Aortic carboxypeptidase-like protein, a novel protein with discoidin and carboxypeptidase-like domains, is up-regulated during vascular smooth muscle cell differentiation

Phenotypic modulation of vascular smooth muscle cells plays an important role in the pathogenesis of arteriosclerosis. In a screen of proteins expressed in human aortic smooth muscle cells, we identified a novel gene product designated aortic carboxypeptidase-like protein (ACLP). The approximately 4-kilobase human cDNA and its mouse homologue encode 1158 and 1128 amino acid proteins, respectively, that are 85% identical. ACLP is a nonnuclear protein that contains a signal peptide, a lysine- and proline-rich 11-amino acid repeating motif, a discoidin-like domain, and a C-terminal domain with 39% identity to carboxypeptidase E. By Western blot analysis and in situ hybridization, we detected abundant ACLP expression in the adult aorta. ACLP was expressed predominantly in the smooth muscle cells of the adult mouse aorta but not in the adventitia or in several other tissues. In cultured mouse aortic smooth muscle cells, ACLP mRNA and protein were up-regulated 2-3-fold after serum starvation. Using a recently developed neural crest cell to smooth muscle cell in vitro differentiation system, we found that ACLP mRNA and protein were not expressed in neural crest cells but were up-regulated dramatically with the differentiation of these cells. These results indicate that ACLP may play a role in differentiated vascular smooth muscle cells.     

Somatostatin receptor subtypes sst1 and sst2 elicit opposite effects on the response to glutamate of mouse hypothalamic neurones: an electrophysiological and single cell RT-PCR study

To clone a new nuclear receptor, we screened a rabbit heart complementary DNA (cDNA) library with degenerate oligonucleotide probes corresponding to the DNA-binding domain of nuclear receptors, which is highly conserved among receptors. One of the cDNA clones, clone 23, encodes a novel protein of 596 amino acids, and predicted molecular mass is 66 kDa. Homology search analysis identified this protein as rabbit TR4 (TR4-0). We also cloned the cDNA encoding a rabbit TR4 isoform (TR4-1), which lacks the putative C-terminal ligand-binding domain (350 amino acids) caused by a 23-bp exon deletion, which probably occurred during messenger RNA (mRNA) splicing. Northern blot analysis showed that TR4s are expressed with two kinds of mRNAs (9.0 kb and 2.8 kb), both of which are relatively abundant in brain, testis, and bone. RT-PCR analysis, using pairs of primers specific for each TR4, showed that both types of receptor express in various tissues. Furthermore, both are present in primary osteoblasts and bone marrow cells, though the mRNA levels of TR4-0 were much higher than those of TR4-1. A functional study, using a transient transfection assay, showed that both receptors suppressed retinoid X receptor (RXR)-retinoid acid receptor, RXR-TR, and RXR-VDR-mediated transactivation significantly in COS-1 and osteosarcoma cells (UMR-106, ROS17/2.8) and that TR4-0 was much more effective than TR4-1. Unexpectedly, we found that the TR4s effectively suppressed estrogen receptor-mediated transactivation in bone cells, but neither in kidney (COS-1) nor breast cancer cells (MCF-7, one of the major target cells of the estrogen action). Thus, the present study shows a novel property of the TR4 orphan receptor, acting as a bone cell-specific repressor in the estrogen receptor-mediated signaling pathway.     

In vitro desaturation or elongation of monotrans isomers of linoleic acid by rat liver microsomes

The mammalian rasGAPs constitute a group of widely expressed proteins involved in the negative regulation of ras-mediated signaling. In this study we have isolated a novel human gene, RASAL (Ras GTPase-activating-like) and its murine ortholog, MRASAL which are most similar to the GAP1 family of rasGAP proteins, based upon the presence and organization of specific conserved domains. Full-length human and murine mRNA sequences are predicted to encode 804 and 799 amino acid polypeptides, respectively. Sequence analysis of these two proteins revealed the presence of two N-terminal calcium-dependent phospholipid binding C2 domains, a conserved GAP related domain (GRD) and a C-terminal pleckstrin homology (PH) domain. Northern blot and mRNA in situ hybridization analyses indicate that RASAL, in contrast to other mammalian rasGAP proteins, has a limited expression pattern; RASAL is highly expressed in the follicular cells of the thyroid and the adrenal medulla and expressed at lower levels in brain, spinal cord and trachea. Human RASAL has been localized by radiation hybrid mapping to chromosome 12q23-24.     

Helix-loop-helix proteins LYL1 and E2a form heterodimeric complexes with distinctive DNA-binding properties in hematolymphoid cells

LYL1 is a basic helix-loop-helix (HLH) protein that was originally discovered because of its translocation into the beta T-cell receptor locus in an acute lymphoblastic leukemia. LYL1 is expressed in many hematolymphoid cells, with the notable exceptions of thymocytes and T cells. Using the yeast two-hybrid system to screen a cDNA library constructed from B cells, we identified the E-box-binding proteins E12 and E47 as potential lymphoid dimerization partners for LYL1. The interaction of LYL1 with E2a proteins was further characterized in vitro and shown to require the HLH motifs of both proteins. Immunoprecipitation analyses showed that in T-ALL and other cell lines, endogenous LYL1 exists in a complex with E2a proteins. A preferred DNA-binding sequence, 5'-AACAGATG(T/g)T-3', for the LYL1-E2a heterodimer was determined by PCR-assisted site selection. Endogenous protein complexes containing both LYL1 and E2a bound this sequence in various LYL1-expressing cell lines and could distinguish between the LYL1 consensus and muE2 sites. These data demonstrate that E2a proteins serve as dimerization partners for the basic HLH protein LYL1 to form complexes with distinctive DNA-binding properties and support the hypothesis that the leukemic properties of the LYL1 and TAL subfamily of HLH proteins could be mediated by recognition of a common set of target genes as heterodimeric complexes with class I HLH proteins.     

Kinesin family in murine central nervous system

In neuronal axons, various kinds of membranous components are transported along microtubules bidirectionally. However, only two kinds of mechanochemical motor proteins, kinesin and brain dynein, had been identified as transporters of membranous organelles in mammalian neurons. Recently, a series of genes that encode proteins closely related to kinesin heavy chain were identified in several organisms including Schizosaccharomyces pombe, Aspergillus niddulans, Saccharomyces cerevisiae, Caenorhabditus elegans, and Drosophila. Most of these members of the kinesin family are implicated in mechanisms of mitosis or meiosis. To address the mechanism of intracellular organelle transport at a molecular level, we have cloned and characterized five different members (KIF1-5), that encode the microtubule-associated motor domain homologous to kinesin heavy chain, in murine brain tissue. Homology analysis of amino acid sequence indicated that KIF1 and KIF5 are murine counterparts of unc104 and kinesin heavy chain, respectively, while KIF2, KIF3, and KIF4 are as yet unidentified new species. Complete amino acid sequence of KIF3 revealed that KIF3 consists of NH2-terminal motor domain, central alpha-helical rod domain, and COOH-terminal globular domain. Complete amino acid sequence of KIF2 revealed that KIF2 consists of NH2-terminal globular domain, central motor domain, and COOH-terminal alpha-helical rod domain. This is the first identification of the kinesin-related protein which has its motor domain at the central part in its primary structure. Northern blot analysis revealed that KIF1, KIF3, and KIF5 are expressed almost exclusively in murine brain, whereas KIF2 and KIF4 are expressed in brain as well as in other tissues. All these members of the kinesin family are expressed in the same type of neurons, and thus each one of them may transport its specific organelle in the murine central nervous system.     

Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens

Unlike mammals, birds, and most other fishes, winter flounder completes spermatogenesis without replacing its germ cell histones with protamines. Instead, during spermiogenesis, these fish produce a family of high molecular weight (80,000-200,000) basic nuclear proteins (HMrBNPs) that bind to sperm chromatin containing the normal complement of histones. These large, basic proteins are built up of tandem iterations of oligopeptide repeats that contain phosphorylatable DNA-binding motifs. Although the HMrBNPs have no obvious homology to histones, protamines, or other sperm-specific chromatin proteins, we report here the isolation of a clone (2B) from a winter flounder genomic DNA library that establishes a link between the HMrBNPs and histone H1. The 2B sequence contains an open reading frame, which, when conceptually translated, encodes a 265-residue protein. At its N terminus the translation product contains numerous simple repeats that match the oligopeptides contained within the HMrBNPs. Unexpectedly, the C terminus of the putative protein shows 66% identity and 76% conservation to the histone H1 globular domain. This connection suggests that the HMrBNPs may have originated from the extended N-terminal tail region of a testis-specific, H1-like linker histone.     

The effect of cycloheximide on the regulation of proenkephalin and prodynorphin gene expressions induced by kainic acid in rat hippocampus

The effect of cycloheximide (CHX), a protein synthesis inhibitor, on the regulation of proenkephalin (proENK) and prodynorphin (proDYN) mRNA levels, proto-oncogenes, such as c-fos, 35-kDa fra and c-jun mRNA, and the levels of their products induced by kainic acid (KA) in rat hippocampus was studied. The proENK and proDYN mRNA levels were markedly increased 4 and 8 h after KA (10 mg/kg i.p.) administration. However, the intracellular proENK protein level was not affected by KA. The elevations of both proENK and proDYN mRNA levels induced by KA were inhibited by pre-administration of CHX (15 mg/kg i.p.). The increases of proENK and proDYN mRNA levels induced by KA were well-correlated with the increases of c-Fos, 35-kDa Fra and c-Jun protein levels. KA administration increased the hippocampal levels of c-Fos, 35-kDa Fra and c-Jun proteins with the time. The increases of c-Fos, 35-kDa Fra and c-Jun protein levels induced by KA administration were also inhibited by CHX pre-administration. KA administration markedly increased both c-fos and c-jun mRNA levels during 1 and 4 h and the increased levels of these proto-oncogene mRNA were further prolonged by the treatment with CHX. In addition, CHX alone increased both c-fos and c-jun mRNA levels although the onset times of induction were different. In electrophoretic mobility shift-assay, both AP-1 and ENKCRE-2 DNA-binding activities were increased by KA. KA-induced increases of AP-1 and ENKCRE-2 DNA-binding activities were also attenuated by CHX. In addition, KA-induced AP-1 and ENKCRE-2 DNA-binding activities were diminished by the antibodies against Fos and Jun family proteins. Furthermore, the cross-competition studies revealed that AP-1 proteins actively participated in ENKCRE-2 DNA domain. The results suggest that KA-induced proENK and proDYN mRNA expressions may require on-going synthesis of proteins, such as c-Fos, c-Jun and 35-kDa Fra, which may have a possible role in the up-regulation of proENK and proDYN gene expression through the binding with AP-1 and ENKCRE-2 DNA-binding motifs.     

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities

Previous studies have suggested that activation of calcium-independent PLA2 (CaIPLA2) is an early event in cell death after hypoxic injury in proximal tubule cells. An approximately 28-kD CaIPLA2 with preferential activity toward plasmalogen phospholipids has been recently purified from rabbit kidney cortex (D. Portilla and G. Dai, J Biol Chem 271, 15,451-15,457, 1996). Their report describes the cloning of a full-length rat cDNA encoding CaIPLA2, using sequences derived from the purified rabbit kidney cortex enzyme. In addition, cDNA from rabbit kidney that encode the rabbit homologue of the enzyme and a closely related isoform were isolated. The rat cDNA is predicted to encode an approximately 24-kD protein, and each cDNA contains the sequence G-F-S-Q-G, which fits the active site consensus sequence G-X-S-X-G of carboxylesterases. Several lines of evidence (DNA sequence comparison, Southern blot analysis, and examination of the expressed sequence tag database) show that CaIPLA2 enzymes are encoded by a multigene family in rats, mice, rabbits, and humans. Northern analysis of various tissues from the rat indicated that the CaIPLA2 gene is ubiquitously expressed, with highest mRNA abundance observed in the kidney and small intestine. The rat CaIPLA2 cDNA, when expressed in a baculovirus expression system, and the purified rabbit kidney cortex protein exhibit both CaIPLA2 and lysophospholipase activities. The cloned CaIPLA2 cDNA are expected to aid in understanding the role of CaIPLA2 in cell death after hypoxic/ischemic cell injury.     

Molecular cloning and characterization of a novel mRNA present in the squid giant axon

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.