Osteonectin mRNA: distribution in normal and transformed cells.

Overlapping cDNA clones encoding bovine osteonectin were isolated from a lambda gt11 expression library constructed from bovine bone cell mRNA. The longest clone, lambda On 17 (insert size 2.0 kb) was studied in detail. The clone was shown to encode osteonectin by hybrid select translation experiments and by DNA sequence analysis. Northern analysis of bone cell RNA showed the length of the osteonectin mRNA to be 2.0 kb. Osteonectin message was found in bone but not in soft tissue (liver and brain) preparations consistent with the distribution of the protein in these tissues. On the other hand, osteonectin message was observed in tendon, a tissue in which little or no osteonectin protein is found in vivo. Hybridization of osteonectin cDNA was detected in cells from a number of species including human, rat, mouse and chick. The level of osteonectin mRNA was drastically decreased in chick embryo fibroblasts transformed by Rous sarcoma virus.     

Tissue distribution and subcellular localization of retinol-binding protein in normal and vitamin A-deficient rats.

Levels of retinol-binding (RBP), the plasma transport protein for vitamin A, were measured by radioimmunoassay in sera and in a large number of tissues from both normal and vitamin A-deficient rats. The tissues included liver, kidney, fat, muscle, brain, eye, salivary gland, thymus, lung, heart, intestine, spleen, adrenal, testes, thyroid, and red blood cells. The RBP levels in tissues other than serum, liver, and kidneys varied from 12 mug/g of tissue for normal spleen to an undetectable level in red blood cells. Much of the RBP in the tissues with low levels may have been due to residual serum in the samples. In general, except for liver, RBP levels were lower in tissues from vitamin A-deficient rats than in those from normal rats. In normal rats, the liver, kidney, and serum levels were 30 plus or minus 4 (mean plus orminus SEM), 151 plus or minus 22, and 44 plus or minus 3 mug/g, respectively. In vitamin A-deficient rats, the liver RBP level was about three times the normal level whereas the kidney and serum levels were about one-fifth the normal values. When normal liver homogenates were fractionated by centrifugation, 67% of the RBP was recovered in the microsomal fraction and only 9% was found in the soluble 105,000 g supernate. In contrast, 76% of the RBP in homogenates of normal kidneys was in the soluble fraction. Similar results were obtained with deficient livers and kidneys. Incubation with deoxycholate released the liver RBP into the soluble fraction. RBP is produced in the liver and removed from the blood by the kidneys. The levels of RBP in normal and deficient liver, serum, and kidney appear to reflect the relative rates of RBP secretion and turnover.     

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

Abstract

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood–brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.     

Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue

An immunohistochemical method was developed, using a polyclonal antibody, to detect the enzyme indoleamine 2,3-dioxygenase (IDO) in normal and malaria-infected tissue. Plasmodium berghei ANKA, a cerebral malaria (CM) model, and P. berghei K173, a non-cerebral malaria (NCM) model, were used. It was found that vascular endothelial cells were the primary site of IDO expression in both models of malaria infection and that this response was systemic, with the vascular endothelium of brain, heart, lung, spleen and uterus all staining positive. These results suggest that IDO is part of a systemic host response to parasite infection. Although high levels of IDO production alone may not cause pathology, it is possible that when its production is combined with other features of CM, such as breakdown of the blood-brain barrier (BBB), metabolites of the kynurenine pathway may be able to influence the otherwise tightly regulated, immunologically privileged site of the CNS and cause some of the symptoms and pathology observed.     

The effect of corticosteroids upon the number and organ distribution of "background" immunoglobulin-secreting cells in mice

The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon the immunoglobulin (Ig)-secreting cells was studied in not intentionally immunized BALB/c mice. This was done for IgM-, IgG-, and IgA-secreting cells in spleen, mesenteric lymph nodes (MLN), and bone marrow (BM). A single injection of DEXA (16 to 144 mg/kg body wt) markedly reduced the number of Ig-secreting cells in spleen and MLN within 1 day, but hardly affected their number in the BM. The decrease was immediately followed by a recovery and, at the highest doses and especially in MLN, by an overshoot. Two weeks after the initial decrease a second decrease was found. When mice were subjected to daily treatment with DEXA during 1 week, initially a recovery pattern was found in spleen and MLN similar to that found after a single injection of a high dose. In this case, however, the effects were less dose dependent, and the overshoot reaction was followed by a period of subnormal numbers of Ig-secreting cells which lasted at least 1 week. This late effect of DEXA not only occurred in spleen and MLN, but also in the BM. The most prominent effect of daily treatment with DEXA was the long-lasting decrease of the number of IgG-secreting cells starting 1 week after withdrawal of treatment. This decrease was associated with a severely decreased serum IgG level.     

Glutathione distribution in normal and oxidatively stressed cells

Glutathione is the most abundant of the low-molecular-mass molecules that provide reducing equivalents that protect cells from oxidative stress. We used immunoelectron microscopy to investigate glutathione distribution in normal and oxidatively stressed cells. Here, for the first time, we show that reduced glutathione is distributed relatively evenly throughout the cell, with the exception of the lumen of the rough endoplasmic reticulum, where little is detected. Oxidant exposure, either to 0.1 mM diamide or ethycrinic acid, eventually caused cellular glutathione depletion. However, despite entering a cell within seconds, both oxidants required hours to dramatically affect glutathione levels in the majority of cells in a population. Interestingly, cells within a homogeneous cell line population lost glutathione at different rates. Structural changes associated with oxidative stress, such as increased vacuolization and membrane blebbing, were correlated with glutathione depletion. Oxidant-exposed cells that appeared normal had higher glutathione levels than those within the same population that appeared stressed. The last reserves of cellular glutathione were found within mitochondria.     

Gammaglobulin treatment and anti-IgA antibodies in IgA-deficient patients.

Antibodies to IgA may cause severe anaphylactic reactions during blood transfusions. Tests for anti-IgA antibodies were carried out on six patients with IgA deficiency (five of whom also had hypogammaglobulinaemia) who had received continuous gammaglobullin treatment for chronic or recurrent infections for three to eight years. Three patients had minute amounts of IgA, and three had none (less than 0.01 microgram/ml). Only one patient had anti-IgA. Her antibody titre did not change during treatment. No patient had any untoward effects of treatment, which relieved the symptoms of infection in every case. IgA determinations should be performed by more accurate methods than radial immunodiffusion when evaluating the risks of giving gammaglobulin to patients with hypogammaglobulinaemia and IgA deficiency. Probably the stimulus provided by intramuscular gammaglobulin in such patients is insufficient for the formation of anti-IgA antibody.     

Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Myosin isoforms in normal and dystrophic chickens

The myosin isoform content in the affected fibers of chickens with inherited muscular dystrophy has been investigated with a new high-performance liquid chromatographic procedure for separation of the tryptic fragments of myosin subfragment 1 (S-1). The results indicate that dystrophic muscle contains substantial amounts of normal adult myosin, together with various myosin species present in normal 5-day posthatch chickens. Confirmation was obtained by comparative peptide mapping of the S-1 tryptic fragments and by N-terminal sequencing of 20-kDa species. Together with data on other contractile proteins and certain metabolic enzymes [Obinata, T., Takano-Ohmura, H., & Matsuda, R. (1980) FEBS Lett. 120, 195-198; Mikasa, T., Takeda, S., Shimizu, T., & Kitaura, T. (1981) J. Biochem. (Tokyo) 89, 1951-1962; Feit, H., & Domke, R. (1982) Cell Motil. 2, 309-315; Cosmos, E. (1966) Dev. Biol. 13, 163-181; Cosmos, E., & Butler, J. (1967) in Exploratory Concepts in Muscular Dystrophy and Related Disorders (Milhorat, A. R., Ed.) pp 197-204, Excerpta Medica, Amsterdam], the results are consistent with the hypothesis that there is a general defect in muscle maturation in avian dystrophy.     

Gamma-irradiation depletes endogenous germ cells and increases donor cell distribution in chimeric chickens

The production of chimeric birds is an important tool for the investigation of vertebrate development, the conservation of endangered birds, and the development of various biotechnological applications. This study examined whether gamma (γ)-irradiation depletes endogenous primordial germ cells and enhances the efficiency of somatic chimerism in chickens. An optimal irradiation protocol for stage X embryos was determined after irradiation at various doses (0, 100, 300, 500, 600, 700, and 2,000 rad). Exposure to 500 rad of γ-irradiation for 73 s significantly decreased the number of primordial germ cells (P < 0.0001). Somatic chimera hatchlings were then produced by transferring blastodermal cells from a Korean Oge into either an irradiated (at 500 rad) or intact stage X White Leghorn embryo. An analysis of feather color pattern and polymerase chain reaction-based species-specific amplification of various tissues of the hatchlings confirmed chimerism in most organs of the chick produced from the irradiated recipient; a lesser degree of chimerism was observed in the non-irradiated control recipient. In conclusion, the exposure of chick embryos to an optimized dose of γ-irradiation effectively depleted germ cells and yielded greater somatic chimerism than non-irradiated control embryos. This technique can be applied to interspecies reproduction or the production of transgenic birds.     

A general method for studying the secretion of macromolecules by single cells. I. Detection of immunoglobulin-secreting cells.

A general method for detecting single cells secreting macromolecules has been developed and applied to the detection of mouse cells secreting antibodies. Secreted Ig molecules were precipitated in the immediate vicinity of an active cell with rabbit anti-mouse Ig antibody. Rapid removal of excess antibody not incorporated into immunoprecipitates was achieved with an electrophoresis technique. The immuno-precipitate surrounding the active cell was then stained with sheep anti-rabbit Ig antibody labeled with horseradish peroxidase. The use of enzyme markers has made the procedure much more convenient and rapid than previous precipitin assays for cell secretion that used radioiodine for detecting immunoprecipitates. Moreover, the availability of several enzyme markers makes possible the detection of cells secreting more than one molecular species. Experiments were also run in which cells producing antibodies specific for horseradish peroxidase (HRP) could be identified among the population of cells producing other immunoglobulins. Presumably, HRP-labeled antigens could be used to identify cells producing other specific antibodies. The generality of this procedure suggests that it may be useful for detecting single T-cells releasing regulatory molecules, since specific antisera are already available for several of these molecules.     

Tumor growth and antibodies after RSV-challenge in normal chickens and in chickens congenitally infected with avian leukosis virus.

Normal chickens and chickens congenitally infected with an avian leukosis virus (ALV) of antigenic subgroup A were challenged with strains of Rous sarcoma virus (RSV) of two different antigenic subgroups (B and C) and tumor induction and growth as well as humoral antibody to viral envelope antigen (VEA) and tumor-specific surface antigen (TSSA) were measured. There was no effect of congenital ALV infection on RSV tumor incidence or latent period but the growth rate and size of the tumors were much higher in congenitally infected birds as compared to controls. Whereas most tumors in the RSV-challenged normal birds regressed, tumors in ALV-infected birds grew progressively. There were no striking differences in the number of birds in either group in the incidence of anti-TSSA or anti-VEA antibodies nor did the presence of either type of antibody reflect the tumor status of the host.     

Construction of EGFP-tagged rBCG of E.tenella and distribution in chickens

Chicken coccidiosis is a major parasitic disease with substantial economic burden to the poultry industry. Enhanced green fluorescent protein (EGFP) tagged recombinant Bacille Calmette-Guerin (rBCG), as a fusion protein with coccidian rhomboid antigen was constructed to track rBCG in vivo in chickens in this study. Immunization of chickens with one dose of rBCG pMV361-Rho/EGFP induced humoral immune response. The colonization of rBCG in liver, spleen, lung, kidney and caecum was observed by laser confocal microscopy. Real-time quantitative RT-PCR showed a rise expression level of rhomboid protein on the 7th day and a peak on the 14th day and disappearance on the 28th day after immunization. These results have significant implications for the development of rBCG vaccines against avian coccidiosis. Supported by Grants from the Foundation of the Jilin Provincial Science and Technology Department of China (Grant No. 20050211-2) and the National Natural Science Foundation of China (Grant No.30170696, 30500370 and 30671580).     

Apparent volumes of distribution of 125-I-lothalamate and inulin in chickens.

The suitability of utilizing 125-I-iothalamate to estimate the volume of extracellular fluid was assessed in ureterally ligated chickens. Subsequent to intravenous administration the movement of labeled iothalamate from the plasma compartment follows closed two-compartment kinetics and equilibration between vascular and extravascular phases is attained in about 20 minutes. The volume of distribution of 125-I-iothalamate prior to and following the influsion of 0.15 M NaCl (equal to 15% of the estimated ECFV) averaged 23.6 plus or minus 0.61 and 28.4 plus or minus 0.22% of the body weight, respectively. The observed postsaline labeled iothalamate space did not differ statistically from the expected value. When administered simultaneously inulin penetrates into an apparent volume that is 75% of the labeled iothalamate space after 60 minutes. The content of 125-I-iothalamate is relatively high in liver and kidney tissue and suggests that these are major sites where removal of the indicator from plasma occur. It is suggested that 125-I-iothalamate, under appropriate conditions, could be used to measure the plasma volume and the extravascular fluid with which plasma is in rapid diffusion equilibrium.     

Tissue specific methylation of c-myc in adult chickens

Methylation of cytosine in DNA has long been correlated with modulation of specific gene expression in eukaryotes. Methylation of the c-myc locus was examined in six tissues from adult Leghorn chickens. The c-myc locus was found to be variably methylated in all examined tissues, except blood, where erythrocytic DNA showed no evidence of significant methylation of c-myc. This is contrasted with the observed sever methylation of the beta actin locus and the generally high methylation patterns found in avian erythrocytic DNA.     

Localization of vitellogenin and serum albumin in hepatic parenchymal cells of normal and estradiol-treated immature chickens

The localization of albumin and vitellogenin was determined in liver sections from control and estradiol-treated chickens by two different immunocytochemical techniques: (1) The sandwich technique with rabbit anti-lipovitellin or rabbit anti-albumin IgG and fluorescent goat anti-rabbit IgG and (2) the mixed aggregation immunocytochemical technique with anti-lipovitellin IgG and fluorescent lipovitellin.The results show that the antibody against albumin bound only to all liver parenchymal cells. Furthermore, the fluorescence intensity was equally strong in the portal, intermediate and central zones of the lobules.The fluorescent stain for vittelogenin was not above background in livers of control chicks but was far above background in estradiol-treated chicks. As with albumin the fluorescent stain was distributed equally among the parenchymal cells.The results were quantitatively the same 2 and 4 days after estradiol treatment. The relative rates of synthesis and the concentrations of albumin and vitellogenin correlate well with values obtained for tissue sections by immunocytochemical techniques.