The ectodomain of the luteinizing hormone receptor interacts with exoloop 2 to constrain the transmembrane region: studies using chimeric human and fly receptors.

Lutropin (LH) and follitropin (FSH) receptors belong to a group of leucine-rich repeat-containing, G protein-coupled receptors (LGRs) found in vertebrates and flies. We fused the ectodomain of human LH or FSH receptors to the transmembrane region of fly LGR2. The chimeric human/fly receptors, unlike their wild type counterparts, exhibited ligand-independent constitutive activity. Because ectodomains likely interact with exoloops to constrain the receptors, individual exoloops of the chimeric receptor containing the ectodomain of the LH receptor and transmembrane region of fly LGR2 was replaced with LH receptor sequences. Chimeric receptors with the ectodomain and exoloop 2, but not exoloop 1 or 3, from LH receptors showed decreases in constitutive activity, but ligand treatment stimulated cAMP production. Furthermore, substitution of key resides in the hinge region of fly LGR2 with LH receptor sequences led to constitutive receptor activation; however, concomitant substitution of the homologous exoloop 2 of the LH receptor decreased G(s) coupling. These results suggest that the hinge region of the LH receptor interacts with exoloop 2 to constrain the receptor in an inactive conformation whereas ligand binding relieves this constraint, leading to G(s) activation.     

Functional Analysis of Tcl1 Using Tcl1-Deficient Mouse Embryonic Stem Cells

Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1’s roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of β-catenin decreased in response to Tcl1 overexpression. We measured the β-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by β-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Molecular cloning of the human histamine H2 receptor.

We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor.     

Downregulation of p‐COUMAROYL ESTER 3‐HYDROXYLASE in rice leads to altered cell wall structures and improves biomass saccharification

p‐Coumaroyl ester 3‐hydroxylase (C3′H) is a key enzyme involved in the biosynthesis of lignin, a phenylpropanoid polymer that is the major constituent of secondary cell walls in vascular plants. Although the crucial role of C3′H in lignification and its manipulation to upgrade lignocellulose have been investigated in eudicots, limited information is available in monocotyledonous grass species, despite their potential as biomass feedstocks. Here we address the pronounced impacts of C3′H deficiency on the structure and properties of grass cell walls. C3′H‐knockdown lines generated via RNA interference (RNAi)‐mediated gene silencing, with about 0.5% of the residual expression levels, reached maturity and set seeds. In contrast, C3′H‐knockout rice mutants generated via CRISPR/Cas9‐mediated mutagenesis were severely dwarfed and sterile. Cell wall analysis of the mature C3′H‐knockdown RNAi lines revealed that their lignins were largely enriched in p‐hydroxyphenyl (H) units while being substantially reduced in the normally dominant guaiacyl (G) and syringyl (S) units. Interestingly, however, the enrichment of H units was limited to within the non‐acylated lignin units, with grass‐specific γ‐p‐coumaroylated lignin units remaining apparently unchanged. Suppression of C3′H also resulted in relative augmentation in tricin residues in lignin as well as a substantial reduction in wall cross‐linking ferulates. Collectively, our data demonstrate that C3′H expression is an important determinant not only of lignin content and composition but also of the degree of cell wall cross‐linking. We also demonstrated that C3′H‐suppressed rice displays enhanced biomass saccharification.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Mutual interactions between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between the bilaterally paired optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording locomotor activity, under constant light or constant red light, after the optic nerve was unilaterally severed.

Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells.

Bafilomycin A1 is known as a strong inhibitor of the vacuolar type H(+)-ATPase in vitro, whereas other type ATPases, e.g. F1,F0-ATPase, are not affected by this antibiotic (Bowman, E.M., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7972-7976). Effects of this inhibitor on lysosomes of living cultured cells were tested. The acidification of lysosomes revealed by the incubation with acridine orange was completely inhibited when BNL CL.2 and A431 cells were treated with 0.1-1 microM bafilomycin A1. The effect was revealed by washing the cells. Both studies using 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine and fluorescein isothiocyanate-dextran showed that the intralysomal pH of A431 cells increased from about 5.1-5.5 to about 6.3 in the presence of 1 microM bafilomycin A1. The pH increased gradually in about 50 min. In the presence of 1 microM bafilomycin A1, 125I-labeled epidermal growth factor (EGF) bound to the cell surface at 4 degrees C was internalized normally into the cells at 37 degrees C but was not degraded at all, in marked contrast to the rapid degradation of 125I-EGF in the control cells without the drug. Immunogold electron microscopy showed that EGF was transported into lysosomes irrespective of the addition of bafilomycin A1. These results suggest that the vacuolar type H(+)-ATPase plays a pivotal role in acidification and protein degradation in the lysosomes in vivo.     

Augmentation of the generation of cell-mediated cytotoxicity in culture by mitomycin C

Summary The effects of mitomycin C (MMC) on the generation of cell-mediated cytotoxicity in primary stimulation culture of human peripheral blood mononuclear cells (PBM) with the B lymphoblastoid Raji cell line were assessed. The cell-mediated cytotoxicity induced in culture was significantly augmented when MMC was added to cultures on day –1 to day 3 for 24 h at concentrations of 2.5×10^–2 g/ml and 2.5×10^–3 g/ml. To identify the cell populations affected by MMC, PBM were separated by adherence to plastic after treatment with MMC for 24 h (day –1). The two populations were recombined with untreated separated cells and stimulated with antigen. The ability to develop an augmented cell-mediated cytotoxicity was associated with the adherent cell fraction of MMC-treated PBM. Therefore, the ability of MMC-treated adherent cells to produce interleukin 1 (IL 1) was examined. Significantly higher levels of IL 1 were produced by treated cells as compared to untreated adherent cells. The results appear to indicate that the selective effects of MMC on the adherent cell fraction, especially the modification of IL 1 production, may be involved in the mechanisms of MMC-induced augmented cell-mediated cytotoxicity.