Molecular cDNA cloning and tissue distribution of mRNA encoding a novel ATP-binding cassette (ABC) half-transporter

After feeding a commercial rodent chow for 8 weeks, tissues from male and female rats were collected and examined for selenium content, glutathione peroxidase (GPX) activities and selenoprotein W (Se-W) levels. There were no differences (P > 0.05) between plasma selenium content, plasma GPX activity, whole blood selenium content, or whole blood GPX activity between male and female rats. There was also no gender effect on selenium concentration in muscle, brain, spleen, and skin, but selenium concentration in liver was higher (P < 0.05) in female than in male rats. Western blots indicated that the tissue distribution of Se-W was similar in male and female rats. Se-W protein level was high in testes of male rats but very low in ovaries of female rats. Muscle and skin from female rats had significantly higher (P < 0.05) Se-W levels than from male rats. Consistent with Se-W content, the Se-W mRNA levels from female skins were significantly higher (P < 0.05) than from male rats. The expression of Se-W was different in various tissues and gender influenced this regulation in some tissues.     

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.     

Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.     

Molecular cloning, characterization and cellular distribution of rat steroidogenic acute regulatory protein (StAR) in the ovary

Rat ovarian genes induced by the treatment of immature rats with pregnant mare serum gonadotropin (PMSG) were isolated by a subtraction cloning method. Amongst them was obtained a probable rat homologue of steroidogenic acute regulatory protein (StAR), which has been recently identified as a protein that is an acute regulator of the rate limiting transfer of cholesterol from the outer to the inner mitochondrial membrane. Structure of rat StAR was determined by nucleotide sequence analysis. Northern blot analysis revealed that StAR mRNA levels were rapidly and strongly increased by PMSG/hCG but not by FSH. In situ hybridization revealed that the expression of StAR mRNA was strongly induced by PMSG in theca interna cells as well as in corpora lutea. These findings indicate that expression of StAR mRNA is restricted to and induced in the ovarian steroidogenic cell types where cholesterol is used as a substrate for synthesis of steroid hormones.     

Molecular cloning and characterization of RGC-32, a novel gene induced by complement activation in oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated Response Genes to Complement (RGC) 1-32. RGC-1 is identical with heat shock protein 105, RGC-2 with poly(ADP-ribose) polymerase, and RGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest that RGC-32 may play a role in cell cycle activation.     

Molecular cloning and tissue expression of a novel orphan G protein-coupled receptor from rat lung

G protein-coupled receptors transduce the signal of a wide variety of hormones, neurotransmitters, cytokines, and other molecules across the cell membrane to elicit the corresponding response inside the target cells. We describe in this paper the molecular cloning and tissue distribution of a novel rat G protein-coupled receptor, GPR41, with highest homology to the human orphan G protein-coupled receptor DRY12. A lower degree of homology was seen with the receptors for bradykinin, angiotensin, and IL8. The expression of GPR41 appears to be the highest in brain and lung tissues, with lesser expression in heart, skeletal muscle, and kidney, as assayed by northern blotting. No GPR41 message was seen in spleen, liver, or testes. GPR41 failed to bind any of the ligands tested.     

Molecular cloning and characterization of a novel form of neuropeptide gene as a developmentally regulated molecule

To examine the molecular basis controlling neuronal differentiation, subtraction library construction and differential screening were used to identify cDNAs whose mRNA levels are regulated in mouse NS20Y cells by dibutyryl cyclic AMP treatment. One of them, N27K, whose mRNA increases transiently during both neuronal differentiation in NS20Y cells and development in mouse brain. The deduced amino acid sequence of N27K comprises 212 amino acid residues and is a novel form of a precursor protein for a new neuropeptide nociceptin/orphanin FQ, which we independently cloned as N23K. That is, the putative protein encoded by N27K is 25 amino acids longer than that encoded by N23K. Using an antibody against a C-terminal peptide of the N27K protein that recognizes a 27-kDa protein in Western blot analysis, a punctate structure in the perinuclear region and areas near the tip of neurites is visualized in neurally differentiating NS20Y cells. The time of maximal expression correlates with periods of neurite extension, and expression decreases as the neuritic network develops. Immunohistochemistry of tissue sections of the mouse central nervous system revealed that reactivity for the anti-N27K protein antibody can detected in early generated neurons at embryonic day 14, in virtually all immature neurons at postnatal day 1, and in subsets of neurons of discrete brain regions such as the hypothalamus and spinal cord in adults. This remarkable redistribution suggests that N27K may be involved in a process in neurite outgrowth and nervous system development.     

Isolation and characterization of a novel connexin gene, Cx-60, in porcine ovarian follicles

Sequences present at the genomic termini of herpesviruses become linked during lytic-phase replication and provide the substrate for cleavage and packaging of unit length viral genomes. We have previously shown that homologs of the consensus herpesvirus cleavage-packaging signals, pac1 and pac2, are located at the left and right genomic termini of human herpesvirus 6 (HHV-6), respectively. Immediately adjacent to these elements are two distinct arrays of human telomeric repeat sequences (TRS). We now show that the unique sequence element formed at the junction of HHV-6B genome concatemers (pac2-pac1) is necessary and sufficient for virally mediated cleavage of plasmid DNAs containing the HHV-6B lytic-phase origin of DNA replication (oriLyt). The concatemeric junction sequence also allowed for the packaging of these plasmid molecules into intracellular nucleocapsids as well as mature, infectious viral particles. In addition, this element significantly enhanced the replication efficiency of oriLyt-containing plasmids in virally infected cells. Experiments revealed that the concatemeric junction sequence possesses an unusual, S1 nuclease-sensitive conformation (anisomorphic DNA), which might play a role in this apparent enhancement of DNA replication--although additional studies will be required to test this hypothesis. Finally, we also analyzed whether the presence of flanking viral TRS had any effect on the functional activity of the minimal concatemeric junction (pac2-pac1). These experiments revealed that the TRS motifs, either alone or in combination, had no effect on the efficiency of virally mediated DNA replication or DNA cleavage. Taken together, these data show that the cleavage and packaging of HHV-6 DNA are mediated by cis-acting consensus sequences similar to those found in other herpesviruses, and that these sequences also influence the efficiency of HHV-6 DNA replication. Since the adjacent TRS do not influence either viral cleavage and packaging or viral DNA replication, their function remains uncertain.     

Molecular cloning of a fish gene encoding a novel seven-transmembrane receptor related distantly to catecholamine, histamine, and serotonin receptors

A genomic DNA fragment encoding a G protein-coupled seven-transmembrane receptor was isolated from Medaka fish, Oryzias latipes. The encoded protein is similar in sequence to other receptors including catecholamine, histamine and serotonin receptors. However, the similarity is much lower than those among members of these receptor subfamilies, thus suggesting this seven-transmembrane receptor to be an orphan receptor whose ligand has not yet been identified. Genomic Southern blot analysis suggested that the fish genome contains additional receptor genes related to the isolated gene, indicating that this novel receptor, possibly with its related receptors, might constitute a novel subfamily of the seven-transmembrane receptor superfamily.     

Molecular cloning and hormonal regulation of PiT-1, a sodium-dependent phosphate cotransporter from rat parathyroid glands

The extracellular concentration of inorganic phosphate (Pi) is an important determinant of parathyroid cell function. The effects of Pi may be mediated through specific molecules in the parathyroid cell membrane, one candidate molecule for which would be a Na+-dependent Pi cotransporter. A complementary DNA encoding a Na+-Pi cotransporter, termed rat PiT-1, has now been isolated from rat parathyroid. The 2890-bp complementary DNA encodes a protein of 681 amino acids that shows sequence identities of 97% and 93% with the type III Na+-Pi cotransporters mouse PiT-1 and human PiT-1, respectively. Expression of rat PiT-1 in Xenopus oocytes revealed that it possesses Na+-dependent Pi cotransport activity. PiT-1 messenger RNA (mRNA) is widely distributed in rat tissues and is most abundant in brain, bone, and small intestine. The amount of PiT-1 mRNA in the parathyroid of vitamin D-deficient rats was reduced compared with that in normal animals and increased markedly after administration of 1,25-dihydroxyvitamin D3. Furthermore, the abundance of PiT-1 mRNA in the parathyroid was much greater in rats fed a low-Pi diet than in those fed a high-Pi diet. Thus, rat PiT-1 may contribute to the effects of Pi and vitamin D on parathyroid function.     

Molecular cloning, expression, and functional characterization of novel mouse sulfotransferases

STUDY OBJECTIVE: The present study was performed to determine the influence of a perioperative myocardial infarction on long-term mortality in patients who have undergone elective vascular surgery. STUDY DESIGN: This was a 4-year follow-up of patients who had undergone elective vascular procedures at a Veterans Affairs Medical Center. Between January 1989 and December 1990, 115 consecutive patients underwent surgery for either an expanding abdominal aortic aneurysm (AAA) (38%) or for pain in the lower extremities (62%). RESULTS: Vital status at 4 years postsurgery was determined for all patients. Thirty-day postoperative mortality was 3%, while estimates at 1, 2, 3, and 4 years were 19%, 26%, 35%, and 39%, respectively. Of the 45 patients who died within 4 years following surgery, the major causes of death were cardiac (40%), cancer (18%), cerebrovascular (13%), and peripheral vascular disease (11%). Univariate predictors of 1-year mortality on preoperative evaluation were an abnormal ECG, moderate or greater sized exercise thallium defect and left ventricular ejection fraction < or =40%, and a perioperative myocardial infarction. Univariate predictors of 4-year mortality were non-AAA surgery and diabetes mellitus. Perioperative myocardial infarction was a marginally significant independent predictor of 1-year mortality (p=0.06), while the need for non-AAA surgery was a strong independent predictor at 4 years. CONCLUSIONS: Cardiac mortality is the major cause of late death among patients undergoing elective vascular surgery. Although preoperative indicators of symptomatic coronary artery disease and nonfatal perioperative myocardial infarction identified those individuals at increased mortality in the first postoperative year, the extent of vascular disease at presentation may be a more important determinant of long-term survival. A randomized trial in such patients is needed to assess the best strategy for treating patients with coexistent coronary artery and vascular diseases.