Localization of expression of KNAT3, a class 2 knotted1-like gene

KNAT3 is a class 2 kn1 -like gene in Arabidopsis thaliana . The RNA expression patterns of KNAT3 were characterized through the use of promoter-GUS fusion analysis and in situ hybridization. KNAT3 is expressed in several tissues and at several times during development. There are three main expression patterns: (1) during early organ development in young leaves, buds and pedicels; (2) at and near the junction between two organs at specific times during development, including the hypocotyl-root boundary in young seedlings, the anther-filament junction in mature flowers, and the ovule-funiculus and peduncle-silique boundaries in elongating siliques; and (3) in maturing tissues such as the style of elongating siliques, the petioles of maturing leaves, and most of the root. The varied expression patterns may indicate that KNAT3 plays several different roles in plants, depending on when and where it is expressed. Previous work on KNAT3 (Serikawa et al. , 1996) indicated that expression of its RNA is regulated by light. Promoter-GUS seedlings were grown under different light conditions (continuous white, red and far-red light) to examine more closely the light regulation of the KNAT3 promoter. Continuous white light resulted in stronger overall GUS staining in the same patterns seen in seedlings grown under long-day conditions (cotyledons, upper hypocotyl and roots). Continuous red light resulted in reduced GUS expression in those same tissues. Continuous far-red light led to seedlings showing stronger staining in the hypocotyl and cotyledons than red light-grown plants but no staining in the roots. Thus, the KNAT3 promoter responds differently to red and far-red light.     

Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes.

The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM-alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes.     

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).     

Isolation of a human B lymphocyte membrane protein with Ia-like properties.

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

HD39 (B3), a B lineage-restricted antigen whose cell surface expression is limited to resting and activated human B lymphocytes

The B cell-restricted antigen HD39, whose cell surface expression is limited to resting and activated human B lymphocytes, is described in this report. The monoclonal antibody HD39 detects a two-chain glycoprotein with apparent molecular weights of 130,000 and 140,000. During B cell ontogeny, HD39 is first expressed in the cytoplasm of bone marrow derived pre-B cells, then appears on the cell surface of sIgM+ B cells, and finally on the majority of sIgM+ sIgD+ resting B cells. After activation in vitro, the expression of HD39 on the cell surface first increases, and then the antigen is lost as cells begin to differentiate. HD39 is weakly expressed on very few non-T cell ALL and B cell CLL, on approximately 50% of B cell lymphomas, and not on Waldenstr?m's macroglobulinemias and myelomas. In contrast, it is strongly expressed on all hairy cell leukemias. Its limited cell surface expression in B cell ontogeny suggests that HD39 may be important in the events that regulate the activation of the human resting B lymphocytes.     

Direct identification of the putative surface IgM receptor-associated molecule encoded by murine B cell-specific mb-1 gene

The B cell-specific mb-1 gene was recently reported to encode a putative surface glycoprotein with CD3-like structural properties. Hombach et al. suggested and presented evidence to show that this mb-1 gene encodes the 34-kDa membrane glycoprotein (B34 or IgM-alpha) associated with IgMR molecule. To identify the mb-1 gene product directly in B cells, affinity-purified MB-1-specific antibody was prepared by immunization of rabbits with synthetic MB-1 oligopeptide. Immunoprecipitation in combination with two-dimensional diagonal gel electrophoresis analysis revealed that this antibody detected a B cell-specific surface glycoprotein that is very similar to the IgM-alpha (B34) protein described by Hombach et al. However, MB-1 protein exists usually as the monomeric form on the surface of B cells, in contrast to IgM-alpha, which was detected as the dimeric (IgM-alpha/IgM-alpha or IgM-alpha/Ig-beta) protein. We also found that MB-1 protein is already expressed on the sIgM- pre-B cell lymphoma, which might suggest an alternative functional role of this B cell-specific MB-1 protein in B cell differentiation. The molecular identity of MB-1 protein and IgM-alpha (B34) is discussed.     

The phosphatase domains of LAR, CD45, and PTP1B: structural correlations with peptide-based inhibitors.

PTP1B is a cytosolic protein tyrosine phosphatase that is a regulator of the kinase activity of the insulin receptor; the two protein tyrosine phosphatases LAR and CD45 are receptor type phosphatases crucially important to cell function. LAR also is involved in regulation of the insulin receptor while CD45 is critical for T-cell activation. Although LAR and CD45 are both transmembrane phosphatases, these enzymes manifest their phosphatase activity through a catalytic cytosolic domain. We have utilized X-ray coordinates of related phosphatases (RPTPalpha and RPTmu) and comparative protein modeling to obtain molecular models of the D1 catalytic domains of CD45 and LAR. The models were tested using established protocols and found to be comparable to low resolution X-ray structures. The structure obtained for LAR was compared with the recently reported X-ray structure. Both the CD45-D1 and LAR-D1 structures were then compared to and contrasted with PTP1B. The active site of pockets of the three enzymes were found to be very uniform in structure and charge distribution. Also, the gross surface topology around the active site was found to be somewhat similar for the 3 phosphatases. However, there were significant differences in surface topology, and, more importantly, large changes in surface charge distribution. The differences between the surface features of these enzymes provide an explanation for the selectivity of inhibition by a number of peptides.     

Epidermal cells synthesize a cytokine with interleukin 3-like properties

Interleukin 3 (IL 3) is produced by T lymphocytes and T cell lines (EL 4), as well as by a monomyelocytic cell line (WEHI 3), and it activates lymphocytes as well as mast cells. Recently we have demonstrated that epidermal cells (EC) perform monocyte/macrophage-like functions through the release of an interleukin 1-like immunomodulating mediator (EC-derived thymocyte activating factor; ETAF. Because mast cells predominantly are located in the skin, in the present study we investigated whether EC in addition to ETAF may produce IL 3. Normal as well as transformed keratinocytes were able to secrete an IL 3-like mediator (EC IL 3) that induces the proliferation of IL 3-dependent cell lines. Furthermore, both EC IL 3 and WEHI IL 3 have a similar m.w. of 30,000. In addition, an antibody against IL 3 also blocked EC IL 3 activity, suggesting that these molecules appear to be very similar. EC IL 3 production was greatly enhanced by the addition of concanavalin A, phorbol myristate acetate, lipopolysaccharide, and silica. Factor production was completely blocked by inhibiting protein synthesis. These findings demonstrate that keratinocytes synthesize an additional cytokine with the biologic and biochemical properties of IL 3, but distinct from ETAF. Thus, through the production of EC IL 3, EC may participate in the activation of mast cells and thereby mediate inflammatory as well as hypersensitivity reactions.