85 were detected by isoelectric focusing of purified galaptin, with the major band having pI 4.85. UV spectral analysis indicated the absence of prosthetic groups and gave A1%(1cm), 280 = 5.5. Circular dichroic analysis suggested the presence of 40% beta structure, considerable random coil, and 10% alpha helix structure. The amino acid composition was very similar to that for human placental galaptin. Amino acid sequence analyses were carried out on V8 protease, CNBr, and iodosobenzoic acid digestion fragments. A total of 94 residues were identified. All sequences determined could be aligned with placental galaptin sequences. We conclude that human splenic galaptin is identical with human placental galaptin. A related polypeptide of molecular mass approximately 14.5 kDa was found to be present in several different mammalian spleens as assessed by Western blot analysis using a monospecific polyclonal anti-human splenic galaptin antiserum.

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.

Macrophage activation by leptospiral lipopolysaccharide.

Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovars copenhageni and hebdomadis were tested for the ability to induce macrophage activation. In-vitro analysis showed that each leptospiral LPS was a potent activator to macrophages. After stimulation with the LPSs, interleukin-1 (IL-1) secretion, interferon (IFN) production and chemiluminescence (CL) response were induced. Intravenous high-dose injection of the leptospiral LPSs induced various lesions such as necrosis of the liver, and the LPSs were detected in macrophages in the liver, spleen and lymphnodes by immunohistochemical examination. Enhancement of macrophage activity in mice inoculated with low doses of leptospiral LPS was recognized. The macrophages of the LPS-treated mice showed a significantly higher bactericidal action than those of control mice. The beta-galactosidase and nitroblue tetrazolium (NBT) positive cells in macrophages of the LPS-treated mice increased significantly. In the NBT reduction test after phagocytosis of latex beads or Salmonella typhimurium, the macrophages of the LPS-treated mice showed a significantly higher activity than those of control mice.

Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells.

We have partially purified and characterized erythropoietin (Epo) receptors of erythroid progenitor cells which were obtained from the spleens of anemia-inducing Friend virus infected mice. Membrane proteins of splenic erythroid progenitor cells were solubilized with 1% Triton X-102. Upon chromatography on DEAE-Sephacel anion-exchange columns, two distinct Epo receptor peak fractions referred to as Peak I and Peak II were identified by 125I-Epo binding assays using the polyethylene glycol precipitation method. The Peak I and Peak II samples were then individually chromatographed on an S-Sepharose column. The S-Sepharose-purified Peak I and Peak II samples were crosslinked with 125I-Epo in the presence and absence of excess unlabeled Epo by disuccinimidyl suberate treatment, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Both Peak I and Peak II samples showed a radiolabeled peptide with a Mr 135K and the labeling was blocked by excess unlabeled Epo. Since the Mr of Epo is about 35K, Epo receptor peptide has a Mr approximately 100K. To determine whether Epo stimulates autophosphorylation of the receptors, the S-Sepharose-purified Peak I and Peak II samples were incubated with or without Epo, and then briefly incubated in the presence of [gamma-32P]ATP and Mn2+. The tyrosine residue phosphorylated protein was isolated by an immunochemical technique, and then analyzed by SDS-PAGE and autoradiography. The result showed that Epo stimulates phosphorylation of a 100-kDa peptide.

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.

Binding characteristics of galactoside-binding lectin (galaptin) from human spleen.

Binding characteristics of human spleen soluble galactoside-binding protein (galaptin) were studied using simple galactosides, galactose-terminated disaccharides, cluster glycosides containing up to 6 terminal lactosyl residues, bovine serum albumin derivatives containing 7 to 40 lactosyl residues, desialylated serum glycoproteins, and glycopeptides derived thereof as inhibitors in a newly developed binding assay. In this assay, aminohexyl lactoside was attached to divinyl sulfone-activated Sepharose, which was then used to bind 125I-galaptin. Similarly derivatized Sepharose containing mannoside served as a control. The assay is sensitive, maintains linearity in the concentration range of 125I-galaptin tested, and has very low nonspecific binding. The following new findings were made. 1) All the alpha-D-galactopyranosides with non-sugar aglycon were better inhibitors than the corresponding beta-D-galactopyranoside. 2) The S-galactosides were better inhibitors than the corresponding O-galactosides, regardless of the anomeric configuration. 3) Many Gal beta 1-4- and Gal beta 1-3-linked disaccharides were tested. Although the galaptin did not appear to recognize N-acetylglucosamine as a monosaccharide, the presence of this sugar penultimate to galactose increased the binding affinity by as much as 500-fold, as was the case for N-acetyllactosamine. Of a particular importance is the presence of an equatorial 3-OH group on this sugar. We synthesized the 3-deoxy derivative of N-acetyllactosamine and found that it had 50-fold lower binding affinity compared to N-acetyllactosamine. 4) The binding sites of this lectin do not seem to be operating in a cooperative fashion, since synthetic lactose-containing divalent ligands with various inter-galactose distances did not increase the binding affinity significantly.

[Histochemical study of proliferating cells in the spleen in mice with acute hepatic injury].

When heat-killed Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS) were injected into mice, liver cell necrosis with infiltrating neutrophils and macrophages were induced. Proliferating cells in the spleen were histochemically investigated. P. acnes injection rapidly produced hyperplasia of neutrophils and macrophages in the red pulp of the spleen. The proportion of Bromodeoxyuridine positive cells reached a peak at 5 days after injection of P. acnes. These results indicate that proliferating cells in the spleen after injection of P. acnes are mainly neutrophils and macrophages, which are the same kinds of infiltrating cells into the liver after injection of P. acnes and LPS.