Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.     

Affinity purification, peptide analysis, and cDNA sequence of the mouse interferon gamma receptor

The receptor for mouse interferon gamma (IFN-gamma) was purified from detergent-solubilized plasma membranes of EL-4, a thymoma cell line which expresses a high number of receptors on its cell surface. The purification was carried out by immunoaffinity chromatography using an anti-receptor monoclonal antibody. The purified receptor was subjected to NH2-terminal sequence analysis as well as sequencing of endopeptidase-generated peptides. One of the peptides was found to be identical to a portion of the published amino acid sequence of the human IFN-gamma receptor deduced from cDNA. This information was utilized to construct a mixed-sequence oligodeoxynucleotide probe which permitted the isolation of a full-length cDNA clone coding for the mouse IFN-gamma receptor. The mouse IFN-gamma receptor cDNA is comprised of 105 base pairs of the 5'-untranslated region, an open reading frame coding for a 477-amino acid serine-rich protein having calculated Mr 52,276, and a 3'-untranslated region of 539 base pairs. The receptor is first synthesized as a pre-protein from which a 25-amino acid signal peptide is cleaved. The receptor contains a hydrophobic transmembrane portion near the center of the molecule. Northern blot analysis of various cell lines showed that each contained a single 2.0-kilobase mRNA. A direct correlation between the amount of IFN-gamma receptor mRNA and the level of receptor expressed on the cell surface was observed. The mouse and human IFN-gamma receptors are structurally similar, showing 51% over-all homology in amino acid sequence. Mouse IFN-gamma receptor cDNA when inserted in a mammalian shuttle vector and transfected into COS-7 monkey cells was able to direct the expression of specific binding activity for mouse IFN-gamma.     

Effect of the administration of prolactin-releasing peptide2 on feeding activity in the intertidal blenny Rhabdoblennius nitidus (Günther, 1861)

Prolactin-releasing peptide2 (PrRP2) was administered intraperitoneally to male intertidal blenny Rhabdoblennius nitidus, a species with male uniparental care of eggs, to investigate the effect on their feeding activity. A significant inhibitory effect on appetite was observed in the breeding season, but not in the nonbreeding season. These results suggest that PrRP2 and PrRP2 receptors are more active during the breeding season. The presence of a mechanism to inhibit feeding activity while parents take care of their offspring may be important for the success of parental care.     

Significant increase in plasma 4β-hydroxycholesterol concentration in patients after kidney transplantation

Several previous studies have shown that renal failure decreases not only renal elimination but also metabolic clearance of drugs, particularly those metabolized by CYP3A. However, whether recovery of renal function results in recovery of hepatic CYP3A activity remains unknown. In this study, we evaluated the effect of renal function on CYP3A activity after kidney transplantation in patients with end-stage renal disease (ESRD) by measuring the change in CYP3A activity using plasma concentration of 4β-hydroxycholesterol as a biomarker. The study enrolled 13 patients with ESRD who underwent the first kidney allograft transplantation. Morning blood samples were collected before and 3, 7, 10, 14, 21, 30, 60, 90, 120, 150 and 180 days after kidney transplantation. Plasma concentration of 4β-hydroxycholesterol was measured using GC-MS. Compared with before kidney transplantation, creatinine clearance increased significantly from day 3 after kidney transplantation and stabilized thereafter. Plasma concentration of 4β-hydroxycholesterol was elevated significantly on days 90 and 180 after kidney transplantation. In conclusion, this study suggests the recovery of CYP3A activity with improvement in renal function after kidney transplantation in patients with ESRD.     

Retrotransposon-mediated restoration of Chlorella telomeres: accumulation of Zepp retrotransposons at termini of newly formed minichromosomes

To elucidate the contribution of LINE-like retrotransposon Zepp elements to the formation and maintenance of chromosomal telomeres, newly formed minichromosomes in irradiated Chlorella vulgaris cells were isolated and structurally characterized. A minichromosome (miniV4) of ~700 kb in size contained a Zepp cluster taking the place of the telomeric repeats on one terminus, whereas the other end of this chromosome consisted of canonical telomeric repeats. The Zepp copies in this cluster were in a tandem array with their poly(A) tails towards the centromere. Another minichromosome Y32 (~400 kb in size) was shown to have several copies of Zepp elements on both termini. On the right arm terminus, two copies of Zepp were found in a tandem array with poly(A) tracts facing towards the chromosomal end. The poly(A) tail and the 3′-end of ~400 bp of the distal copy were replaced by the telomeric repeats. On the 5′-side of the proximal copy was another Zepp element in the reverse orientation. These newly formed telomeric structures are very similar to those previously found in the left arm of chromosome I and the terminus of an unidentified chromosome and support the model of Zepp-mediated restoration and maintenance of Chlorella telomeres.     

Comparative genomic structure of human, dog, and cat MHC: HLA, DLA, and FLA

Comparisons of the genomic structure of 3 mammalian major histocompatibility complexes (MHCs), human HLA, canine DLA, and feline FLA revealed remarkable structural differences between HLA and the other 2 MHCs. The 4.6-Mb HLA sequence was compared with the 3.9-Mb DLA sequence from 2 supercontigs generated by 7x whole-genome shotgun assembly and 3.3-Mb FLA draft sequence. For FLA, we confirm that 1) feline FLA was split into 2 pieces within the TRIM (member of the tripartite motif) gene family found in human HLA, 2) class II, III, and I regions were placed in the pericentromeric region of the long arm of chromosome B2, and 3) the remaining FLA was located in subtelomeric region of the short arm of chromosome B2. The exact same chromosome break was found in canine DLA structure, where class II, III, and I regions were placed in a pericentromeric region of chromosome 12 whereas the remaining region was located in a subtelomeric region of chromosome 35, suggesting that this chromosome break occurred once before the split of felid and canid more than 55 million years ago. However, significant differences were found in the content of genes in both pericentromeric and subtelomeric regions in DLA and FLA, the gene number, and amplicon structure of class I genes plus 2 other class I genes found on 2 additional chromosomes; canine chromosomes 7 and 18 suggest the dynamic nature in the evolution of MHC class I genes.     

Gene conversion between mammalian CCR2 and CCR5 chemokine receptor genes: a potential mechanism for receptor dimerization

The chemokine receptor genes of the CCR cluster on human chromosome 3p21 play important roles in humoral and cellular immune responses. Several of these receptors have been shown to influence human immunodeficiency virus infection and progression to AIDS, and their homologues may play a role in feline immunodeficiency virus infection. We report the isolation and sequencing of a 150-kb domestic cat BAC clone containing the feline CCR genes CCR1, CCR2, CCR3, and CCR5 to further analyze these four receptor genes within the family Felidae. Comparative and phylogenetic analyses reveal evidence for historic gene conversion between the adjacent CCR2 and CCR5 genes in the Felidae and in three independent mammalian orders (Primates, Cetartiodactyla, and Rodentia), resulting in higher than expected levels of sequence similarity between the two paralogous genes within each order. The gene conversion was restricted to the structural (transmembrane) domains of the CCR2 and CCR5 genes. We also discovered a recent gene conversion event between the third extracellular loop of CCR2 and CCR5 genes that was fixed in Asian lions and found at low frequency in African lions (Panthera leo), suggesting that this domain may have an important functional role. Our results suggest that ongoing parallel gene conversion between CCR2 and CCR5 promotes receptor heterodimerization in independent evolutionary lineages and offers an effective adaptive strategy for gene editing and coevolution among interactive immune response genes in mammals.