A genetic linkage map of the MSM Japanese wild mouse strain with restriction landmark genomic scanning (RLGS)

A high-resolution genetic map of the Mus musculus molossinus (MSM) Japanese wild mouse strain was constructed with restriction landmark genomic scanning (RLGS) and compared with that of the laboratory strain C3H. MSM is phylogenetically 1 million years apart from common laboratory mouse strains and is distinctly resistant to chemical carcinogenesis. Since it exhibits frequent genetic polymorphisms with laboratory mice but can still be easily crossed with laboratory strains, hybrids between MSM and carcinogen-sensitive laboratory mouse strains provide excellent materials for analysis of modifier genes and genetic changes during carcinogenesis. We have generated MSM backcross progeny with the C3H strain, which is extremely sensitive to hepatocarcinogenesis, to construct the present map. RLGS profiles with two combinations of restriction enzymes (NotI–PvuII–PstI, NotI–PstI–PvuII) yielded more than 2000 spots each. The polymorphism rate was about 39.2%, and of a total of 1732 polymorphic spot loci identified, 1371 could be assigned to specific chromosomes by comparison with 79 microsatellite marker loci. Thus, 1450 loci, on all chromosomes except for Y, effectively mapped 90% of the genome (1431.7 cM length). Although some spots might be derived from the same NotI site, each NotI site potentially generating two fragments, the presence of at least 515 loci groups with different progeny distribution patterns dispersed through the genome with an average spacing of 3 cM, means that this genetic map should be useful for analysis of various biological phenomena, including carcinogenesis and ontogenesis, at the gene level. Received: 25 August 1999 / Accepted: 20 December 1999     

Structure–activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure–activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.     

Evaluation of a soybean lectin-based method for the enrichment of erythroblasts.

We performed a comparative study of the enrichment of erythroblasts by a soybean agglutinin galactose-specific lectin method and a standardized magnetic cell-sorting (MACS) protocol. Blood samples, obtained from 11 pregnant women at between 11 and 40 weeks of gestation, were split and examined by each method in parallel. The number of erythroblasts recovered by the lectin method was approximately eightfold higher than the number obtained by MACS. Our data suggest that the lectin-based method may provide a better approach for the enrichment of rare fetal erythroblasts from maternal blood.     

Appearance of prolactin-releasing peptide-producing cells in the area postrema of postnatal rats

Growth hormone–releasing hormone (GHRH) is secreted by the hypothalamus and upon binding to specific GHRH receptors in the pituitary stimulates growth hormone production and release. In addition to its neuroendocrine action GHRH plays a role in tumorigenesis. Consistently with this latter role, the splice variant 1 (SV1) of GHRH receptor, which is widely expressed in non-pituitary normal tissues and cancers, can mediate the proliferative effects of GHRH and even in the absence of GHRH is capable of eliciting mitogenic signals in the tissues in which it is expressed. The aim of the present study was to investigate the expression of GHRH and its tumoral receptor SV1 in primary human melanomas and dysplastic nevi by immunohistochemistry. None of the specimens tested expressed GHRH. Only 1 of 12 (8%) dysplastic nevi expressed SV1 but 14 of 23 (61%) melanomas showed moderate or strong staining for SV1 (association p < 0.005). This is the first report demonstrating the involvement of SV1 in the pathogenesis of melanomas. Our work implies that the progression from a state of dysplasia into malignancy is accompanied by expression of SV1 receptor. Our findings also suggest that treatment with GHRH antagonists should be further explored for the management of malignant melanomas.     

Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils

The neutral glycosphingolipid lactosylceramide (LacCer) forms lipid rafts (membrane microdomains) coupled with the Src family kinase Lyn on the plasma membranes of human neutrophils; ligand binding to LacCer activates Lyn, resulting in neutrophil functions, such as superoxide generation and migration (Iwabuchi and Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100, 1454–1464, 2002 and Sato et al. Induction of human neutrophil chemotaxis by Candida albicans-derived beta-1,6-long glycoside side-chain-branched beta glycan, J. Leukoc. Biol. 84, 204–211, 2006). Neutrophilic differentiated HL-60 cells (D-HL-60 cells) express almost the same amount of LacCer as neutrophils. However, D-HL-60 cells do not have Lyn-associated LacCer-enriched lipid rafts and lack LacCer-mediated superoxide-generating and migrating abilities. Here, we examined the roles of LacCer molecular species of different fatty acid compositions in these processes. Liquid chromatography-mass spectrometry analyses revealed that the very long fatty acid C24:0 and C24:1 chains were the main components of LacCer (31.6% on the total fatty acid content) in the detergent-resistant membrane fraction (DRM) from neutrophil plasma membranes. In contrast, plasma membrane DRM of D-HL-60 cells included over 70% C16:0-LacCer, but only 13.6% C24-LacCer species. D-HL-60 cells loaded with C24:0 or C24:1-LacCer acquired LacCer-mediated migrating and superoxide-generating abilities, and allowed Lyn coimmunoprecipitation by anti-LacCer antibody. Lyn knockdown by siRNA completely abolished the effect of C24:1-LacCer loading on LacCer-mediated migration of D-HL-60 cells. Immunoelectron microscopy revealed that LacCer clusters were closely associated with Lyn molecules in neutrophils and C24:1-LacCer-loaded D-HL-60 cells, but not in D-HL-60 cells or C16:0-LacCer-loaded cells. Taken together, these observations suggest that LacCer species with very long fatty acids are specifically necessary for Lyn-coupled LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration. This study was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16017293) to K.I., by COFIN-PRIN 2004 to A.P., and by “High-Tech Research Center” Project for Private Universities: matching fund subsidy. An erratum to this article can be found at     

Differential antagonism of endomorphin-1 and endomorphin-2 supraspinal antinociception by naloxonazine and 3-methylnaltrexone

To determine if different subtypes of mu-opioid receptors were involved in antinociception induced by endomorphin-1 and endomorphin-2, the effect of pretreatment with various mu-opioid receptor antagonists beta-funaltrexamine, naloxonazine and 3-methylnaltrexone on the inhibition of the paw-withdrawal induced by endomorphin-1 and endomorphin-2 given intracerebroventricularly (i.c.v.) were studied in ddY male mice. The inhibition of the paw-withdrawal induced by i.c.v. administration of endomorphin-1, endomorphin-2 or DAMGO was completely blocked by the pretreatment with a selective mu-opioid receptor antagonist beta-funaltrexamine (40 mg/kg), indicating that the antinociception induced by all these peptides are mediated by the stimulation of mu-opioid receptors. However, naloxonazine, a mu1-opioid receptor antagonist pretreated s.c. for 24h was more effective in blocking the antinociception induced by endomorphin-2, than by endomorphin-1 or DAMGO given i.c.v. Pretreatment with a selective morphine-6 beta-glucuronide blocker 3-methylnaltrexone 0.25mg/kg given s.c. for 25 min or co-administration of 3-methylnaltrexone 2.5 ng given i.c.v. effectively attenuated the antinociception induced by endomorphin-2 given i.c.v. and co-administration of 3-methylnaltrexone shifted the dose-response curves for endomorphin-2 induced antinociception to the right by 4-fold. The administration of 3-methylnaltrexone did not affect the antinociception induced by endomorphin-1 or DAMGO given i.c.v. Our results indicate that the antinociception induced by endomorphin-2 is mediated by the stimulation of subtypes of mu-opioid receptor, which is different from that of mu-opioid receptor subtype stimulation by endomorphin-1 and DAMGO.     

Maturation of the activities of recombinant mite allergens Der p 1 and Der f 1, and its implication in the blockade of proteolytic activity

Recombinant pro-Der p 1 expressed in yeast Pichia pastoris was convertible into the prosequence-removed mature Der p 1 with full activities of cysteine protease and IgE-binding with or without N-glycosylation of the mature sequence as well as pro-Der f 1. The active recombinant variants will be the basis for various future studies. The major N-terminus of pro-Der p 1 with low proteolytic activity was the putative signal-cleavage site, while that of pro-Der f 1 contained not only the equivalent site but also 21 residues downstream, and pro-Der f 1 retained significant activity. Contribution of the N-terminal region of the Der p 1 prosequence including an N-glycosylation motif on effective inhibition of proteolytic activity of pro-Der p 1 was suggested.     

Disruption of lolCDE, encoding an ATP-binding cassette transporter, is lethal for Escherichia coli and prevents release of lipoproteins from the inner membrane

ATP-binding cassette transporter LolCDE was previously identified, by using reconstituted proteoliposomes, as an apparatus catalyzing the release of outer membrane-specific lipoproteins from the inner membrane of Escherichia coli. Mutations resulting in defective LolD were previously shown to be lethal for E. coli. The amino acid sequences of LolC and LolE are similar to each other, but the necessity of both proteins for lipoprotein release has not been proved. Moreover, previous reconstitution experiments did not clarify whether or not LolCDE is the sole apparatus for lipoprotein release. To address these issues, a chromosomal lolC-lolD-lolE null mutant harboring a helper plasmid that carries the lolCDE genes and a temperature-sensitive replicon was constructed. The mutant failed to grow at a nonpermissive temperature because of the depletion of LolCDE. In addition to functional LolD, both LolC and LolE were required for growth. At a nonpermissive temperature, the outer membrane lipoproteins were mislocalized in the inner membrane since LolCDE depletion inhibited the release of lipoproteins from the inner membrane. Furthermore, both LolC and LolE were essential for the release of lipoproteins. On the other hand, LolCDE depletion did not affect the translocation of a lipoprotein precursor across the inner membrane and subsequent processing to the mature lipoprotein. From these results, we conclude that the LolCDE complex is an essential ABC transporter for E. coli and the sole apparatus mediating the release of outer membrane lipoproteins from the inner membrane.     

Preparation and biological activity of 13-substituted retinoic acids

13-Demethyl or 13-substituted all-E- and 9Z-retinoic acids were synthesized using a palladium-catalyzed coupling reaction of enol triflates and tributylstannylolefins. Their biological activities were then measured. The 13-ethyl analogs exhibited approximately one-half of the antiproliferative and differentiation-inducing activity of ATRA in HL-60 cells. In contrast, in the 9Z-derivatives, all analogs, except for the 13-butyl derivatives, showed apoptosis-inducing activity.