Thyroid hormone beta receptor activation has additive cholesterol lowering activity in combination with atorvastatin in rabbits, dogs and monkeys.

BACKGROUND AND PURPOSE: Thyroid hormone receptor (TR) agonists are in clinical trials for the treatment of hypercholesterolaemia. As statins are the standard of clinical care, any new therapies must have adjunctive activity, when given in combination with statins. As already known for the statins, the cholesterol lowering effect of TR activation involves increased expression of the low-density lipoprotein receptor. Using animal models, we tested whether TR activation would have additive cholesterol lowering activity in the presence of effective doses of a statin. EXPERIMENTAL APPROACH: We evaluated the activity of a liver-targeted prodrug, MB07811, of a novel TH receptor beta agonist, MB07344, as monotherapy and in combination with atorvastatin in rabbits, dogs and monkeys. KEY RESULTS: In rabbits, MB07344 (i.v.) decreased total plasma cholesterol (TPC) comparable to that achieved with a maximally effective dose of atorvastatin (p.o.). The addition of MB07344 to atorvastatin resulted in a further decrease in TPC. Similarly, the addition of MB07811 (p.o.) to atorvastatin treatment decreased TPC beyond the level achieved with either agent as monotherapy. In dogs and monkeys, atorvastatin and MB07811 were administered as monotherapy or in combination. Consistent with the rabbit studies, the combination treatment caused a greater decrease in TPC than either MB07811 or atorvastatin administered as monotherapy. CONCLUSIONS AND IMPLICATIONS: We conclude that the effects of MB07811 and atorvastatin in lowering cholesterol are additive in animals. These results would encourage and support the demonstration of similarly improved efficacy of combination versus monotherapy with such agents in the clinic.

Distribution of a novel hypothalamic neuropeptide Y receptor gene and it's absence in rat.

A recently reported Y receptor that has been confusingly referred to as both Y5 and Y2b has now been designated as Y6 by the IUPHAR organization. Using random primed Y6 coding sequence as a hybridization probe we examined the mRNA expression pattern and gene distribution of the Y6 receptor in a variety of species. We detail the relative abundance of Y6 message in mouse and human tissues and report the apparent absence of message for this receptor in any rat tissues tested. We also document the presence of the Y6 gene in chicken, rabbit, cow, dog, mouse, monkey and human, but the complete absence of the Y6 gene in rat.

Expression cloning of a rat hypothalamic galanin receptor coupled to phosphoinositide turnover.

The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a 125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of 125I-pGAL to transiently expressed GALR2 receptors was saturable (KD = 0.15 nM) and displaceable by galanin peptides and analogues in rank order: porcine galanin approximately M32 approximately M35 approximately M40 >/= galanin-(1-16) approximately M15 approximately [D-Trp2]galanin-(1-29) > C7 >> galanin-(3-29). This profile resembles that of the rat GALR1 receptor with the notable exception that [D-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C.

A receptor subtype involved in neuropeptide-Y-induced food intake.

Neuropeptide Y (NPY) is a powerful stimulant of food intake and is proposed to activate a hypothalamic 'feeding' receptor distinct from previously cloned Y-type receptors. This receptor was first suggested to explain a feeding response to NPY and related peptides, including NPY2-36, that differed from their activities at the Y1 receptor. Here we report the expression cloning of a novel Y-type receptor from rat hypothalamus, which we name Y5. The complementary DNA encodes a 456-amino-acid protein with less than 35% overall identity to known Y-type receptors. The messenger RNA is found primarily in the central nervous system, including the paraventricular nucleus of the hypothalamus. The extent to which selected peptides can inhibit adenylate cyclase through the Y5 receptor and stimulate food intake in rats correspond well. Our data support the idea that the Y5 receptor is the postulated 'feeding' receptor, and may provide a new method for the study and treatment of obesity and eating disorders.

Cloning and expression of a novel neuropeptide Y receptor.

The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

Expression cloning of a human B1 bradykinin receptor.

A cDNA clone encoding a human B1 bradykinin receptor was isolated from a human embryonic lung fibroblast cDNA library by expression cloning. The photoprotein aequorin was utilized as an indicator of the ability of the B1 receptor agonist [des-Arg10]kallidin to mediate Ca2+ mobilization in Xenopus laevis oocytes injected with RNA. A clone was isolated with a 1307-nucleotide insert which contains an open reading frame encoding a 353-amino acid protein with the characteristics of a G-protein-coupled receptor. The amino acid sequence of the B1 bradykinin receptor is 36% identical to the amino acid sequence of the B2 bradykinin receptor. The cloned B1 bradykinin receptor expressed in mammalian cells exhibits high affinity binding for 3H-labeled [des-Arg10]kallidin and low affinity for bradykinin. The B1 receptor antagonist [des-Arg10,Leu9]kallidin effectively displaces 3H-labeled [des-Arg10]kallidin from the cloned receptor, whereas the B2 receptor antagonist Hoe-140 (D-Arg0-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin, where Thi is L-[3-(2-thienyl)alanyl], Tic is D-(1,2,3,4-tetrahydroisoquinolin-3-yl-carbonyl), and Oic is L-[(3aS, 7aS)-octahydroindol-2-yl-carbonyl]) does not. Therefore, the expressed receptor has the pharmacological characteristics of the B1 receptor subtype. The availability of both the cloned human B1 and B2 bradykinin receptors should allow the elucidation of the relative contributions of these two receptor subtypes in acute and chronic inflammatory processes.

Conversion of cysteine to serine residues alters the activity, stability, and heparin dependence of acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is a broad spectrum mitogen that is stabilized by complexation with heparin and heparan proteoglycans. The monomeric human protein contains 3 reduced cysteine residues of unknown function, the first 2 of which are conserved among all seven known fibroblast growth factors. The influence of these free sulfhydryl groups on the level, stability, and heparin dependence of the mitogenic activity at physiological temperature and pH is characterized using a complete set of site-directed mutants in which either any 1, 2, or all 3 of the cysteine residues are converted to serines. Mutants of aFGF in which either any 2 or all 3 cysteine residues are substituted by serines are more active, have longer activity half-lives, and are less heparin dependent than wild-type aFGF. In contrast, wild-type aFGF and the three mutants that each retain 2 cysteine residues inactivate more rapidly in the absence of heparin by a nonproteolytic mechanism but are markedly stabilized by heparin. This cysteine-mediated destabilization of aFGF not only diminishes its activity in the absence of heparin in tissue culture but also could functionally restrict its activity in vivo to the vicinity of mast cell-derived heparins and heparan proteoglycans associated with cell surfaces and basement membranes.

Expression in Escherichia coli of rat liver cytosolic glutathione S-transferase Yc cDNA.

An expression plasmid, pKK-GTB2, containing the complete coding sequence of a rat liver glutathione S-transferase Yc subunit was constructed and expressed in Escherichia coli. The entire Yc cDNA sequence from plasmid pGTB42 (Telakowski-Hopskins et al., 1985, J. Biol. Chem. 260, 5820-5825) was amplified by the polymerase chain reaction, subcloned into modified expression vector A6316 (Schoner et al., 1986, Proc. Natl. Acad. Sci. USA 83, 8506-8510 and Linemeyer et al., 1987, Bio/Technology 5, 960-965) and transformed into E. coli strain AB1899. The colonies were screened by hybridization to pGTB42 and the production of Yc subunit was detected by immunoblot analysis. The purified recombinant Yc subunit was active in the conjugation and peroxidation reactions, and appeared homogeneous as judged by sodium dodecyl sulfate gel electrophoresis. Amino acid sequencing of the expressed Yc subunit revealed that about 40% of the expressed protein was blocked at the N-terminus. Approximately 25% of the sequenceable protein (15% of total protein) contained the initiation methionine residue at the amino terminus whereas the rest of the sequenceable protein had proline as the N-terminus. In contrast, only one molecular species with Pro as the first amino acid was identified when the inducer isopropyl-beta-D-thiogalactopyranoside was omitted in the growth medium. Our observation indicated that under certain growth conditions, the enzymes responsible for protein maturation were not able to complete the processing of the overproduced recombinant Yc in E. coli.

Disulfide bonds are neither required, present, nor compatible with full activity of human recombinant acidic fibroblast growth factor.

Human acidic fibroblast growth factor (aFGF) is a potent broad-spectrum mitogen that contains three Cys residues within its monomeric structure. We have found that site-directed mutants in which any one of these Cys residues is converted to serine remain highly active, although variably dependent on heparin, so none of the three possible intramolecular disulfide bonds that can be formed are required for mitogenic activity. Furthermore, a dispensable disulfide bond that might stabilize the active conformation is not present since all three Cys residues are accessible to chemical modification in recombinant as well as brain-derived aFGFs. Finally, formation of a disulfide bond between the two Cys residues conserved among all seven known members of the FGF family results in a virtually inactive product that can subsequently be reactivated by reduction. Thus, despite the extracellular function of aFGF, its Cys residues do not form intramolecular disulfide bonds in the active conformation.

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.

Molecular cloning of biologically active proviral DNA of the anemia-inducing strain of spleen focus-forming virus.

Previously, we have molecularly cloned proviral DNA of a polycythemia-inducing strain of the spleen focus-forming virus (SFFVp). In this paper, we report that unintegrated proviral DNA of the anemia-inducing strain of SFFV (SFFVA) has been molecularly cloned into pBR322. This molecularly cloned DNA retains the biological activity of SFFVA, as infectious SFFV can be recovered from the DNA clone by marker rescue using a previously described two-stage cotransfection assay (Linemeyer et al., J. Virol. 35:710-721, 1980). The recovered SFFV retains an important property of the initial SFFVA which distinguishes SFFVA from SFFVP, namely, the ability of SFFVA to cause proliferation of erythroid cells in which hemoglobin synthesis is erythropoietin dependent. By utilizing a marker rescue technique, the splenomegaly and anemia characteristic of SFFVA-induced disease have been traced to a DNA fragment of SFFVA containing sequences coding for the env gene product. gp52. The results suggest that the differences in pathogenicity between SFFVP disease and SFFVA disease are an intrinsic property of the env gene products of these two variants of Friend virus, and future studies with the molecular clones of each strain should allow us to map regions of each env gene responsible for common and distinctive features of the erythroproliferative diseases induced by each virus.

Envelope gene sequences which encode the gp52 protein of spleen focus-forming virus are required for the induction of erythroid cell proliferation.

A series of insertion-deletion mutants was constructed in a molecularly cloned DNA copy of the Friend strain of spleen focus-forming virus (SFFV). The mutants were produced by inserting a synthetic oligonucleotide linker containing the recognition sequence of SalI endonuclease into several different locations of the SFFV DNA. Three classes of mutants were isolated: insertion-deletion mutants in the 5' half of the SFFV genome, in the long terminal repeat of the SFFV genome, and in the env gene of the SFFV genome. The env gene mutant has a deletion of sequences shared in common between the env gene of SFFV and the env genes of mink cell focus-inducing murine leukemia viruses. From analyses of the biological activity of the various mutants and a biologically active subgenomic SFFV DNA fragment described herein, we can deduce that the coding sequence encompassing the env gene of SFFV is required for the biological activity. This region, required for the pathogenic phenotype, cannot be larger than 1.5 kilobase pairs, a size only slightly more than that sufficient to encode the nonglycosylated precursor of the gp52 env gene product.

Genomes of murine leukemia viruses isolated from wild mice.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.

Biological activity of the spleen focus-forming virus is encoded by a molecularly cloned subgenomic fragment of spleen focus-forming virus DNA.

A biologically active subgenomic DNA fragment of a polycythemia-inducing strain of the replication-defective spleen focus-forming virus (SFFV) has been molecularly cloned. The SFFV DNA fragment includes 2.0 kilobase pairs (kbp) from the 3' end of SFFV, the long terminal repeat sequences of SFFV, and 0.4 kbp from the 5' end of SFFV. The fragment contains the previously described env-related gene of SFFV. All the properties associated with SFFV can be assigned to this SFFV DNA fragment by using a two-stage DNA transfection assay with infectious helper virus DNA. The virus recovered from the transfection assays can induce erythroblastosis, splenic foci, and polycythemia in infected mice. Fibroblast cultures transfected with the SFFV DNA fragment synthesize gp52, the known intracellular product of the env-related gene of SFFV. gp52 can also be detected in spleens from diseased mice infected with the virus recovered in the two-stage transfection. The results are consistent with the hypothesis that the env-related gene sequences of SFFV and their product gp52 are required for the initiation of SFFV-induced disease.

Recovery of biologically active spleen focus-forming virus from molecularly cloned spleen focus-forming virus-pBR322 circular DNA by cotransfection with infectious type C retroviral DNA.

The genome of the Lilly-Steeves strain of spleen focus-forming virus (SFFV) was molecularly cloned in the plasmid vector pBR322. Infectious SFFV could be recovered by releasing the SFFV DNA from the vector, transfecting the released DNA onto NIH 3T3 cells, and rescuing the SFFV either by superinfection with helper virus or by cotransfection with molecularly cloned infectious helper viral DNA. By using transfections with SFFV DNA still attached to the plasmid vector, infectious SFFV activity could also be recovered with either method of rescue. Studies performed with these latter types of transfections indicated that only a portion of the SFFV genome was required for biological activity. Since gp52, a marker protein for SFFV, could be detected in all cultures from which adequate titers of biologically active SFFV were recovered, the results are consistent with the hypothesis that gp52 is necessary for SFFV-induced erythroblastosis and polycythemia.