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PubMed1990

Adhesion of human T-lymphoid cells to fibronectin is mediated by two different fibronectin domains.

Lymphocyte adhesion to components of extracellular matrices (i.e. fibronectin) is important for their proper localization in tissues and inflammatory sites. We have studied the attachment of the human cell line HUT-78 (mature T lymphocytes) to fibronectin and to several tryptic fragments of fibronectin. HUT-78 cells effectively adhered to surfaces coated with two Hep II domain-containing fragments of 38,000 and 58,000 MW derived from the A and B chains of fibronectin, respectively. Cells also bound to an 80,000 MW fragment containing the RGDS sequence of fibronectin. Cell adhesion to the 38,000 MW fragment was completely inhibited (100%) by cell preincubation with the soluble 38,000 MW fragment; it was partially inhibited (30-37%) by preincubation with the 58,000 MW fragment or with a synthetic peptide CS-1, comprising the first 25 amino acid residues of the alternatively spliced connecting segment (IIICS), which is present in the A chain of fibronectin and in the 38,000 MW fragment. Cell preincubation with RGDS-containing synthetic peptides or with the 80,000 MW fragment, did not affect attachment to 38,000 MW-coated surfaces. Moreover, preincubation of HUT-78 cells with 38,000 MW fragment had no effect on cell adhesion to 80,000 MW-coated wells, while preincubation with 80,000 MW fragment completely inhibited cell attachment to these surfaces. These results strongly suggest the involvement of two different cell surface receptors which recognize the Hep II/IIICS site and the RGDS site independently. Preincubation with either 38,000 or 80,000 MW fragments prevented cell attachment to fibronectin, indicating that adhesion to the intact molecule requires interaction with both regions. Therefore T-lymphocyte adherence to fibronectin-containing matrices may be regulated by the co-expression of both receptors at the cell surface.

Cell Adhesion↗

PubMed1990

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells.

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.

Animals↗

PubMed1990

Nonadrenergic [3H]idazoxan binding sites are physically distinct from alpha 2-adrenergic receptors.

We have recently demonstrated that the alpha 2-adrenergic radioligand [3H]idazoxan also labels additional sites that do not recognize catecholamines but bind with high affinity several chemically distinct drugs previously assumed to be highly selective for alpha 2-adrenergic receptors [Mol. Pharmacol. 35:324-330 (1989)]. We now have used three approaches to distinguish the nonadrenergic [3H]idazoxan sites from alpha 2-adrenergic receptors. (a) No nonadrenergic [3H]idazoxan binding sites were found in COS-7 cells transfected with the genes for the two known alpha 2-adrenergic receptor subtypes. (b) The ratio of alpha 2-adrenergic and nonadrenergic [3H]idazoxan sites in human platelet membranes varied considerably between various donors. (c) Highly purified platelet plasma membranes were enriched for alpha 2-adrenergic receptors but did not contain any nonadrenergic [3H]idazoxan binding sites. We conclude that the nonadrenergic [3H]idazoxan binding sites are not co-expressed with alpha 2-adrenergic receptors and at least in human platelets may be located in an intracellular compartment.

Animals↗

PubMed1990

Down-regulation of KOLT-2 antigen (CD28) by interleukin 2; role of IL-2R (p70).

Using KOLT-2 monoclonal antibody (mAb) recognizing a 44,000 molecular weight (MW) T-cell activation antigen (CD28), we observed the co-expression of KOLT-2 (CD28) antigen and Tac (CD25) antigen, associated with a 55,000 MW chain of the interleukin-2 receptor (IL-2R) complex. IL-2R (p55), on several T-cell lines transformed by human T-lymphotropic virus-I (HTLV-I), as well as several subclones of the natural killer (NK)-like cell line YT. When YT cells (YTC3T, YT5.1) and IL-2-dependent HTLV-I+ T-cell line cells (ED, ATL35C) expressing both IL-2R (p55) and IL-2R (p70) chains were cultured with recombinant IL-2 (rIL-2), KOLT-2 antigen was down-regulated in 24 hr. However, KOLT-2 antigen on HPB-ALL (Kurume) cells expressing neither of the IL-2R chains was unaffected by IL-2. IL-2 also failed to down-regulate KOLT-2 antigen on MT-1 cells bearing IL-2R (p55)/Tac without IL-2R (p70). To clarify the role of IL-2R (p70) in the IL-2-induced down-regulation of KOLT-2 antigen, we analysed the effect of IL-2 on a Tac- subclone of YT (YT2C2) that expresses IL-2R (p70). As was the case with Tac+ YT cells, KOLT-2 antigen was down-regulated by IL-2, and this down-regulation was inhibited by anti-IL-2R (p70) mAb but not by anti-Tac mAb. These results show that the signalling through the IL-2/IL-2R system down-regulates KOLT-2 (CD28) antigen by way of the interaction between IL-2 and IL-2R (p70), irrespective of IL-2R (p55)/Tac. Possible involvement of the IL-2/IL-2R system in the CD28-mediated mitogenic mechanism is discussed.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Transphosphorylation as a possible mechanism for insulin and epidermal growth factor receptor activation.

Fully functional chimeric receptors, consisting of major epidermal growth factor and insulin receptor domains, were co-expressed with kinase-negative epidermal growth factor and insulin receptor mutants in human kidney fibroblasts. Under these conditions, homologous extracellular and cytoplasmic domains mediated association of receptors and their precursors. The significance of receptor-receptor interaction was confirmed by transphosphorylation of kinase-negative receptors by ligand-activated chimeric receptors, which was observed between receptors sharing the same cytoplasmic domain as well as between receptors bearing only the same extracellular domain and containing heterologous kinases. Furthermore, the impaired ligand internalization capacity of a kinase-deficient insulin receptor was partially restored by transphosphorylation. Our experiments suggest interreceptor transphosphorylation and transactivation as a possible mechanism for signal amplification.

Cell Line↗

PubMed1990

Lectin histochemical studies on renal tumors.

Histochemical characteristics on lectin binding of 16 cases of nephroblastoma and of 57 cases of renal cell carcinoma were studied on those of six embryonal and fetal kidneys in 4 to 24 gestational weeks and in six normal adult kidneys using four biotinylated lectins (PNA, LTA, SBA and UEA-1), which show segment specific binding for normal nephron. In normal nephrogenesis, Gal beta 1-3 GalNAc (PNA receptor) appeared at first in developing tubuli of the metanephros at 10 gestational weeks, and remained positive in proximal tubuli of 13 gestational weeks, where the Lewis X antigen (LTA receptor) emerged. In adult kidneys, Gal beta 1-3 GalNAc distributed along distal tubuli and collecting ducts and not in proximal tubuli. Type 2 H antigen (UEA-1 receptor) was confined to collecting ducts. In nephroblastomas, positive reaction to these lectins were almost exclusively confined to tubular structures. Gal beta 1-3 GalNAc was constantly positive there. Intensely positive stain of Lewis X antigen was observed in three cases of histologically well differentiated type. Lewis X antigen positive tubuli co-expressed Gal beta 1-3 GalNAc. There were six type 2 H antigen positive cases showing differentiation toward ureteric bud or collecting ducts. In view of the cell surface glycosylation, regular differentiation of fetal renal tubuli was also retained in nephroblastomas according to their degree of histological differentiation. In renal cell carcinoma, Gal beta 1-3 GalNAc was positive in 49% (28/57) and Lewis X antigen was positive in 54% (31/57). Type 2 H antigen was completely negative. In contrast to the nephroblastoma, there was no correlation between histological features and lectin binding patterns in renal cell carcinoma.

Adult↗

PubMed1990

Specificity of interactions of receptors and effectors with GTP-binding proteins in native membranes.

Individual G-proteins are highly similar in primary sequence. It is thus pertinent to ask what degree of specificity of interaction each of these display with the various receptors and effector systems. Many of the identified G-proteins are co-expressed in a single tissue or cell. As the extreme C-terminus of the alpha-subunit of each G-protein appears to be a key domain for the interactions of receptors and G-proteins, we have generated a series of G-protein-selective anti-peptide antisera against this region and then have used these antisera to attempt to interfere with receptor-G-protein coupling. With this approach, we have demonstrated that delta-opioid receptor-mediated inhibition of adenylate cyclase in neuroblastoma x glioma (NG108-15) cell membranes is transduced specifically by Gi2 and in the same cell that alpha 2-adrenergic inhibition of Ca2+ currents is transduced by G0.

Animals↗

PubMedDate not supplied

[Pathogenetic and clinical significance of the adhesion molecule expression on T cells of the lung in sarcoid alveolitis].

A double immunofluorescence analysis of CD4+ cell population from bronchoalveolar lavage (BAL) fluid samples of patients with active pulmonary sarcoidosis was carried out. The results showed that, unlike BAL and peripheral blood CD4+ cells of healthy subjects, almost all BAL CD4+ cells of the patients highly express, besides CDw29 antigen, LFA-1 and ICAM-1 adhesion molecules. The co-expression of these molecules on BAL CD4+ cells during high intensity sarcoid alveolitis could represent a marker of immunological memory. The relevant pathogenetic and clinical implications of this observation are discussed.

Adhesiveness↗

PubMed1990

Alternative splicing of the human c-myb gene.

Two cDNA clones of the human c-myb gene have been isolated from a CCRF-CEM leukemia cell cDNA library and sequenced in their entirety. These sequences, when compared with those previously reported for the human c-myb gene, reveal an alternative splicing process that generates at least four forms of the c-myb message. Three of these forms co-migrate on Northern blots and are co-expressed in several human hematopoietic cell types. Data on sequence comparisons with mouse and chicken homologues of c-myb coupled with oligonucleotide hybridization to genomic clones of the human c-myb gene indicate that this alternative splicing process utilizes three closely spaced splice donor sites and two unique exons present between viral defined exons 5 and 6. In one clone, the alternative splicing would generate a predicted myb protein with a three amino acid deletion in the region involved in transcription activation. In the other clone, incorporation of a new exon leads to introduction of a translation stop codon leading to loss of the entire carboxy terminus of the protein. This includes loss of a portion of the region involved in transcription activation as well as a separate highly conserved domain. The effect of these changes on protein function is currently unknown.

Base Sequence↗

PubMed1990

Selective down-regulation of the pro-enkephalin gene during differentiation of a multiple neuropeptide-co-expressing cell line.

Regulation of co-expression of three neuropeptide genes, i.e. genes encoding enkephalin, cholecystokinin, and gastrin-releasing peptide, was studied in human neuroepithelioma cells. In nondifferentiated state, the continuous cell line SK-N-MC displayed an equally high level of expression of the enkephalin, cholecystokinin, and gastrin-releasing peptide genes. By culturing in medium containing endothelial cell growth supplement the SK-N-MC cells differentiated morphologically into a cell type with neurite-like processes. After 3 days the expression of the enkephalin gene in endothelial cell growth supplement-differentiated cells was significantly reduced by 75% as compared to the nondifferentiated cells, while there was no change in the expression of the cholecystokinin and gastrin-releasing peptide genes during differentiation. The results show that the enkephalin gene is selectively down-regulated during differentiation of neuroepithelioma cells. It is suggested that the down-regulation is related to the transient expression of the enkephalin gene in developing brain and other organs. Thus the neuroepithelioma cell line may provide a cellular model to study the underlying molecular mechanism.

Blotting, Northern↗

PubMed1990

A raf/myc virus immortalized macrophage cell line which supports the growth of B-cell and B-cell hybridomas.

Using a combination of raf and myc oncogenes co-expressed by the recombinant retrovirus J-2 we have generated and characterized a cell line which very efficiently supports the growth of B-cells and B-cell hybridomas. Murine spleen cells were cultured under in vitro immunization conditions favoring the short term proliferation of splenic B lymphocytes and infected with J-2 virus. Screening of immortalized spleen cell pools for the capability to support long term B cell growth in vitro led to the selection of a clonal cell line termed alpha ChyJ2. The presence of macrophage specific features and surface markers suggest that alpha ChyJ2 belongs to the macrophage lineage. alpha ChyJ2 cells constitutively produce low levels of IL-1 like activity and high levels of IL-6. Expression of specific mRNAs as well as production of IL-1 alpha, IL-1 beta and IL-6 are inducible with LPS. Expression or production of other cytokines including IL-2, IL-3, IL-4, IL-5, TGF beta and GM-CSF could not be detected. As the biological effects of alpha ChyJ2 supernatant cannot be fully explained by the described pattern of cytokine production, participation of other, yet uncharacterized, factors is possible. Using alpha ChyJ2 as feeder cells for in vitro as well as in vivo immunizations increased the number of antibody secreting B-cell clones 2 to 15 fold, respectively.

B-Lymphocytes↗

PubMed1990

Mixed-lineage leukemia and asynchronous antigen expression.

Considerable confusion exists regarding the definition of acute mixed-lineage leukemia. We have proposed a list of strict criteria, limiting the term acute mixed-lineage leukemia to those patients whose blast cells co-express lymphoid and myeloid characteristics. This system includes cytochemical, immunologic, molecular, and cytogenetic characteristics that are strongly associated with either lymphoid or myeloid lineages. As more information becomes available, the criteria for mixed-lineage leukemia will undoubtedly change. Identification of patients with mixed-lineage leukemia and metachronous leukemia (lineage switch) is important for determining the prognostic implications of these findings. Care must be taken in identifying cases of metachronous leukemia because of the increased incidence of second malignancies following aggressive therapy. Evidence of a recurrence of the original clone must be obtained before metachronous leukemia can be diagnosed. As with mixed-lineage and metachronous leukemias, the potential clinical and prognostic implications of lymphoid leukemias with antigenic asynchrony should be identified. The asynchronous antigen expression in leukemic lymphoblasts may provide a means for detecting minimal residual disease. Detection of minimal residual leukemia is possible because these blasts differ from the predominant population of normal lymphoid cells in their expression of cell surface markers. Study of the mechanisms that lead to these unusual leukemias may result in better understanding of the processes that underlie both normal hematopoietic differentiation and leukemogenesis. An understanding of these leukemias may also permit identification of cases that are destined to fail current therapies so that more intensive or selective therapy can be instituted for such children. Curing the 30% of children with ALL that relapse despite our best efforts should be one of the top priorities for pediatric oncologists.

Antigens, Neoplasm↗

PubMed1990

Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

Animals↗

PubMed1990

The CD4 associated tyrosine protein kinase p56lck is positively regulated through its site of autophosphorylation.

Antibody-mediated aggregation of the CD4 T-cell surface antigen activates bound p56lck molecules and can result in increased intracellular tyrosine protein phosphorylation. To evaluate the basis of the CD4 induced tyrosine phosphorylation signal, we have studied the ability of CD4 to regulate the function of p56lck when these two molecules are co-expressed in non-lymphoid cells. Our studies indicate that cross-linking of CD4 is capable of activation of p56lck in fibroblasts in a manner analogous to that previously reported for T-lymphocytes. They also demonstrate that replacement of the major site of autophosphorylation of p56lck (tyrosine 394) by a phenylalanine residue abolishes the ability to activate p56lck by CD4 cross-linking, implying that this residue is critical for the positive regulation of the Lck tyrosine kinase activity by CD4. Contrary to what we have previously reported for an antigen-dependent murine T-cell clone, as well as murine thymocytes, the CD4 induced activation of p56lck observed in fibroblasts does not result in marked changes in Lck tyrosine phosphorylation, suggesting that other lymphoid specific components may be required for these tyrosine phosphorylation changes.

Animals↗

PubMed1990

Immunohistochemical study of cat Pacinian corpuscles: co-localization of vimentin- and S-100 protein-like in the inner core.

The presence of vimentin and S-100 protein in cat Pacinian corpuscles of cat mesentery has been investigated immunohistochemically (streptavidin-biotin method) using monoclonal antibodies. A positive reaction for both vimentin- and S-100 protein-like was found only in the lamellae of the inner core. The presence of vimentin and the co-expression of vimentin/S-100 protein-like in sensory corpuscles is reported for the first time. The authors discuss the origin of the inner core and capsule of sensory corpuscles on the basis of their immunohistochemical characteristics.

Animals↗

PubMed1990

Premature translation termination of the pre-E1 alpha subunit of the branched chain alpha-ketoacid dehydrogenase as a cause of maple syrup urine disease in Polled Hereford calves.

Maple syrup urine disease in man and cattle is an inborn metabolic error caused by the deficiency of the branched chain alpha-ketoacid dehydrogenase. We have studied the molecular basis of the disease in Polled Hereford calves. The E1 component of branched chain alpha-ketoacid dehydrogenase was virtually undetectable by Western blot analysis of fibroblasts from an affected calf. Northern blot analysis failed to detect the E1 alpha mRNA species in the fibroblasts. Nevertheless, it was readily demonstrated by reverse transcription of RNA followed by polymerase chain reaction that the mRNA for the E1 alpha subunit was present in the cells, albeit at very low concentrations. Sequencing of the polymerase chain reaction-generated cDNA for the entire coding region of the E1 alpha subunit revealed a single base substitution at codon -6 (CAG to TAG). This mutation introduces a stop codon in the leader peptide of the E1 alpha subunit, resulting in the premature termination of translation. The mutation was verified by hybridization of the amplified cDNA fragments from two affected calves with allele-specific oligonucleotides. This finding explains the pathogenesis of maple syrup urine disease in this breed of cattle, which provides the only known animal model for the human disease. In addition, the results provide evidence for the effect of premature translation termination on reducing the steady-state mRNA level and the dependence of E1 beta protein stability on the co-expression of the E1 alpha.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1990

Stimulation of the antigen and interleukin-2 receptors on T lymphocytes activates distinct tyrosine protein kinases.

The T cell antigen receptor complex (TCR) and the interleukin 2 (IL-2) receptor are responsible for signal transduction that results in T lymphocyte activation and proliferation. Stimulation of either the TCR or the IL-2 receptor induces an increase in tyrosine phosphorylation of several cellular proteins indicating that signal transduction by both of these receptors involves the activation of a tyrosine protein kinase. Although the tyrosine protein kinases activated by these receptors have not yet been characterized the receptors themselves are known not to contain a tyrosine protein kinase domain. To determine if these receptors are coupled to the activation of similar or distinct tyrosine protein kinases we examined the patterns and kinetics of tyrosine phosphorylation induced by stimulation of these receptors on a cloned cell line. Hut 78.3 cells co-express the TCR and the p75 IL-2 receptor. These cells were stimulated with either OKT3 antibodies, specific for the TCR, or with IL-2. Signal transduction by these receptors was found to increase the tyrosine phosphorylation of a set of proteins unique to each stimulus. The kinetics of the tyrosine phosphorylation induced by OKT3 antibodies also differed from that induced by IL-2. The OKT3-dependent tyrosine phosphorylation reached maximal levels within 2.5 min and began to decline by 5 min after stimulation. In contrast, the IL-2-induced tyrosine phosphorylation did not achieve maximal levels until 15 min after the addition of IL-2 and the proteins remained phosphorylated even after 60 min of incubation. In addition the tyrosine phosphorylations induced by OKT3 and IL-2 were not affected by prior stimulation with the other agent. These results demonstrate that the TCR and IL-2 receptor are coupled to different signal transduction pathways responsible for the independent activation of distinct tyrosine protein kinases.

Antibodies, Monoclonal↗

PubMed2025

Whole genome duplication drives transcriptome reprogramming in response to drought in alfalfa.

Genome doubling did not enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape. Whole genome duplication (WGD) has been shown to enhance stress tolerance in plants. Cultivated alfalfa is autotetraploid, but diploid wild relatives are important sources of genetic variation for breeding. Investigating how WGD affects gene expression in stress conditions could provide better understanding for use of diploid genetic resources. In this work, we compared the drought response of neotetraploid plants obtained by bilateral sexual polyploidization with diploid full sibs, by measuring physiological and biochemical traits and RNA-seq. Without drought, 4x plants had lower photosynthetic potential than 2x plants per unit leaf area, but larger leaves allowed them to outperform the per leaf photosynthetic potential of 2x plants. Physiological and biochemical traits were significantly affected by drought in both 2x and 4x plants, but the differences between ploidies were small and nonsignificant. Proline levels were higher in 4x than 2x plants, both in control and drought conditions, indicating that larger cells with higher volume-to-surface ratio of 4x  plants require a higher osmolyte concentration. RNA-seq and gene network analyses showed that more genes were affected by drought at 4x than at 2x level, with downregulation of hundreds of genes involved in photosynthesis and stomatal movement at 4x level, suggesting that WGD made the 4x plants more responsive to drought. Genes involved in proline, phytormone and cell wall functions were also transcriptionally affected by drought in 4x plants. We conclude that WGD did not immediately enhance drought tolerance in alfalfa, but may set the stage for long-term adaptation to drought through a novel transcriptional landscape.

Medicago sativa↗