Tsuneaki Sakata

Presence of pancreatic-type phospholipase A2 mRNA in rat gastric mucosa and lung.

The content of mRNA for a pancreatic-type phospholipase A2 present in rat gastric mucosa was much greater than that in pancreas. In lung the mRNA for this pancreatic-type phospholipase A2 was also detected, but less than in pancreas. Nucleotide sequence analysis showed that these pancreatic-type phospholipase A2 cDNAs derived from rat gastric mucosa and lung were completely identical to that from rat pancreas (Ohara et al. (1986) J. Biochem. 99, 733-739). This demonstrates that the pancreatic-type phospholipase A2 present in gastric mucosa and lung does not originate from pancreas.

Constitutive expression of interleukin-7- mRNA and production of IL-7 by a cloned murine thymic stromal cell line

Mouse interleukin‐7 (IL‐7) cDNA was cloned from mouse thymic stromal cell clone MRL104.8a using a polymerase chain reaction (PCR) technique and expressed in COS‐7 cells. The resulting recombinant interleukin‐7 (rlL‐7) supported the proliferation of mouse antigen‐specific helper T cell (Th) clone 9–16 in the absence of IL‐2 and antigen as well as mouse pre‐B cell line DW34. It was also found that high levels of the mRNA for IL‐7 were constitutively expressed in the MRL104.8a cells, and a potent amount of IL‐7 was produced in its culture supernatant. These results provide the evidence for constitutive expression of IL‐7 mRNA and for production of IL‐7 by thymic stromal cells that have a critical role in intrathymic T cell development. The results are discussed in the context of the functional and molecular relationship between IL‐7 and the previously described cytokines produced by thymic stromal cells.

Ca2(+)-dependent interaction of N-copine, a member of the two C2 domain protein family, with OS-9, the product of a gene frequently amplified in osteosarcoma.

N‐copine is a novel two C2 domain protein that shows Ca2+‐dependent phospholipid binding and membrane association. By using yeast two‐hybrid assays, we identified OS‐9 as a protein capable of interacting with N‐copine. We further revealed that the second C2 domain of N‐copine bound with the carboxy‐terminal region of OS‐9. Their interaction in vivo was also confirmed by co‐immunoprecipitation from 293E cells co‐expressing transfected N‐copine and OS‐9. In vitro binding assays showed that this interaction was Ca2+‐dependent. By Northern blot analysis, N‐copine and OS‐9 were co‐expressed in the same regions of human brain. These results reveal that OS‐9 is a potential target of N‐copine.

Arthritis and pneumonitis produced by the same T cell clones from mice with spontaneous autoimmune arthritis

SKG mice, a newly established model of rheumatoid arthritis (RA), spontaneously develop autoimmune arthritis accompanying extra-articular manifestations, such as interstitial pneumonitis. To examine possible roles of T cells for mediating this systemic autoimmunity, we generated T cell clones from arthritic joints of SKG mice. Two distinct CD8(+) clones were established and both showed in vitro autoreactivity by killing syngeneic synovial cells and a variety of MHC-matched cell lines. Transfer of each clone to histocompatible athymic nude mice elicited joint swelling and histologically evident synovitis accompanying the destruction of adjacent cartilage and bone. Notably, the transfer also produced diffuse severe interstitial pneumonitis. Clone-specific TCR gene messages in the inflamed joints and lungs of the recipients gradually diminished, becoming hardly detectable in 6-11 months; yet, arthritis and pneumonitis continued to progress. Thus, the same CD8(+) T cell clones from arthritic lesions of SKG mice can elicit both synovitis and pneumonitis, which chronically progress and apparently become less T cell dependent in a later phase. The results provide clues to our understanding of how self-reactive T cells cause both articular and extra-articular lesions in RA as a systemic autoimmune disease.

Glycosylation of human CRLR at Asn123 is required for ligand binding and signaling.

Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs.

Scanning gene expression during neuronal cell death evoked by nerve growth factor depletion

Depletion of nerve growth factor (NGF) from differentiated, neuronal PC12 cells causes a form of programmed cell death that stems from the attenuation of NGF receptor signaling and the resultant expression of certain genes required for cell death. To better understand the associated molecular events, we surveyed the changes in gene expression in PC6-3 cells, a subline of PC12, caused by depletion of NGF. Using restriction landmark cDNA scanning, we assessed the expression patterns of as many as 15,000 gene species, and 30 genes were isolated whose expression was altered in the absence of NGF. Of the 20 genes up-regulated in the absence of NGF, including transcription factor LRF-1/ATF3, most were also up-regulated during the programmed death of cortical neurons caused by Ca2+ ionophore. Their function may thus be a general feature of programmed neuronal cell death. In contrast, with one exception, expression of down-regulated genes was NGF-dependent and therefore diminished in the absence of NGF but unaffected by Ca2+ ionophore. These findings confirm that global investigation of the features of up- and down-regulated genes should add substantially to our understanding of the regulation of programmed neuronal cell death and the mechanisms involved.

Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification

Human adrenomedullin (hAM) is a 52-amino-acid regulatory peptide containing a six-membered ring structure and an amidated C-terminus, features that are essential for its biological activity. Here, we describe a simple and effective protocol for producing large quantities of highly pure, functional recombinant hAM. A peptide precursor (hAM-Gly) was expressed in Escherichia coli as a fusion protein with thioredoxin and collected as inclusion bodies. The fusion protein was then digested with BLase, a glutamate-specific endopeptidase, to prepare hAM-Gly. The essential ring structure formed spontaneously, while the terminal amide was generated by conversion of the added glycine residue using peptidylglycine alpha-amidating enzyme. The low solubility of hAM-Gly enabled the use of a selective precipitation/extraction method to generate a product that was 80-90% pure, which was sufficient to proceed with the alpha-amidating enzyme reaction. The resultant hAM was then purified further by column chromatography. The final yield was about 82 mg/L of bacterial culture, and the purity, determined by reverse phase HPLC, was >99.5%. The recombinant hAM was biologically active, eliciting concentration-dependent increases in cAMP in CHO-K1 cells expressing a specific hAM receptor and hypotensive responses when intravenously injected into rats. This new approach to the synthesis of hAM is simpler and more cost-effective for large-scale production than chemical synthesis. It therefore represents a new powerful tool that has the potential to facilitate analysis of the structure and function of hAM, as well as the development of new therapeutic protocols for the treatment of ailments such as hypertension.

TrkB mutant lacking the amino-terminal half of the extracellular portion acts as a functional brain-derived neurotrophic factor receptor

A series of mutants with deletion in the extracellular portion of TrkB were expressed transiently and stably in mammalian cells to examine the brain-derived neurotrophic factor (BDNF)-binding properties of TrkB. We found that these binding activities were retained by the TrkB deletion mutant (TrkBDelta4) lacking most of the extracellular portion, cysteine-rich cluster 1 and 2, leucine-rich motif and most of the first immunoglobulin-like domain (Ig1). Furthermore, the results of the neurotrophin selectivity, the equilibrium binding constant, auto-phosphorylation and BDNF dependent cell survival indicate that TrkBDelta4 acts as a functional BDNF receptor comparable to wild-type TrkB. Thus, our findings showed that only the carboxyl-terminal half of the extracellular portion of TrkB, which includes the Ig2 domain, is essential for the functional BDNF receptor.

Production of granulocyte-macrophage colony-stimulating factor or GM-CSF-like growth factor by rat thymic epithelial cell line

An epithelial cell line, IT‐45R1, has been established from a thymus of normal Wistar rat and was found to produce growth factor which stimulated the proliferation of bone marrow cells. This growth factor induced the formation of colonies composed of macrophages, granulocytes, or both, in semi‐solid medium and stimulated the proliferation of an interleukin 3 (IL3)/granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent clone. Neither IL3‐dependent clone nor IL6‐dependent clone responded to IT‐45R1 factor. Additionally, IT‐45R1 factor acted on both rat and mouse bone marrow cells but not on human bone marrow cells. Molecular weight of IT‐45R1 factor was 30 kD and its isoelectric point was 4.5. To determine whether IT‐45R1 factor is GM‐CSF or not, Northern blot analysis and neutralization with anti‐mouse GM‐CSF antibody were carried out. IT‐45R1 expressed GM‐CSF mRNA, but neither M‐CSF nor IL6 transcripts. However, antiserum specific for mouse GM‐CSF did not neutralize IT‐45R1 factor. Taken together, a rat thymic epithelial cell line, IT‐45R1, constitutively produces GM‐CSF or GM‐CSF‐like growth factor.

A simple method for identification of cDNA clone the microinjection of cloned vector into the nuclei of Xenopus laevis oocytes

Abstract When human interferon (HuIFN)-α2 and -β1 cDNAs cloned in the cloning vector of Okayama-Berg or pBR322 plasmid vector were injected directly into the nuclei of Xenopus laevis oocytes, the antiviral activity of HuIFN was detected in the incubation medium of the oocytes 48 hr after injection. These data demonstrated that direct microinjection of the vector plasmid, which contains cloned cDNA, into the nuclei of Xenopus oocytes is a useful technique to identify the cloned DNA that contains the nucleotide sequence sufficient to show the biological activity. We used this technique to identify a novel subtype of HuIFA-α cDNA.