Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8⁺ T or CD4⁺ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18. Received: 7 May 1998 / Accepted: 27 May 1999     

Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding d-glutamate racemase

The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.     

Suppression of Interleukin-11–mediated bone resorption by cyclooxygenases inhibitors

We previously found that human melanoma (A375M) and human breast cancer (MDA-MB-231) cells formed osteolytic bone metastasis in vivo. These cancer cells produced interleukin-11 (IL-11) by themselves and stimulated its production from osteoblasts. Interleukin-11 could increase the number of osteoclasts and raise the calcium concentration in the medium of neonatal murine calvaria organ culture, indicating bone resorption in vitro. Therefore, IL-11 could play an important role in the promotion of osteolysis at the site of bone metastasis. In the present study, we used the calvaria culture system to try to clarify the mechanisms of IL-11–mediated bone resorption. The murine calvaria expressed both the specificity-determining α subunit and the signal–transducing β subunit (gp130) of the IL-11 receptor. When IL-11 was added to the calvaria culture, the concentrations of prostaglandin E₂ (PGE₂) was elevated. Pretreatment of calvaria with cyclooxygenases inhibitors (e.g., indomethacin, NS-398, and dexamethasone) suppressed the production of PGE₂ and the bone resorption induced by IL-11. Addition of exogenous PGE₂ overcame the inhibitory effect of cyclooxygenases inhibitors and promoted bone resorption. These results indicate that IL-11 promotes bone resorption through a PGE₂ synthesis–dependent mechanism and that cyclooxygenases inhibitors could be interesting drugs to suppress IL-11–mediated osteolytic bone metastasis of cancer cells. J. Cell. Physiol. 175:247–254, 1998. © 1998 Wiley-Liss, Inc.     

Adult bengalese finches (Lonchura striata var. domestica) require real-time auditory feedback to produce normal song syntax

Songbirds develop their songs by imitating songs of adults. For song learning to proceed normally, the bird's hearing must remain intact throughout the song development process. In many species, song learning takes place during one period early in life, and no more new song elements are learned thereafter. In these so-called close-ended learners, it has long been assumed that once song development is complete, audition is no longer necessary to maintain the motor patterns of full song. However, many of these close-ended learners maintain plasticity in overall song organization; the number and the sequence of song elements included in a song of an individual vary from one utterance to another, although no new song elements are added or lost in adulthood. It is conceivable that these species rely on continued auditory feedback to produce normal song syntax. The Bengalese finch is a close-ended learner that produces considerably variable songs as an adult. In the present study, we found that Bengalese finches require real-time auditory feedback for motor control even after song learning is complete; deafening adult finches resulted in development of abnormal song syntax in as little as 5 days. We also found that there was considerable individual variation in the degree of song deterioration after deafening. The neural mechanisms underlying adult song production in different species of songbirds may be more diverse than has been traditionally considered. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 343–356, 1997     

Properties of primary murine stroma induced by macrophage colony-stimulating factor

The ability of purified human macrophage colony-stimulating factor (M-CSF) to accelerate the formation of stromal cells from murine bone marrow cells was investigated. The liquid culture of the marrow cells with M-CSF resulted in the formation of monolayers of macrophages on day 7. When the M-CSF was removed on that day and the residual adherent cells were cultured in the absence of M-CSF for an additional 7 days, many colonies appeared with cells that were morphologically distinguishable from M-CSF-derived macrophages. The appearance of the colonies was dependent on the concentration of M-CSF used at the beginning of the culture. Each colony was isolated as a single clone and analyzed. All clones were negative for esterase staining. These cells did not express M-CSF receptor mRNA and did not show a mitogenic response to M-CSF. On the contrary, these cells could be stimulated to proliferate by fibroblast growth factor and platelet-derived growth factor. The polymerase chain reaction analysis of these cells demonstrated constitutive expression of mRNA for M-CSF, stem cell factor, and interleukin (IL)-1, but not IL-3. Some clones expressed mRNA for granulocyte/M-CSF and IL-6. We also examined the ability of the cells to maintain murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in a coculture system. Most of the clones showed a significant increase in total HPP-CFC numbers after 2 weeks of coculture, although the extent of stimulation differed among clones. These results suggested that the colonies established by M-CSF were composed of functional stromal cells that were phenotypically different from macrophages. J. Cell. Physiol. 173:1–9, 1997. © 1997 Wiley-Liss, Inc.     

Isolation and partial characterization of crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009

Crispacin A, a cell-associated bacteriocin produced by Lactobacillus crispatus JCM 2009, was purified from culture broth by ammonium sulfate precipitation, followed by ion exchange and reversed-phase chromatography. Crispacin A was also purified from the cells of L. crispatus JCM 2009 by acid extraction and reversed-phase chromatography. Purified crispacin A was determined to be 5393 Da by mass spectrometry and found not to show sequence homology with other bacteriocins from lactic acid bacteria.     

A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana

A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.     

Rational design,synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor

(?)-Dehydroxymethylepoxyquinomicin ((?)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-?-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (?)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.