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PubMed2005

GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing.

Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein-coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid-induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid-induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D(2) (PGD(2)) and prostaglandin E(2) (PGE(2)) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G-deficient mice restored the nicotinic acid-induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid-induced flushing and that this effect involves release of PGE(2) and PGD(2), most likely from immune cells of the skin.

Adipocytes↗

PubMed1990

Oocyte recovery, maturation and fertilization in vitro in the puma (Felis concolor).

Eight female pumas were treated i.m. with 1000 (N = 5) or 2000 (N = 3) i.u. PMSG followed 84 h later by 800 i.u. hCG. Eggs were recovered 24-26 h after hCG from ovarian follicles by using laparoscopy and transabdominal aspiration. Mature eggs were inseminated in vitro 4-6 h later whereas immature eggs were cultured for 24 h and then inseminated. Electroejaculates from 3 pumas were diluted with mKRB before insemination to evaluate the influence of sperm concentration on fertilization. Seven of 8 pumas responded with follicle development, and 140 eggs were recovered from 145 follicles (96.6%; 77 mature, 43 immature, 20 degenerate eggs; mean +/- s.e.m., 20.0 +/- 5.9 eggs/female). Overall fertilization rate was 43.5% (total eggs fertilized = 40) despite using inseminates containing 82-99% pleiomorphic spermatozoa. Of the 36 immature oocytes matured in vitro and inseminated, 12 were fertilized even though 50% of the inseminating spermatozoa contained an acrosomal defect. Fertilization rate of mature oocytes collected from follicles appeared unrelated (P greater than 0.05) to PMSG dose or number of spermatozoa/inseminate. This study demonstrates that a high proportion of follicular eggs can be recovered laparoscopically from adult pumas treated with PMSG and hCG. These gametes are capable of being fertilized in vitro (immediately or after maturation in vitro) even with low quality semen with a high incidence of sperm pleiomorphisms.

Animals↗

PubMed2005

Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma.

In response to DNA damage, the p53 tumor suppressor can elicit either apoptosis or cell-cycle arrest and repair, but how this critical decision is made in specific cell types remains largely undefined. We investigated the mechanism by which the transcriptional repressor Slug specifically rescues hematopoietic progenitor cells from lethal doses of gamma radiation. We show that Slug is transcriptionally induced by p53 upon irradiation and then protects the damaged cell from apoptosis by directly repressing p53-mediated transcription of puma, a key BH3-only antagonist of the antiapoptotic Bcl-2 proteins. We established the physiologic significance of Slug-mediated repression of puma by demonstrating that mice deficient in both genes survive doses of total-body irradiation that lethally deplete hematopoietic progenitor populations in mice lacking only slug. Thus, Slug functions downstream of p53 in developing blood cells as a critical switch that prevents their apoptosis by antagonizing the trans-activation of puma by p53.

Animals↗

PubMedDate not supplied

Large-scale quantitative proteomic study of PUMA-induced apoptosis using two-dimensional liquid chromatography-mass spectrometry coupled with amino acid-coded mass tagging.

By coupling two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) with amino acid-coded mass tagging (AACT), we have greatly increased the analytical throughput and sequence coverage of MS-based methods for proteome-wide quantitation. The dynamic range and reproducibility of this 2D-LC-AACT quantitative approach were evaluated by profiling the mixtures with different ratios of E. coli cells grown in either regular or AACT medium. A SQL-based high thoughput MASCOT data analysis tool was developed for proteomic data sorting and mining. We investigated the early stage of apoptosis by inducing the p53 upregulated modulator of apoptosis (PUMA) through the analyses of the relative ratios of the pairwise isotope signals that were originated from the control and labeled PUMA-induced cells. In 20-hour 2D-LC-MS/MS run, 480 proteins were conclusively identified, and more than half of them were quantified. A noteworthy change in the quantitative profile was that histones and a ubiquitin conjugate protein UBC9, which are involved in DNA double-strand break (DSB) repair were significantly down-regulated in the PUMA-overexpressing apoptotic cells, suggesting the detection of DSB in the apoptotic process. The quantitative profiling efficiency of this approach was compared with the gel-based quantitative analysis scheme.

Amino Acids↗

PubMed2005

Chronic lymphocytic leukemia cells display p53-dependent drug-induced Puma upregulation.

We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.

Antineoplastic Agents↗

PubMed2005

(D)-beta-Hydroxybutyrate inhibits adipocyte lipolysis via the nicotinic acid receptor PUMA-G.

As a treatment for dyslipidemia, oral doses of 1-3 grams of nicotinic acid per day lower serum triglycerides, raise high density lipoprotein cholesterol, and reduce mortality from coronary heart disease (Tavintharan, S., and Kashyap, M. L. (2001) Curr. Atheroscler. Rep. 3, 74-82). These benefits likely result from the ability of nicotinic acid to inhibit lipolysis in adipocytes and thereby reduce serum non-esterified fatty acid levels (Carlson, L. A. (1963) Acta Med. Scand. 173, 719-722). In mice, nicotinic acid inhibits lipolysis via PUMA-G, a Gi/o-coupled seven-transmembrane receptor expressed in adipocytes and activated macrophages (Tunaru, S., Kero, J., Schaub, A., Wufka, C., Blaukat, A., Pfeffer, K., and Offermanns, S. (2003) Nat. Med. 9, 352-355). The human ortholog HM74a is also a nicotinic acid receptor and likely has a similar role in anti-lipolysis. Endogenous levels of nicotinic acid are too low to significantly impact receptor activity, hence the natural ligands(s) of HM74a/PUMA-G remain to be elucidated. Here we show that the fatty acid-derived ketone body (D)-beta-hydroxybutyrate ((D)-beta-OHB) specifically activates PUMA-G/HM74a at concentrations observed in serum during fasting. Like nicotinic acid, (D)-beta-OHB inhibits mouse adipocyte lipolysis in a PUMA-G-dependent manner and is thus the first endogenous ligand described for this orphan receptor. These findings suggests a homeostatic mechanism for surviving starvation in which (D)-beta-OHB negatively regulates its own production, thereby preventing ketoacidosis and promoting efficient use of fat stores.

3-Hydroxybutyric Acid↗

PubMed2003

Domestic cats infected with lion or puma lentivirus develop anti-feline immunodeficiency virus immune responses.

Attenuated live viral strains have afforded significant protection against virus challenge in HIV vaccine models. Although both cellular and humoral immunity are assumed to be vital for protection, specific parameters consistently associated with control of infection have been elusive. Our previous studies have shown that lentiviruses from 2 nondomestic feline species--lion (Pathera leo) and puma (Felis concolor)--persistently but nonpathogenetically infect domestic cats (Felis domestica). Moreover, infection with either the puma lentivirus (PLV) or lion lentivirus (LLV) conferred partial protection against superinfection with virulent feline immunodeficiency virus (FIV), the feline equivalent of HIV. To determine whether domestic cats infected by the lentiviruses of pumas or lions generate cross-reactive immune responses, we infected groups of 5 domestic cats with PLV, LLV, or a sham control and then monitored virus load, hematologic parameters, antibody protection, proliferative responses, and the ability of blood mononuclear cells to inhibit LLV, PLV, and FIV replication in vitro. All cats inoculated with LLV or PLV developed persistent infection, and low-level cell-associated viremia has been previously described. Infected cats also generated robust antibody titers and lymphocytes that proliferated in response to viral antigens and downregulated PLV, LLV, and FIV replication in vitro. This latter activity was CD8 cell associated for PLV and LLV inhibition but not for FIV inhibition. Thus, cats infected with the phylogenetically more ancient and less pathogenic feline lentiviruses generated humoral and cell-mediated immune responses reactive against both the homologous viruses and the heterologous FIV of domestic cats, which correlated with decreased viral load. These results are analogous to protection studies with attenuated primate immunodeficiency viruses and provide a system by which to examine adaptation, interference, and cross protection among lentiviruses.

Animals↗

PubMed1978

Evidence for an in vivo conformational change in ribulose bisphosphate carboxylase-oxygenase from Puma rye during cold adaptation.

Ribulose bisphosphate carboxylase-oxygenase (RUBPCase) from leaves of cold-hardened and unhardened Puma rye was purified by gel filtration and ion exchange chromatography. Chemical properties that might be associated with a previously demonstrated difference in molecular charge of purified RUBPCase from cold-hardened and unhardened Puma rye were investigated. Amino acid analyses indicated no significant differences in amino acid composition or average hydrophobicity per residue. Enzymes from hardened and unhardened rye were reversibly cold inactivated at 0 degrees C. However, the former was more stable at this temperature than the latter. Titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) indicated the presence of fast and slow titrating sulfhydryl groups in both enzyme preparations but there were 50% fewer SH groups titrated in the enzyme from hardened rye in 30 min that in the enzyme from unhardened rye. Activation of both enzymes by HCO-3 enhanced the reactivity of sulfhydryl groups to titration with DTNB. According to the kinetics of the slow titrating SH groups, the enzyme from hardened rye was less susceptible to denaturation by sodium dodecyl sulfate than was the same enzyme from unhardened rye. It is concluded that the tertiary structure of RUBPCase from Puma rye is affected during low-temperature adaptation.

Adaptation, Physiological↗

PubMed2006

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma.

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

Animals↗

PubMed2004

p53 activation domain 1 is essential for PUMA upregulation and p53-mediated neuronal cell death.

The p53 tumor suppressor gene has been implicated in the regulation of apoptosis in a number of different neuronal death paradigms. Because of the importance of p53 in neuronal injury, we questioned the mechanism underlying p53-mediated apoptosis in neurons. Using adenoviral-mediated gene delivery, reconstitution experiments, and mice carrying a knock-in mutation in the endogenous p53 gene, we show that the transactivation function of p53 is essential to induce neuronal cell death. Although p53 possesses two transactivation domains that can activate p53 targets independently, we demonstrate that the first activation domain (ADI) is required to drive apoptosis after neuronal injury. Furthermore, the BH3-only proteins Noxa and PUMA exhibit differential regulation by the two transactivation domains. Here, we show that Noxa can be induced by either activation domain, whereas PUMA induction requires both activation domains to be intact. Unlike Noxa, the upregulation of PUMA alone is sufficient to induce neuronal cell death. We demonstrate, therefore, that the first transactivation domain of p53 is indispensable for the induction of neuronal cell death.

Amino Acid Motifs↗

PubMed1994

Gonadotrophin dose and timing of anaesthesia for laparoscopic artificial insemination in the puma (Felis concolor).

Ovarian response to equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG), the effect of timing of anaesthesia relative to hCG injection and the use of laparoscopic intrauterine artificial insemination were examined in the puma (Felis concolor). In Expt 1, females were treated with 100 (n = 6) or 200 (n = 8) IU eCG (i.m.) followed 80 h later by 100 IU hCG (i.m.) and were then anaesthetized 40-43 h after hCG injection for ovarian assessment. Although there was no difference (P > 0.05) in the number of unovulated ovarian follicles, females treated with 200 IU eCG had more (P < 0.05) corpora lutea per female and more corpora lutea as a percentage of the total number of ovarian structures. In Expt 2, all females were treated with 200 IU eCG and 80 h later with 100 IU hCG, and then anaesthetized either 31-39 h (Group A; n = 8) or 41-50 h (Group B; n = 6) after hCG injection for ovarian assessment. All Group B pumas ovulated compared with only three (37.5%) Group A females (P < 0.05). Compared with Group A, Group B pumas had more corpora lutea per female, more corpora lutea as a percentage of the total number of ovarian structures, and fewer unovulated follicles (P < 0.05). One of nine post-ovulatory females laparoscopically inseminated in utero with 16 x 10(6) motile spermatozoa became pregnant and delivered a healthy cub.(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, General↗

PubMed2003

Evaluation of an indirect enzyme-linked immunosorbent assay for the detection of feline lentivirus-reactive antibodies in wild felids, employing a puma lentivirus-derived synthetic peptide antigen.

An enzyme-linked immunosorbent assay (ELISA) using a puma lentivirus-derived synthetic peptide as coating antigen was evaluated as a diagnostic test for infection with feline immunodeficiency virus (FIV) or related lentiviruses in free-ranging lions. The sensitivity and specificity of the ELISA was determined using two approaches. In the first approach, the results were standardized according to certain statistical criteria, and in the second, the puma lentivirus western blot was used as the gold standard. The sensitivity of the test when compared with the standardized results was 85.4% and the specificity 100%. The sensitivity of the test when using the western blot as the gold standard was 78.6% and the specificity 100%. The test would therefore be well-suited to the screening of populations of wild felids in which FIV or related lentiviruses are endemic. The results also indicate that in spite of genetic divergence between lentiviruses isolated from Panthera and Felis spp., puma lentivirus-derived antigens can be used in immunoassays for the detection of antibodies in Panthera spp. reactive to FIV or related lentiviruses. The results also indicate that the lion population in the Hluhluwe-Umfolozi Game Reserve, South Africa is lentivirus negative.

Animals↗

PubMed2006

Ectoparasites of free-ranging pumas and jaguars in the Paraguayan Chaco.

Ectoparasites were collected from seven puma (Puma concolor) and seven jaguar (Panthera onca) live-captures (each representing six different animals) in the Paraguayan Chaco from 2002 to 2004. The same five species of ectoparasites were recovered from both host species: the flea, Pulex simulans (total on both hosts combined=30 male, 49 female), and the ticks, Amblyomma cajennense (1 male, 4 female, 46 nymphs, 241 larvae), Amblyomma parvum (42 male, 25 female), Amblyomma tigrinum (1 male, 7 female, 34 larvae) and Amblyomma triste (4 male). There were no statistical differences between the prevalences (percent of hosts infested) for any of these ectoparasite species between the two host species, and only P. simulans showed a significantly higher mean intensity (mean no. of each ectoparasite species per infested host) on one of the host species (jaguar). Clearly, these two large carnivores share similar ectoparasite faunas in the Paraguayan Chaco. However, both carnivores occupy large geographical distributions in the New World and this study, combined with the few previous studies, suggests that their ectoparasite faunas differ slightly in different parts of their respective ranges.

Animals↗

PubMed2005

Puma lentivirus is controlled in domestic cats after mucosal exposure in the absence of conventional indicators of immunity.

A high percentage of free-ranging pumas (Felis concolor) are infected with feline lentiviruses (puma lentivirus, feline immunodeficiency virus Pco [FIV-Pco], referred to here as PLV) without evidence of disease. PLV establishes productive infection in domestic cats following parenteral exposure but, in contrast to domestic cat FIV, it does not cause T-cell dysregulation. Here we report that cats exposed to PLV oro-nasally became infected yet rapidly cleared peripheral blood mononuclear cell (PBMC) proviral load in the absence of a correlative specific immune response. Two groups of four specific-pathogen-free cats were exposed to PLV via the mucosal (oro-nasal) or parenteral (i.v.) route. All animals were PBMC culture positive and PCR positive within 3 weeks postinfection and seroconverted without exhibiting clinical disease; however, three or four oro-nasally infected animals cleared circulating proviral DNA within 3 months. Antibody titers reached higher levels in animals that remained persistently infected. PLV antigen-induced proliferation was slightly greater in mucosally inoculated animals, but no differences were noted in cytotoxic T-lymphocyte responses or cytokine profiles between groups. The distribution of virus was predominantly gastrointestinal as opposed to lymphoid in all animals in which virus was detected at necropsy. Possible mechanisms for viral clearance include differences in viral fitness required for crossing mucosal surfaces, a threshold dose requirement for persistence, or an undetected sterilizing host immune response. This is the first report of control of a productive feline or primate lentivirus infection in postnatally exposed, seropositive animals. Mechanisms underlying this observation will provide clues to containment of immunodeficiency disease and could prompt reexamination of vaccine-induced immunity against human immunodeficiency virus and other lentiviruses.

Animals↗

PubMed1994

Nucleotide sequence analysis of puma lentivirus (PLV-14): genomic organization and relationship to other lentiviruses.

The complete nucleotide sequence of an isolate of puma lentivirus (PLV-14) was obtained by an inverse polymerase chain reaction (I-PCR) technique and confirmed by conventional PCR. Both methods were used to amplify overlapping regions of proviral DNA, for cloning and sequencing, from genomic DNA isolated from PLV-14 infected Florida puma (Felis concolor coryi) peripheral blood mononuclear cells (PBMC). The provirus has a total length of 9100 nucleotides and the genomic organization of presumed protein coding regions are similar to those seen in other members of the lentivirus family, i.e., three large open reading frames gag, pol, and env as well as smaller intergenic regions that apparently encode regulatory proteins vif and 3' rev by positional and sequence similarity to those seen in other lentiviruses. Two additional open reading frames were identified in the env region and their function (if any) is unknown. The length of the PLV-14 long terminal repeat (LTR) was found to be shorter than the LTRs of feline immunodeficiency virus (FIV). The sequence homology between PLV-14 and other lentiviruses demonstrates that PLV-14 is most closely related to FIV from domestic cats. However, the extent of sequence divergence of each retroviral gene segment is large (e.g., percentage sequence similarity between FIV and PLV-14 env is 8% amino acid and 37% nucleotide similarity), indicating relatively ancient divergence of these feline lentiviral genomes.

Amino Acid Sequence↗

PubMed1997

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Animals↗

PubMed2003

Purification and characterization of an extracellular non-aspartyl acid protease (pumAe) from Ustilago maydis.

The proteinase pumAe was purified to homogeneity from haploid U. maydis FB1 growing on acid mineral medium. The purification procedure consisted of ammonium sulfate fractionation and gel filtration chromatography, resulting in a 7.7% recovery and a 15.1-fold increase in specific activity. The molecular weight of the enzyme was estimated to be 72 kDa and 74 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 4.0 and at 45 degrees C toward hemoglobin, and the pI was determined to be 5.5. The effects of six protease inhibitors on pumAe were tested, and no inhibitory effect was observed. The pure enzyme degraded gelatin and albumin, but casein and collagen were not degraded. The Km value was 3.5 microM, and the Vmax value was 11,430 micromol h(-1) mg(-1) for Suc-R-P-F-H-L-L-V-Y-MCA.

Albumins↗

PubMed2005

Slugging it out: fine tuning the p53-PUMA death connection.

In response to DNA damage, the tumor suppressor p53 elicits a complex cellular response. In this issue of Cell, Wu et al. (2005) show that the transcription factor SLUG is induced by p53 and protects hematopoietic progenitor cells from apoptosis triggered by DNA damage. SLUG exerts this protective role by repressing Puma, a proapoptotic target of p53. PUMA is also a key coordinator of apoptosis mediated by both nuclear and cytoplasmic functions of p53 (Chi-puk et al., 2005).

Animals↗