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Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3--4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses.

We have previously shown that acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) protein content increases significantly during the human monocyte-macrophage differentiation process. To gain further insight, we used undifferentiated human monocytic THP-1 cells as a model system with which to examine whether ACAT-1 mRNA and protein content can be increased by treating cells with 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] or with 9-cis-retinoic acid (9-cis-RA), two agents known to upregulate the expression of various genes during the monocyte-macrophage differentiation process. Immunoblot analysis with anti-human ACAT-1 antibodies revealed that ACAT-1 protein was increased by 2.6-fold, using 1,25-(OH)(2)D(3) at a physiological concentration (100 pM). ACAT-1 protein was also increased when using 9-cis-RA, but only at relatively high concentrations (0.1;-1 microM). Northern blot analysis revealed that among the four ACAT-1 mRNA transcripts (2.8, 3.6, 4.2, and 7.0 kb) examined, only the 2.8- and 3.6-kb transcripts were selectively increased. On the basis of enzyme assays in vitro, ACAT activity was increased 3.0-fold by using 100 nM 1,25-(OH)(2)D(3), and 1.8-fold by using 1 microM 9-cis-RA. Together, our results suggest that 1,25-(OH)(3) participates in ACAT-1 gene expression during the monocyte-macrophage differentiation process.

Previously, we mapped the novel CC chemokine myeloid progenitor inhibitory factor 2 (MPIF-2)/eotaxin-2 to chromosome 7q11.23 (Nomiyama, H., Osborne, L. R., Imai, T., Kusuda, J., Miura, R., Tsui, L.-C., and Yoshie, O. (1998) Genomics 49, 339-340). Since chemokine genes tend to be clustered, unknown chemokines may be present in the vicinity of those mapped to new chromosomal loci. Prompted by this hypothesis, we analyzed the genomic region containing the gene for MPIF-2/eotaxin-2 (SCYA24) and have identified a novel CC chemokine termed eotaxin-3. The genes for MPIF-2/eotaxin-2 (SCYA24) and eotaxin-3 (SCYA26) are localized within a region of approximately 40 kilobases. By Northern blot analysis, eotaxin-3 mRNA was constitutively expressed in the heart and ovary. We have generated recombinant eotaxin-3 in a baculovirus expression system. Eotaxin-3 induced transient calcium mobilization specifically in CC chemokine receptor 3 (CCR3)-expressing L1.2 cells with an EC(50) of 3 nM. Eotaxin-3 competed the binding of (125)I-eotaxin to CCR3-expressing L1.2 cells with an IC(50) of 13 nM. Eotaxin-3 was chemotactic for normal peripheral blood eosinophils and basophils at high concentrations. Collectively, eotaxin-3 is yet another functional ligand for CCR3. The potency of eotaxin-3 as a CCR3 ligand seems, however, to be approximately 10-fold less than that of eotaxin. Identification of eotaxin-3 will further promote our understanding of the control of eosinophil trafficking and other CCR3-mediated biological phenomena. The strategy used in this study may also be applicable to identification of other unknown chemokine genes.

The primary structure of pig mitochondrial aspartate aminotransferase (mAspATase; L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) precursor was deduced from its cDNA sequence. A library of cDNA clones was constructed from pig liver poly(A)+ RNA by applying the vector/primer method of Okayama and Berg [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170]. The library was screened for pig mAspATase sequences by using a mixture of eight oligodeoxyribonucleotides as a probe. The sequences of the probe were deduced from the known amino acid sequence of pig mAspATase residues 196-201. Two recombinant plasmids containing inserts of about 2500 and 2600 base pairs were selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the pig mAspATase precursor consists of the mature enzyme of 401 amino acid residues and an amino-terminal segment of 29 amino acid residues called the "presequence" that contains four basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The sequence of this 29-amino acid mAspATase precursor segment was compared with the presequences of other mitochondrial enzymes.

The Physarum polycephalum 26S ribosomal RNA gene contains two intervening sequences (introns). The DNA sequence of one of these introns was analyzed together with that of its flanking regions (exons). In addition, the nucleotide sequence of the corresponding region of the reverse transcript of 26S rRNA was determined, and from comparisons of both sequences the precise location and size of the intron were determined. Our findings, when compared with those for Tetrahymena and Chlamydomonas rRNA gene introns, led to the conclusion that certain characteristics exist near the ends of these introns. (i) An exon ends in T at the exon/intron junction and an intron ends in G at the intron/exon junction in all cases. (ii) For each intron, direct repeats several nucleotides long are present 5 to approximately 30 nucleotides upstream from both the exon/intron and intron/exon junctions.

By searching the Expressed Sequence Tag (EST) data base, we identified partial cDNA sequences potentially encoding a novel human CC chemokine. We determined the entire cDNA sequence which encodes a highly basic polypeptide of 134 amino acids total with a putative signal peptide of 23 amino acids. The predicted mature protein of 111 amino acids has the four canonical cysteine residues and shows 21-33% identity to other human CC chemokines, but has a unique carboxyl-terminal extension of about 30 amino acids which contains two extra cysteine residues. The mRNA was expressed strongly in tissues such as the lymph nodes, Appendix, and spleen. The recombinant protein, which was produced by the baculovirus system and purified to homogeneity, was a highly efficient chemoattractant for certain human T cell lines and a highly potent one for freshly isolated peripheral blood lymphocytes and cultured normal T cells expanded by phytohemagglutinin and interleukin 2. Unlike most other CC chemokines, however, this novel chemokine was not chemotactic for monocytes or neutrophils, suggesting that it is specific for lymphocytes. From these results, we designated this novel CC chemokine as SLC from secondary lymphoid-tissue chemokine. SLC fused with the secreted form of alkaline phosphatase (SLC-SEAP) was used to characterize the SLC receptor. Binding of SLC-SEAP to freshly isolated lymphocytes was blocked by SLC (IC50, 0.12 nM) but not by any other CC chemokine so far tested, suggesting that resting lymphocytes express a class of receptors highly specific for SLC. By using somatic cell hybrids, radiation hybrids, and selected yeast and bacterial artificial chromosome clones, we mapped the SLC gene (SCYA21) at chromosome 9p13 and between chromosomal markers, D9S1978(WI-8765) and AFM326vd1, where the gene for another novel CC chemokine termed ELC from EBI1-ligand chemokine (SCYA19) also exists. Collectively, SLC is a novel CC chemokine specific for lymphocytes and, together with ELC, constitutes a new group of chemokines localized at chromosome 9p13.

Liver and activation-regulated chemokine (LARC) is a recently identified CC chemokine that is expressed mainly in the liver. LARC functions as a selective chemoattractant for lymphocytes that express a class of receptors specifically binding to LARC with high affinity. To identifiy the receptor for LARC, we examined LARC-induced calcium mobilization in cells stably expressing five CC chemokine receptors (CCR1-CCR5) and five orphan seven-transmembrane receptors. LARC specifically induced calcium flux in K562 cells as well as 293/EBNA-1 cells stably expressing an orphan receptor GPR-CY4. LARC induced migration in 293/EBNA-1 cells stably expressing GPR-CY4 with a bi-modal dose-response curve. LARC fused with secreted alkaline phosphatase (LARC-SEAP) bound specifically to Raji cells stably expressing GPR-CY4 with a Kd of 0.9 nM. Only LARC but not five other CC chemokines (MCP-1, RANTES, MIP-1alpha, MIP-1beta, and TARC) competed with LARC-SEAP for binding to GPR-CY4. By Northern blot analysis, GPR-CY4 mRNA was expressed mainly in spleen, lymph nodes, Appendix, and fetal liver among various human tissues. Among various leukocyte subsets, GPR-CY4 mRNA was detected in lymphocytes (CD4(+) and CD8(+) T cells and B cells) but not in natural killer cells, monocytes, or granulocytes. Expression of GPR-CY4 mRNA in CD4(+) and CD8(+) T cells was strongly up-regulated by IL-2. Taken together, GPR-CY4 is the specific receptor for LARC expressed selectively on lymphocytes, and LARC is a unique functional ligand for GPR-CY4. We propose GPR-CY4 to be designated as CCR6.

By searching the expressed sequence tag (EST) data base, we identified partial cDNA sequences encoding a novel human CC chemokine. We determined the complete cDNA sequence that encodes a highly basic polypeptide of a total 98 amino acids with 20 to 30% identity to other human CC chemokines. We termed this novel chemokine from EBI1-Ligand Chemokine as ELC (see below). The ELC mRNA was most strongly expressed in the thymus and lymph nodes. Recombinant ELC protein was expressed as a fusion protein with the Flag tag (ELC-Flag). For receptor-binding assays, recombinant ELC protein fused with the secreted form of alkaline phosphatase (SEAP) was used. By stably expressing five CC chemokine receptors (CCR1 to 5) and five orphan receptors, ELC-SEAP was found to bind specifically to an orphan receptor EBI1. Only ELC-Flag, but not MCP-1, MCP-2, MCP-3, eotaxin, MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed and secreted), thymus and activation-regulated chemokine (TARC), or liver and activation-regulated chemokine (LARC), competed with ELC-SEAP for EBI1. ELC-Flag-induced transient calcium mobilization and chemotactic responses in EBI1-transfected cells. ELC-Flag also induced chemotaxis in HUT78 cells expressing endogenous EBI1 at high levels. By somatic hybrid and radiation hybrid analyses, the gene for ELC (SCYA19) was mapped to chromosome 9p13 instead of chromosome 17q11.2 where the genes for CC chemokines are clustered. Taken together, ELC is a highly specific ligand for EBI1, which is known to be expressed in activated B and T lymphocytes and strongly up-regulated in B cells infected with Epstein-Barr virus and T cells infected with herpesvirus 6 or 7. ELC and EBI1 may thus play roles in migration and homing of normal lymphocytes, as well as in pathophysiology of lymphocytes infected with these herpesviruses. We propose EBI1 to be designated as CCR7.

EBI1-ligand chemokine (ELC) is a CC chemokine constitutively expressed in various lymphoid tissues and a high-affinity functional ligand for EBI1/CCR7, a seven transmembrane G-protein-coupled receptor originally identified as an Epstein-Barr virus (EBV)-inducible gene. Here we examined chemotactic activity of ELC on peripheral blood leukocytes. ELC attracted both CD4⁺ and CD8⁺ T cells, particularly efficiently after activation with IL-2 or with phytohaemagglutinin (PHA) plus IL-2, as well as CD19⁺ B cells, but not CD16⁺ NK cells, CD14⁺ monocytes or neutrophils. Among CD3⁺ T cells, ELC attracted both CD45RO^- naive and CD45 RO⁺ memory subsets. ELC also induced vigorous calcium mobilization in T cells stimulated with IL-2 with an ED₅₀ of 3 nM. ELC fused with the secreted form of alkaline phosphatase (ELC-SEAP) specifically bound to lymphocytes and this binding was blocked only by ELC among 10 CC chemokines so far tested. Notably, lymphocytes stimulated with IL-2 or T cells expanded by PHA plus IL-2 showed much higher levels of binding than fresh lymphocytes. Consistently, CCR7 mRNA was detected in CD4⁺ and CD8⁺ T cells as well as B cells, but not in NK cells, monocytes or neutrophils, and was dramatically increased in T cells upon treatment with IL-2 or with PHA plus IL-2. Like ELC mRNA, CCR7 mRNA was expressed in various lymphoid tissues. By in situ hybridization, ELC and CCR7 mRNA were detected in the parafollicular and inner cortical regions of a lymph node, and in the parafollicular regions of an appendix. Collectively, ELC and CCR7 may be involved in the trafficking of a broad spectrum of lymphocytes, especially activated T cells, into and within various lymphoid tissues.

The complete nucleotide sequences of the 5.8S and 26S rRNA genes of Physarum polycephalum and the transcribed spacer between them were determined. Comparison of the sequences with those of the Escherichia coli 23S rRNA and yeast 26S rRNA genes showed that there are 16 highly homologous regions in the sequences of Physarum and E. coli and that eukaryotes have some eukaryote-specific extra sequences. The sequence immediately following the 5.8S-like region of E. coli 23S rRNA was found to be highly homologous to the 5' terminus of Physarum 26S rRNA, indicating that the eukaryote-specific 5.8S rRNA gene is derived from the 5'-terminal region of the prokaryote large rRNA gene.

The nucleotide sequences of mRNAs for the mouse mitochondrial and cytosolic aspartate aminotransferase isoenzymes (mAspAT and cAspAT) (EC 2.6.1.1) were determined from complementary DNAs. The mAspAT mRNA comprises minimally 2460 nucleotides and codes for a polypeptide of 430 amino acid residues corresponding to the precursor form of the mAspAT (pre-mAspAT). The cAspAT mRNA comprises minimally 2086 nucleotides and codes for a polypeptide of 413 amino acid residues. The region coding for the mature mAspAT and that for the cAspAT show about 53% overall homology. The former shares 49% and the latter 48% of homology, respectively, with that of the Escherichia coli aspC gene, which has been shown to code for the E. coli AspAT (Kuramitsu, S., Okuno, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 1259-1262). When the deduced amino acid sequence of the mouse pre-mAspAT was compared with that of the pig pre-mAspAT polypeptide, we found that they share a 94% homology and that the mouse pre-mAspAT yields a presequence consisting of 29 amino acid residues and a mature mAspAT, consisting of 401 amino acid residues. These numbers and the amino acid residues present at the putative cleavage site are all in complete agreement in these two species. The deduced amino acid sequence of the mouse cAspAT shares 91% homology with that of the pig cAspAT. Comparisons of the nucleotide and deduced amino acid sequences between the mouse and E. coli AspATs suggest that the mammalian mAspAT gene is more closely related to the E. coli aspC gene than is the mammalian cAspAT gene.

Partial overlapping cDNA sequences likely to encode a novel human CC chemokine were identified from the GenBank Expressed Sequence Tag data base. Using these sequences, we isolated full-length cDNA encoding a protein of 96 amino acid residues with 20-28% identity to other CC chemokines. By Northern blot, this chemokine was mainly expressed in liver among various tissues and strongly induced in several human cell lines by phorbol myristate acetate. We thus designated this chemokine as LARC from Liver and Activation-Regulated Chemokine. We mapped the LARC gene close to the chromosomal marker D2S159 at chromosome 2q33-q37 by somatic cell and radiation hybrid mappings and isolated two yeast artificial chromosome clones containing the LARC gene from this region. To prepare LARC, we subcloned the cDNA into a baculovirus vector and expressed it in insect cells. The secreted protein started at Ala-27 and was significantly chemotactic for lymphocytes. At a concentration of 1 microg/ml, it also showed a weak chemotactic activity for granulocytes. Unlike other CC chemokines, however, LARC was not chemotactic for monocytic THP-1 cells or blood monocytes. LARC tagged with secreted alkaline phosphatase-(His)6 bound specifically to lymphocytes, the binding being competed only by LARC and not by other CC or CXC chemokines. Scatchard analysis revealed a single class of receptors for LARC on lymphocytes with a Kd of 0.4 nM and 2100 sites/cell. Collectively, LARC is a novel CC chemokine, which may represent a new group of CC chemokines localized on chromosome 2.

Cytokine LD78 is a member of a newly identified cytokine superfamily. We cloned the third human gene for the LD78, termed LD78γ and the sequence analysis showed that it is a 5'-truncated pseudogene. Exons 2 and 3 and the intron between them are highly homologous to those of the LD78β gene, hence, the γ gene was probably derived from the β gene. Southern blot analysis of humanxmouse somatic hybrid cell DNAs and in situ hybridization experiments mapped all the three gene loci on human chromosome 17q21.1-q21.3. Analysis of DNAs from family members supports our previous finding that the β and γ genes on each of the paired chromosome 17 vary in copy number and that the LD78α gene is presumably a single copy. In our analyses of cosmid clones, the LD78β gene and the second gene for AT744, which is also a member of the superfamily are closely linked in a head-to-head arrangement. The mechanism of generation of the three LD78 genes is discussed.