High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.

In vitro of adenosine on lymphocytes and erythrocytes from horses with combined immunodeficiency.

The effect of adenosine on the mitogenic response of peripheral blood lymphocytes (PBL) and on the nucleotide pools of erythrocytes from normal horses, horses heterozygous for the combined immunodeficiency (CID) trait (carriers), and foals with CID was studied. When PBL from normal, carrier, and CID horses were stimulated by phytohemagglutinin (PHA), concanavalin A, or pokeweed mitogen, [3H]thymidine uptake was inhibited by adenosine (0.1 microM) to 1.0 mM) in a dose-dependent manner. Adenosine (100 microM) mediated inhibition of [3H]thymidine uptake was prevented in both normal and carrier horse PBL by incubation with uridine. Uridine had no sparing effect on PBL from horses with CID. Differences were detected between human and horse PBL in response to adenosine and erythro-9(2-hydroxy-3-nonyl) adenine (EHNA), a competitive inhibitor of adenosine deaminase. In the first assay, mitogen-stimulated PBL from horses were more sensitive to adenosine. In the second assay, adenosine was added to PBL cultures at various times after PHA addition. Adenosine inhibited mitogenesis in horse PBL if added within the first 24 h. In human PBL cultures, adenosine inhibited mitogenesis only if added within the first 4 h. The third assay measured capacity of PHA-stimulated human and horse lymphocytes to escape inhibition by adenosine or EHNA. At the end of a 72-h culture period, horse PBL were still inhibited of mitogenesis in both human and horse PBL. With prolonged incubation (72 h), synergistic inhibition was detected only in horse PB. With high-pressure liquid chromatography, nucleotide levels in erythrocytes of normal, carrier, and CID horses were found to be similar. Incubation with adenosine produced a 1.5- to 2-fold increase in total adenine nucleotide pools in erythrocytes from all horses. However, these increases were accompanied by alterations in the relative amounts of the nucleotide components. This was seen as a significant decrease in the ATP:(AMP plus ADP plus ATP) ratio and energy charge in erythrocytes from normal horses. In contrast, the ATP:(AMP plus ADP plus ATP) ratio decreased only slightly in erythrocytes from CID horses, whereas no change in the energy charge was detected. The data from these studies indicate a difference in adenosine metabolism exists between human and horse lymphoyctes, and an abnormality may exist in purine metabolism or in an interconnecting pathway in horses with CID.

Elevated pim-1 and c-myc proto-oncogene induction in B lymphocytes from BLV-infected cows with persistent B lymphocytosis.

Bovine leukemia virus (BLV) induces a non-malignant, polyclonal, persistent lymphocytosis (PL) of circulating, CD5 B lymphocytes in cattle, with variable progression to CD5 B cell leukemia or lymphoma. We analyzed the expression of two proto-oncogenes, pim-1 and c-myc, proto-oncogenes deregulated in some human B cell leukemias and lymphomas, in peripheral blood mononuclear leukocytes (PBML) from BLV-infected PL cows. Results demonstrate that pim-1 and c-myc mRNA levels are elevated in unfractionated stimulated PBML from a sample of PL cows naturally infected with BLV. Results confirm that pim-1 is constitutively expressed, but not inducible in normal bovine peripheral blood B lymphocytes, but can be induced in the predominantly CD5 B lymphocytes from BLV-infected PL cows. Results further demonstrate that c-myc is inducible in bovine B and T lymphocytes regardless of BLV status, but the amount of induction is greater in B lymphocytes from BLV-infected PL cows than in B lymphocytes from noninfected control cows. These results suggest that pim-1 and c-myc are upregulated in B lymphocytes from BLV-infected PL cows and that deregulation of proto-oncogene expression is not limited to completely transformed cells, but can also characterize a naturally occurring, pre-neoplastic lymphocytic state.

Rapid induction of pim-1 expression by prolactin and interleukin-2 in rat Nb2 lymphoma cells.

The lactogen-dependent Nb2 lymphoma line (Nb2-11) represents a useful pre-T cell model for investigation of early molecular events coupled to PRL-stimulated cell cycle progression. Expression of pim-1, a protooncogene that encodes a conserved cytosolic serine/threonine protein kinase, is rapidly induced in hematopoietic cells upon mitogen stimulation and is thought to be important for lymphocyte activation. The present study was conducted to determine whether mitogen stimulation in Nb2-11 or lactogen-independent Nb2-SFJCD1 cells provokes pim-1 gene expression. The pim-1 transcript was undetectable in control growth-arrested Nb2-11 cultures; however, PRL rapidly stimulated its expression in a biphasic manner. Peak expression occurred within 2-4 h (> 40-fold) and was followed by a second elevation at 12 h. The effect of PRL and IL-2 to induce pim-1 at 2 h was concentration dependent and not inhibited by cycloheximide. In Nb2-SFJCD1 cells, pim-1 messenger RNA was expressed in control cultures and augmented by PRL stimulation. Results from stability studies indicated that the t1/2 values for the pim-1 transcript were 79 and 81 min in PRL-stimulated Nb2-11 cells at 2 and 12 h. However, in the lactogen-treated Nb2-SFJCD1 line, it was nearly 3-fold more stable (219 min) at 2 h compared to that determined at either 12 h or in unstimulated cultures. In other experiments, PRL-stimulated expression of the pim-1 protein was evaluated in [35S]methionine-labeled cells by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In Nb2-11 cells, enhanced [35S]pim-1 expression paralleled its messenger RNA transcription through 8 h. Elevated [35S]pim-1 was detected within 1 h and peaked by 2-4 h. Therefore, pim-1 represents an immediate early gene induced by PRL stimulation in Nb2-11 cells. Its initial peak of transcription occurs early during G1 cell cycle progression, whereas a second elevation is coincident with the G1/S transition. These results demonstrate that mitogen-induced expression of pim-1 is a rapid event in Nb2 lymphoma cells and suggest that it may be associated with cell cycle progression.

Prolactin regulation of pim-1 expression: positive and negative promoter elements.

The lactogen-dependent rat Nb2 lymphoma is a useful model to investigate PRL signaling pathways that lead to regulation of gene transcription. A primary mechanism coupled to PRL receptor (PRLR) activation in Nb2 cells involves phosphorylation by Jak-family tyrosine kinases of one or more signal transducers and activators of transcription (Stat) factors which subsequently bind to gamma-interferon activation sequences (GAS) within promoter regions of target genes. However, it is presently unclear whether this mechanism is operative as a means for regulating PRL-induced gene expression to the exclusion of other signaling pathways. Previously, we reported that PRL directly stimulated rapid expression of the protooncogene, pim-1, at the mRNA and protein levels in lactogen-dependent Nb2-11 cells. In the present study, experiments were conducted to evaluate signaling mechanisms by which PRL regulates transcription of pim-1. Toward this end, a 1,268-bp segment upstream of the transcription initiation site of the 5'-pim-1 promoter and a series of deletion mutants were ligated upstream of the chloramphenicol acetylase transferase (CAT) gene in an expression vector that was introduced into FDC/Nb2 cells, a premyeloid line that stably expresses the intermediate form of the PRLR. Analysis of PRL-treated cultures indicated that two elements [distal (DE), -427 to -336 bp and proximal (PE), - 104 to -1] but not several GAS or GAS-like sequences were required for hormone activation of the pim-1 promoter. Moreover, treatment of Nb2-11 cells with PRL activated protein binding to these elements assessed by gel mobility shift assay. Deoxyribonuclease I (DNase I) protection experiments revealed a motif containing a nuclear factor-1 (NF-1, -224 to -217 bp) half-site that was hydrolyzed when exposed to extracts from PRL-treated cells but protected by proteins from unstimulated cells. Gel mobility shift analysis of this sequence showed decreased protein binding after PRL stimulation. It is concluded that the PRLR initiates pim-1 transcription by a mechanism that involves transcriptional activation by factors that stimulate the DE- and PE-sites and derepress a NF-1-containing element. Moreover, this mechanism appears to be independent of an interaction between Stat transcription factors and GAS-like elements present within the promoter.

Correction of equine severe combined immunodeficiency by bone marrow transplantation.

A 32-day-old horse with severe combined immunodeficiency was transplanted with equine bone marrow cells in an attempt to establish immunologic responsiveness. A histocompatible, mixed-leukocyte-culture-nonreactive, sex-matched, full sibling was used as the donor. Recipient total lymphocyte count, T and B lymphocyte numbers, and response of peripheral blood mononuclear cells to phytolectin stimulation increased by 14 days following transplantation. Circulating lymphocytes exceeded 1000 cells/microliter blood by 40 days posttransplantation, and by 170 days following transplantation, T and B lymphocyte numbers had reached normal values. The foal demonstrated significant primary and secondary antibody responses when immunized with bacteriophage phi X 174 at 100 and 142 days posttransplantation. Concentrations of IgG and IgM remained within the normal range following cessation of i.v. plasma therapy 156 days after transplantation. More than 300 days following transplantation, the foal remains healthy and is growing normally. At no time during the posttransplant period was there detectable evidence of graft-versus-host disease.

Trypanosoma brucei: peptide mapping of partially homologous variable surface glycoproteins.

Antigenic variation in Trypanosoma brucei is caused by amino acid sequence changes in a major surface glycoprotein. Each trypanosome may contain between 100 and 1000 genes coding for this glycoprotein. Some of these genes are members of partially homologous gene families. In addition, segments of different genes may combine to form 'hybrid' or 'mosaic' genes. Thus, surface glycoproteins exist containing varying amounts of amino acid sequence homology. For investigations of molecular mechanisms of antigenic diversity it is important to identify sub-sets of trypanosomes expressing related surface glycoproteins. We describe here a simple method based on trypanosome surface labeling followed by peptide mapping to indicate homologous peptides present in one sub-set of T. brucei surface glycoproteins.

Suppression of lymphocyte proliferation by proteins secreted by cultured Sertoli cells.

Secreted proteins from cultured rat Sertoli cells were assessed for effects on phytolectin-stimulated rat splenic lymphocytes. Sertoli cell proteins (SCP) suppressed DNA, RNA and protein synthesis in stimulated rat splenic lymphocytes whether added at 0, 4, 24 and 48 h after culture initiation. SCP preparations were not toxic to cells. SCP suppressive activity was heat stable but was not associated with the carbohydrate component of SCP preparations. SCP also suppressed the proliferation of lymphoid and non-lymphoid cell lines from several different animal species but did not inhibit proliferation-independent lysis of YAC-1 target cells by rat natural killer cells. These results suggest that Sertoli cells synthesize inhibitory factors that might be secreted into seminal plasma. Furthermore, our results demonstrate that one mode of action of these factors is suppression of cell proliferation.

Effects of conjugated dienoic derivatives of linoleic acid and beta-carotene in modulating lymphocyte and macrophage function.

The in vitro effects of conjugated dienoic derivatives of linoleic acid (CLA) in combination with beta-carotene on lymphocyte and macrophage function was studied. Porcine blood lymphocytes and murine peritoneal macrophages were incubated with 0 (control), 1.78 x 10(-5), 3.57 x 10(-5) and 7.14 x 10(-5) M CLA and 0 (control), 10(-9), 10(-8) and 10(-7) M beta-carotene. CLA alone stimulated mitogen-induced lymphocyte proliferation, lymphocyte cytotoxic activity and macrophage bactericidal activity. In contrast, CLA inhibited interleukin-2 production by lymphocytes and suppressed the phagocytic activity of macrophages. beta-Carotene alone stimulated the cytotoxicity of lymphocytes and increased superoxide production by peritoneal macrophages. When present together, CLA and beta-carotene interacted in an additive manner to further enhance lymphocyte cytotoxicity and spontaneous lymphocyte proliferation. In addition, beta-carotene was able to negate the inhibitory action of CLA on the phagocytic activity of macrophages. Also, CLA and beta-carotene together seemed to suppress mitogen-induced lymphocyte proliferation. Therefore, CLA and beta-carotene; alone and in concert, act to modulate different aspects of cellular host defense.

A rapid method for the large scale preparation of bovine leukemia virus antigen.

Bovine leukemia virus (BLV) is the etiologic agent responsible for enzootic bovine leukosis. Detection of cattle seropositive for BLV and laboratory studies of BLV require the preparation of large quantities of BLV antigen. A convenient and economical method for concentrating virus from large volumes of cell culture medium involves the precipitation of the virus in the cold by polyethylene glycol (PEG). The present work demonstrates the feasibility of rapidly precipitating BLV with 10% PEG and subsequently separating the resuspended virus from the residual PEG on a disposable desalting column.

Biochemical and functional characterization of lymphocytes from a horse with lymphosarcoma and IgM deficiency.

Neoplastic lymphocytes from a horse with lymphosarcoma and IgM deficiency were analyzed for ability to grow in culture; surface and cytoplasmic IgM; functional activity in blastogenesis, cytoxicity, and suppressor assays; and activities of six enzymes involved in purine and pyrimidine metabolism. The cells lacked surface and cytoplasmic IgM. They had elevated activity of adenosine deaminase and reduced activity of purine nucleoside phosphorylase. Neoplastic cells were nonresponsive in blastogenesis assay and did not kill allogeneic lymphocyte target cells or YAC-1 targets in a lectin-dependent cytotoxicity assay, however, the cells were active in a suppressor assay. They were grown for 16 weeks in cultures supplemented with interleukin 2, during which time the cells retained suppressive activity. These results are consistent with a T cell lymphoma of suppressor cell origin, and may explain the deficiency of IgM observed in some horses with lymphoreticular neoplasms.

cDNA cloning, sequencing and characterization of bovine pim-1.

The cDNA clone of bovine pim-1 has been isolated from phorbol-12-myristate-13-acetate (PMA) and concanavalin A (ConA)-activated peripheral blood lymphocytes (PBLs). The full-length cDNA contains a 411bp 5' untranslated region (5'-UTR), followed by a 939bp coding region and a 3' untranslated region (3'-UTR) that contains 1403bp. Comparison of the bovine pim-1 coding sequence with the human, rat, mouse, frog and zebrafish counterparts reveals 94, 90, 89, 67 and 40% homology at the nucleotide level, respectively. The predicted amino acid sequence of bovine Pim-1 shares 98.7, 97.1, 93.3, 68.8, and 52.4% similarity with the sequences of human, rat, mouse, frog, and zebrafish, respectively. The 5'-UTR of bovine pim-1 shares high sequence similarity to the human and mouse counterparts and is G/C-rich (75%) which may promote a high degree of secondary structure. The 3'-UTR of bovine pim-1 contains two potential polyadenylation sites and an A/T-rich motif which has been shown to decrease the stability of polyA mRNA molecules. Southern blot results indicate that a single copy of the gene exists in the bovine genome. Northern blot results show that PMA stimulation of PBLs increases the expression of the pim-1 mRNA. In addition, examination of Pim-1 protein expression in PBLs stimulated with a variety of mitogens including ConA, PMA, anti-CD3 and purified protein derivative (PPD) from Mycobacterium tuberculosis, reveals two different types of expression patterns during the course of a 24h period of stimulation. ConA and PPD gave a biphasic pattern of expression while PMA and anti-CD3 gave single transient pattern of expression suggesting that expression is controlled by more than one signaling pathway.

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Immunologic reconstitution of foals with combined immunodeficiency.

Thirty-eight foals with combined immunodeficiency (CID) received transplanted fetal liver cells, fetal liver and thymus cells, histocompatible bone marrow cells, or equine lymphocyte antigen (ELA) haploidentical bone marrow cells in an attempt to reconstitute their deficient immune systems. Engraftment was infrequent, partial, and unpredictable when fetal cells were employed. Three of five CID foals receiving ELA haploidentical bone marrow cells demonstrated partial reconstitution, but engraftment was only temporary. Administration of histocompatible bone marrow cells resulted in rapid, full and sustained engraftment.

Characterization of heteromyeloma fusion partners which promote the outgrowth of porcine hybridomas.

Production of porcine monoclonal antibodies for use in research and immunotherapy has been hampered by the lack of suitable fusion partners which promote high efficiencies of hybridoma out-growth and immunoglobulin synthesis. To overcome these obstacles, five heteromyeloma fusion partners (HM-1,2,3,4 and 5) were constructed by successively fusing porcine lymphocytes with murine myeloma cells or murine x bovine heteromyeloma cells. Following section of hypoxanthine/aminopterin/thymidine (HAT)-sensitive mutants, karyotypes, growth rates and surface phenotypes of the heteromyelomas were determined. Karyotyping revealed an increase in the mean number of chromosomes present in HM-1,4 and 5 cells. Peak doubling times of the parental and HM cells ranged between 12.2 and 17.4 h. Uisng flow microfluorimetry and monoclonal antibodies specific for class I/II major histocompatability antigens, it was determined that the surface phenotype of HM-1,2,3,4 and 5 resembled that of the parental murine X63 myeloma cells. HM 1,2,3,4 and 5 were evaluated for their abilities to serve as fusion partners. Highest percentages of hybrid outgrowth (37%) and immunoglobulin synthesis (52%) were observed when HM-1 was fused with procine lymphocytes. When cloned, percentage of outgrowth and immunoglobulin synthesis increased if HM-1 and HM-2 were used as fusion partners. Cryopreservation of HM-1 and HM-2 did not adversely affect their abilities to promote hybrid outgrowth or immunoglobulin synthesis. During the first week following fusion of porcine lymphocytes with heteromyelomas, murine thymocytes were found to be essential for survival of the nascent hybrids. To confirm that immunoglobulin secreted by hybridomas was of porcine and not murine or bovine origin, culture supernates were subjected to SDS gel electrophoresis, electroblotted and identified. using species-specific isotyping reagents. Two of four cell lines tested secreted porcine light chains and one of four cell lines secreted whole IgM molecules. This paper is the first to describe porcine heteromyelomas for use as fusion partners. Similar to findings of human and bovine studies, our data suggest that heteromyeloma fusion partners perform better than rodent myelomas for creating hybridomas synthesizing porcine immunoglobulin.

Composition of a polysaccharide from mycoplasma (F-38) recognised by antibodies from goats with contagious pleuropneumonia.

A polysaccharide was extracted by warm aqueous phenol from the F-38 strain of mycoplasma which causes contagious caprine pleuropneumonia (CCPP). After acid hydrolysis, the polysaccharide was found to be composed of the neutral sugars glucose, galactose, mannose and fucose and the amino sugars galactosamine and glucosamine. All the sugars were present in approximately equal quantities. Unmodified goat erythrocytes bound the polysaccharide readily and the sensitised cells reacted with antibodies in sera from goats with experimental or natural CCPP. The unique composition of the F-38 polysaccharide and the specific reactivity of polysaccharide-sensitised red cells with antibodies from CCPP infected animals suggests that the polysaccharide should be useful for identification of F-38 organisms and diagnosis of the disease.

Total plasma corticosteroid concentrations in horses with combined immunodeficiency.

Plasma corticosteroid concentrations of seven Arabian foals with combined immunodeficiency (CID) and five non-CID Arabian foals were measured. Plasma corticosteroid concentrations were quantitated throughout gestation for ten mares heterozygous for the CID trait and pregnant with CID foals, as well as for 20 mares heterozygous for the CID trait and pregnant with non-CID foals. Five nonpregnant mares heterozygous for the CID trait also were tested during the same period. Concentrations of plasma corticosteroids in foals with CID (34.4 +/- 5.2 ng/ml) were not different from those of non-CID foals (37.6 +/- 8.4 ng/ml). Significant differences in corticosteroid concentrations were not detected between mares pregnant with CID foals (61.5 +/- 16.4 ng/ml) and mares pregnant with non-CID foals (61.5 +/- 12.& ng/ml) or between pregnant mares and nonpregnant mares (77.9 +/- 10.7 ng/ml).

In vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes from ponies.

The in vitro and in vivo effects of corticosteroids on peripheral blood lymphocytes (PBL) from ponies were studied. Prednisolone inhibited lymphocyte stimulation by phytohemagglutin (PHA) in a dose-dependent manner, without inducing lysis even at large doses. The PBL from horses heterozygous for the combined immunodeficiency trait responded to corticosteroid treatment the same as did PBL from normal ponies. Removal of the corticosteroid after incubation with PBL from normal ponies partially restored responsiveness of these cells to PHA. Chronic in vivo treatment of ponies with corticosteroids caused a marked decrease in the absolute numbers of circulating lymphocytes. Most remaining lymphocytes had detectable surface immunoglobulin and C3 receptors, suggesting a greater decrease in the T-lymphocyte population. In spite of this, there was little change in the in vitro PHA- or keyhole limpet hemocyanin-sensitized ponies. In general, the corticosteroid effects of lysis, as well as the mitogenic and antigenic responses of PBL from ponies, were similar to those previously reported for human lymphocytes.

Pim-1: a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis.

Pim-1 belongs to a family of serine/threonine protein kinases that are highly conserved through evolution in multicellular organisms. Originally identified from moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice, Pim-1 kinase is involved in the control of cell growth, differentiation and apoptosis. Expression of Pim-1 kinase can be stimulated by a variety of growth factors and regulated at four different levels: transcriptional, post-transcriptional, translational and post-translational. Several signal transduction pathways may be associated with the regulation of Pim-1's expression; accumulating data support that the expression of Pim-1 protein is mediated through activation of JAK/STATs. Recent studies of Pim family kinases indicate that Pim-1 kinase plays important roles outside of the hematopoietic system as well.