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T Leanderson

Publications and source records attributed to T Leanderson.

At least 55 records · Page 3Linked to original sources
PubMed1995

Multiple ligand interactions for bacterial immunoglobulin-binding proteins on human and murine cells of the hematopoetic lineage.

A group of bacterial Ig-binding surface proteins were studied: protein H and M1 are from Streptococcus pyogenes and interact with IgG, protein L is expressed by Peptostreptococcus magnus and shows affinity for Ig light chains, whereas protein LG is a chimeric construction combining the binding properties of protein L with the IgG-binding activity of protein G from group C and G streptococci. Proteins L and H coupled to Sepharose were mitogenic for human peripheral blood lymphocytes (PBL) and mouse splenic B cells, but not when added in soluble form. Differentiation to Ig secretion was induced by protein H-Sepharose in mouse splenic B cells but not in human PBLs. In FACS analysis FITC-labelled protein H stained virtually all CD19+ cells in human peripheral blood as well as a majority of the CD3+ population. Protein L bound the majority of the CD19+ population, but also a fraction of the CD19-/CD3 population. Protein M1 was not mitogenic but stained the entire CD19+ population and 70% of the CD3+ population. Identical staining patterns were observed with mouse splenocytes using B220 and T-cells receptor as lineage markers. The chimeric protein LG was a potent mitogen for mouse splenic B cells when added either coupled to Sepharose or in soluble form. In addition, protein LG induced differentiation to Ig secretion of the responding mouse splenic B cells. In FACS analysis, protein LG stained the entire CD19+ and the majority of the CD19-/CD3 lymphocyte population as well as all B220+ mouse splenocytes and a fraction of the splenic T cells. These data indicate that the bacterial proteins studied interact with surface structures of several leucocyte populations and can hence interfere with the immune system at multiple levels.

Animals↗
PubMed1995

Pentadecamer-binding proteins: definition of two independent protein-binding sites needed for functional activity.

The SP6 kappa-promoter pentadecamer (pd) element was found to be unable to stimulate transcription when present in one copy as the only promoter element in a minimal promoter but showed weak stimulatory activity when present as a multimer (four copies). One copy of the pd element acted synergistically with an octamer element, but not with a SP1 site, to stimulate transcription. The effect was orientation dependent with regard to the pd element. Gel shift analysis showed that pd-binding proteins were expressed in transformed as well as nontransformed B lymphocytes, irregardless of their differentiation stage, and in HeLa cells. Two major complexes, binding to different sites within the pd element, were observed in gel shifts. A low-molecular-weight form dominated in resting cells, while a higher-molecular-weight form appeared after mitogenic stimulation. Southwestern analysis showed that the low-molecular-weight pd-binding protein had a molecular mass of 35 kDa, which was confirmed by fractionation by denaturating polyacrylamide gel electrophoresis and molecular sieving. The higher-molecular-weight complex was sensitive to detergent treatment, while the low-molecular-weight complex was not. Mutation analysis showed that the two pd-binding complexes interacted with distinct sites within the element and that dual occupancy was required for functional activity. The functional synergy between the pd element and the octamer was more pronounced in plasmacytomas than in B-cell lymphomas.

Animals↗
PubMed1994

Superinduction of cytokine gene transcription by ciprofloxacin.

In addition to their antibacterial properties, certain antibiotics are also biologic response modifiers. The fluorinated 4-quinolone, ciprofloxacin, for example, up-regulates IL-2 and IFN-gamma production in PBLs stimulated in vitro. In the present study, ciprofloxacin was shown to increase the levels of mRNA for IL-1 alpha, IL-2 and its receptor, IFN-gamma, IL-3, IL-4, granulocyte-macrophage/CSF, TNF-alpha, and lymphotoxin. As investigated with different T cell mitogens and alloantigens, the up-regulation of IL-2 production by ciprofloxacin was found to be independent of the mode of stimulation. Analysis of transcription activity showed that ciprofloxacin enhances IL-2 gene induction. The concentrations of nuclear factor of activated T cells (NF-AT-1) and AP-1 were also found to be increased by ciprofloxacin. Thus, ciprofloxacin interferes with a regulative pathway common to several cytokines.

Ciprofloxacin↗
PubMed1994

Positive transcriptional control elements within the SP6 kappa promoter decamer 3' flanking sequence.

The SP6 kappa promoter decamer 3' flanking sequence was shown to contain several positive control elements for transcriptional stimulation which together increased transcriptional stimulation one order of magnitude. One was mapped to an A nucleotide immediately 3' of the decamer consensus motif. The mechanism was to increase the binding affinity of Oct2 for the decamer core motif by a direct interaction between the Oct2 protein and the template, since in vitro transcribed and translated Oct2 showed an identical binding preference when compared to the corresponding gene products in nuclear extracts. By mutation analysis it was shown that the functional activity of the SP6 kappa promoter 3' flanking sequence with regard to transcriptional stimulation was dependent on the presence of such a high affinity Oct binding site. Deletion analysis showed that the POU-Homeo domain of Oct2 sufficed for the flanking sequence mediated affinity increase for decamer elements and that amino or carboxy terminal variations in Oct2 did not influence this interaction. The flanking sequence element also increased Oct1 binding to the same decamer motif. The A 3' of the decamer increased affinity of Oct2 binding to a consensus as well as a mutated decamer. These data illustrate how flanking sequence elements can compensate mutations in the decamer core motif present in a majority of kappa promoters.

B-Lymphocytes↗
PubMed1994

Transcriptional regulation of immunoglobulin gene expression by anti-Ig.

When transfected into mouse splenic B cells stimulated with lipopolysaccharide (LPS) the expression of DNA vectors containing the chloramphenicol acetyl transferase gene under the control of a SP6 kappa promoter and the Ig heavy chain intron enhancer could be down-regulated 5- to 10-fold by treatment of the cells with anti-Ig prior to transfection. Exchanging the SP6 kappa promoter by minimal promoters consisting of an octamer or a SP1 motif linked to TATA box did not impair the anti-Ig induced down-regulation while inserting a rabbit beta-globin promoter did. The transcriptional regulation could be observed after replacing the Ig heavy chain intron enhancer with a SV40 enhancer, or duplicated minimal Ig heavy chain enhancers containing or lacking the octamer element. The down-regulation was not dependent on the level of transcriptional stimulation observed. A difference in Oct2 expression could neither be detected at the RNA nor protein level after treatment of LPS stimulated B cells with anti-Ig or phorbol-dibutyrate. Anti-Ig treatment, but not phorbol-di-butyrate treatment, induced increased levels of AP1 and NF kappa B transcription factors. Thus, either differentiation specific transcriptional control of Ig genes is exerted via transcription factors common to several distinct enhancers or via transcriptional adaptor molecules that can interact with several distinct DNA binding proteins.

Animals↗
PubMed1994

Analysis of Oct2-isoform expression in lipopolysaccharide-stimulated B lymphocytes.

Oct2-isoform expression in splenic B cells stimulated with lipopolysaccharide or lipopolysaccharide plus phorbol-di-butyrate was analysed by cDNA cloning. The frequency of Oct2-positive clones was 1/15,000 in both libraries. Two new isoforms were found that generate novel amino- or carboxy-terminal sequences. An isoform lacking exon 11 destroyed the carboxy-terminal leucin-zipper region and introduced a frame shift creating a novel, proline-rich carboxy terminus. A new exon containing a highly basic region (4c) was characterized, between exons 4 and 5. This exon was inserted between glutamine-rich regions 2 and 3, carboxy terminal of a tentative leucine-zipper structure. In addition, a new combination isoform containing Oct2a's amino terminal insert (exon 7a) and Oct2b's carboxy terminal insert (exon 13) was found that created a novel large isoform, Oct2ab. More frequent use of the classical Oct2a and Oct2b isoforms was observed in the lipopolysaccharide-stimulated B cells, while a preference for the Oct2ab and Oct2ba isoforms was observed in lipopolysaccharide plus phorbol-di-butyrate-treated cells.

Amino Acid Sequence↗
PubMed1994

Kinetics of somatic mutation in lymph node germinal centres.

Somatic mutation activity in immunoglobulin V kappa genes during the response to the hapten 2-phenyl-5-oxazolone was measured in lymph node B-cell populations at various timepoints after footpad immunization. When the V kappa Ox1 genes rearranged to the J kappa 5 segment were amplified from genomic DNA using the polymerase chain reaction and sequenced, somatic mutations could be detected as early as day 4 after immunization. Somatic mutations were also detected after sequencing RNA from oxazolone-specific hybridomas derived from lymph node cells at day 4 after immunization. These early mutations were found mostly in cells with a germinal centre phenotype. No indication of selection at the population level by apoptosis was detected until day 7 after immunization. These results suggest somatic mutations can be induced very early during the immune response in lymph node cells, prior to the peak of clonal expansion and selection with regard to antigen binding.

Animals↗
PubMed1994

Ectopic expression of myc or myn down-regulates immunoglobulin transcription.

Co-transfection of expression vectors for c-myc or myn down-regulated the expression from a reporter plasmid containing the chloramphenicol-acetyl-transferase (CAT) gene, under the control of an immunoglobulin kappa promoter and the immunoglobulin heavy-chain enhancer. The effect was dose-dependent and an additive effect was seen when both expression vectors were transfected simultaneously. Deletion mutants lacking the leucine zipper region of c-myc were unable to down-regulate the transcription from the reporter construct. Reporter genes where the immunoglobulin enhancer had been exchanged with a SV40 enhancer or an immunoglobulin minimal enhancer were still down-regulated by a cotransfection with c-myc expression vectors. A minimal promoter containing an octamer motif responded to myc expression while a minimal promoter containing a SP1 motif did not. No direct interactions between myc, myn and Oct proteins could be detected either by band-shift analysis, or by co-translation in vitro followed by precipitation with SP6 kappa promoter bound to magnetic carriers. Furthermore, B cells from mice transgenic for myc showed aberrant expression of transcription factors.

Animals↗
PubMed1993

Analysis of somatic mutation activity in multiple V kappa genes involved in the response to 2-phenyl-5-oxazolone.

We have studied somatic mutation activity early in a response to 2-phenyl-5-oxazolone coupled to ovalbumin (phOx-OVA). Although the V kappa Ox1 gene rearranged to J kappa 5 is known to predominate in this response, other closely related V kappa genes are involved. We compared the introduction of point mutations into V kappa Ox1 genes and into a set of related V kappa genes rearranged to the same J kappa segment at two time points after primary immunization. The result showed that quantitation of mutations in a single rearrangement substrate leads to an underestimation of the total mutational activity. There is pronounced somatic mutation activity early within genes that may be absent later in the response. We also show that multiple somatic mutations can be detected in B cells from draining lymph nodes after foot-pad injection with phOx-OVA already at day 7 after immunization. The data suggest a system in which mutation acts early in the response on a wide range of substrates and that selection and expansion of high affinity paratopes occurs later.

Animals↗
PubMed1993

Characterization of the joining chain (J-chain) promoter.

A 300 b.p. promoter from the mouse joining chain (J-chain) gene was studied with regard to functional activity and protein/DNA interactions. The promoter only stimulated expression of a chloramphenicol-acetyl-transferase (CAT) reporter gene when an enhancer was present in the construct, regardless of whether the construct was transfected into cell lines that did or did not express an endogenous J-chain. Furthermore, deletion mutants lacking the 5' portion of the promoter were transcribed at a higher rate than the intact promoter in both J-chain positive and J-chain negative B-cell lines but not in untransformed B lymphocytes stimulated by lipopolysaccharide, indicating the presence of a negative control element in the 5' portion of the J-chain promoter active in tumour cells only. The octamer element in the J-chain promoter was found to bind Oct proteins, albeit with a low affinity. The penta-deca (p.d.) element in the J-chain promoter bound proteins in extracts from untransformed B cells but not in the tested cell lines. The protein binding to the J-chain p.d. element did not compete efficiently with a p.d. element from the SP6 kappa promoter. A protein binding to the 5' portion of the J-chain was expressed in some cell lines but not in others; neither a negative nor a positive correlation to J-chain expression could be seen. It was concluded that the J-chain promoter is equivalent to a kappa promoter and that differentiation-specific J-chain expression is governed by distal, positive control elements located outside the analysed region.

Animals↗
PubMed1991

Functional modularity in the SP6 kappa promoter.

The requirements of the SP6 kappa promoter for transcriptional activation were studied in nontransformed murine B lymphocytes stimulated with lipopolysaccharide. Three different DNA motifs, besides the TATA-box, were needed for restoration of transcriptional activation to the same magnitude as seen with the native SP6 kappa promoter. The decamer motif (TNCTTTGCAT) was found to induce transcription alone and point-mutation of this element reduced transcription to negligible levels, although the other two required elements were present. The penta-decamer element (TGCAG/CCTGTGNCCAG) did not stimulate transcription alone, but activated transcription synergistically in conjunction with the decamer motif. This synergism required the presence of a third pyrimidine rich element (CCCT) in the decamer 3' flanking sequence. The pyrimidine rich element could partly be substituted for by an E-box core motif (CANNTG) 3' of, but not by the kappa Y motif (CTTCCTTA) 5' of, the decamer. Proteins interacting specifically with the penta-decamer element were detected by band-shift assay. The decamer 3' flanking sequence of the SP6 kappa promoter was found to modify the binding of endogenous Oct2 isoforms to the decamer motif i B lymphocytes, but not in CHO cells transfected with various Oct2 isoforms. Thus, complex protein/DNA interactions can be observed in the SP6 kappa promoter which correlate functionally with a synergism in transcriptional activation.

Animals↗
PubMed1991

Immunoglobulin heavy chain cDNA from the teleost Atlantic cod (Gadus morhua L.): nucleotide sequences of secretory and membrane form show an unusual splicing pattern.

Rabbit antibodies to Atlantic cod (Gadus morhua L.) immunoglobulin were affinity purified and used to screen cDNA libraries from spleen and head kidney mRNA. cDNA clones for both the secretory and membrane-bound heavy (H) chain were isolated, the nucleotide and deduced amino acid sequences of which are reported here. Comparisons of the cod secretory H chain amino acid sequence show 24%, 27%, 30% identity to the mu chain of Mus, Xenopus and Ictalurus, respectively. The highest degree of identity was observed in the CH4 domain. The cDNA encoding the transmembrane form shows a novel splicing pattern where the TM1 exon is spliced directly onto the CH3 domain and not to the CH4 domain as in other animal groups. Southern blot analyses with VH and C probes on genomic DNA from cod erythrocytes indicate that there is a unique C gene but several V genes in the cod immunoglobulin H chain locus.

Amino Acid Sequence↗
PubMed1991

'E-boxes' as promoter elements in B cell lines and untransformed B lymphocytes.

The penta-deca (pd) promoter element from the SP6 kappa promoter and the muE2 box from the Ig heavy chain intron enhancer were analysed in an electrophoretic mobility shift assay (EMSA), utilising cell extracts from total mouse splenic B cells before and after stimulation with lipopolysaccharide. With both probes a changed EMSA pattern was observed after stimulation of the cells, where higher molecular weight DNA/protein complexes became dominant. When the pd and muE2 sequence elements were cloned in front of a TATA box and analysed for their transcriptional promoting activity in lipopolysaccharide stimulated B cells, they were inactive. On the contrary, the same constructs were transcriptionally active in some but not all B cell lines. Thus, E-boxes display functional heterogeneity as transcriptional activators depending on host cell.

Animals↗
PubMed1991

Regulation of immunoglobulin transcription.

B lymphocytes express membrane-bound immunoglobulin molecules as antigen-specific, clonally distributed, cell surface receptors. Molecules with the same fine specificity can be secreted in large amounts into the body fluids by progeny of the clone, subsequent to antigenic challenge and appropriate stimulation into terminal differentiation. While this review is limited to the regulation of immunoglobulin transcription, it should be noted that immunoglobulin expression is regulated at the level of DNA, RNA and protein. A DNA recombination process determines the specificity of the antibody to be expressed, the immunoglobulin RNA level is regulated qualitatively and quantitatively, as is the mature immunoglobulin protein.

Animals↗