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PubMed2003

No PUMA, no death: implications for p53-dependent apoptosis.

More than a decade ago, it was found that one of the two essential physiological functions of p53 is to selectively destroy stressed cells through apoptosis. Despite the large number of studies describing p53-dependent apoptosis since then, how p53 turns on the apoptotic switch has remained enigmatic. In this issue of Cancer Cell, Jeffers et al. report that knockout of PUMA, a recently identified BH3-only Bcl-2 family protein, recapitulates virtually all apoptotic deficiency in p53 knockout mice. Their results indicate that PUMA is an essential mediator of p53-dependent and -independent apoptosis in vivo.

Animals↗

PubMed2006

Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways.

The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well-known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.

Adenovirus E1A Proteins↗

PubMed1992

Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye.

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.

Journal Article↗

PubMed1978

A skeletal growth defect in the puma (Felis concolor).

Clinical and radiological signs shown by a young deformed puma cub, and the pathological findings following its euthanasia at eight months, are presented. These findings do not resemble those in the recognised nutritional bone diseases of the growing carnivore, but closely resemble an hereditary disease of the dog and, at least in one respect, an hereditary disease in man. This case may be an example of hereditary chondrodysplastic dwarfism in the puma. An unusual finding was the presence of iron in the cartilage of abnormal growth plates.

Animals↗

PubMed2005

Historical and other patterns of monomethyl and inorganic mercury in the Florida panther (Puma concolor coryi).

Since the late 1980s, elevated levels of mercury have been reported in the tissues of the Florida panther (Puma concolor coryi) from the Florida Everglades. The extent, degree, and length of time of mercury contamination in the Florida panther are unknown. The objective of this study was to determine the historical and other patterns of monomethyl and inorganic mercury in the Florida panther by analysis of mercury in panther hair from museum collections. In addition, this study evaluated the effects of preservation of skins on mercury concentrations in hair and the representativeness of museum collections for evaluating historical trends of contamination in the Florida panther. Hair from 42 Florida panther specimens collected from 1896 to 1995 was analyzed for both monomethyl and inorganic mercury. Monomethyl mercury (MMHg) and inorganic mercury (IHg) were found in all specimens. Monomethyl mercury in hair from untanned skins was significantly higher than MMHg in hair from tanned skins. For untanned specimens, the mean MMHg concentration in hair was 1.62 +/- 1.87 mug/g (range 0.11 to 6.68 mug/g, n = 16). Monomethyl mercury accounted for 88% of the total mercury in untanned Florida panther hair. No sexual or geographical differences were found. Although MMHg is generally stable in hair, the tanning process appears to reduce the amount of MMHg in hair. In addition, exogenous IHg contamination of the panther hair was found in museum specimens, especially in older specimens. The implication of these and other factors in interpreting results of museum studies is discussed. The presence of MMHg in panther hair since the 1890s indicates long-term and widespread exposure of the Florida panther to mercury. Levels of MMHg are significantly greater in the 1990s than the 1890s. When combined with field studies of mercury in the Florida panther, considerable individual variability is observed, reflecting short-term changes in exposure of individual panthers to mercury. Although museum specimens showed a significant increase in MMHg over the last 100 years, they did not show the magnitude of increase that field populations of Florida panthers did. A number of Florida panthers appeared to be at risk from mercury over their lifetimes, especially individuals from the early 1990s.

Animals↗

PubMed2005

Suspected secondary thiafentanil intoxication in a captive mountain lion (Puma concolor).

Inadvertent ingestion of thiafentanil oxalate by a captive adult female mountain lion (Puma concolor) caused a prolonged clinical syndrome that included sedation and depression, muscle tension, and myopathy that was incompletely antagonized by naltrexone HCl. A serum chemistry profile revealed markedly elevated creatinine phosphokinase (CK; 490,450 IU/l), alanine aminotransferase (ALT; 1,896 IU/l), and aspartate aminotransferase (AST; 4,321 IU/l) 2 days after onset. The affected animal's condition gradually improved over the next 15 days in response to supportive therapy that included diazepam (5 mg as needed), Normasol R (3 l/day), dexamethasone (tapering dose starting at 1 mg/kg), and ketoprofen (1 mg/kg). She eventually recovered completely. Based on these observations, carcasses of animals immobilized with thiafentanil should be marked and disposed of properly to preclude opportunities for secondary exposure and potential intoxication in scavenging species. In addition, caution is advised when using thiafentanil in animals that could be preyed upon before full metabolism of the drug.

Alanine Transaminase↗

PubMed2006

Gastrointestinal helminths of free-ranging Florida panthers (Puma concolor coryi) and the efficacy of the current anthelmintic treatment protocol.

Thirty-five Florida panthers (Puma concolor coryi [Bangs, 1899]) collected from six counties in southern Florida between 1978 and 2003 were examined at necropsy for gastrointestinal helminths. The panthers were placed into two groups: 1) treated with anthelmintics (n = 17), and 2) untreated (n = 18). Nine species of helminths (one trematode, six nematodes, and two cestodes) were identified in the untreated panthers. The most prevalent helminths were Alaria marcianae (LaRue, 1917) (100%), Spirometra mansonoides (Mueller, 1935) (91%), and Ancylostoma pluridentatum (Alessandrini, 1905) (89%). Ancylostoma caninum (Ercolani, 1859) is reported from the Florida panther for the first time. The intensities of helminths with prevalences >10% did not differ between untreated panthers collected in 1978-1983 and 1996-2003. Treated panthers had helminth faunas similar to those of untreated panthers. The current anthelmintic treatment being used reduced the intensity of both A. marcianae and A. pluridentatum in panthers < or =6 mo posttreatment (PT); however, treated panthers between 6 and 9 mo PT, and >9 mo PT were similar to untreated panthers. Treatment was less effective on S. mansonoides and Taenia omissa Lühe, 1910. Treated panthers had slightly lower intensities of S. mansonoides at < or =6 mo PT; however, between 6 and 9 mo PT and >9 mo PT they had significantly higher intensities than untreated panthers. At all periods PT, the intensity of T. omissa for the treated panthers was similar to that of untreated panthers. We suggest that Mesocestoides sp. may not be present in the Florida panther population as reported earlier by Forrester et al. (1985), due to parasite misidentification by those authors.

Animals↗

PubMed1994

Recombinant polypeptides transplanted from pUMA vector-derived cRNAs are translocated through microsomal membranes and exported out of frog oocytes.

pSP64 derivatives were constructed to obtain cloning vectors suitable for in vitro transcription and subsequent in vitro synthesis of recombinant proteins equipped with N-terminal signal sequences. The amino acid sequence of the signal peptide was adapted and slightly modified from the one occurring in the neural cell adhesion molecule, NCAM. Its ability to direct recombinant proteins into secretory pathways was tested by in vitro translation in microsomal membrane-containing reticulocyte lysates and by injection of the pUMA-derived cRNAs into Xenopus laevis oocytes.

Amino Acid Sequence↗

PubMed2005

DeltaNp73 antisense activates PUMA and induces apoptosis in neuroblastoma cells.

The p73 gene codes for various different protein isoforms. They include proteins expressed under the control of the P1 promoter that contain a transactivation domain and are similar in function to p53 (TAp73 isoforms), as well as proteins regulated by the P2 promoter that lack this domain and function as dominant negative inhibitors of TAp73 and p53 (DeltaNp73 isoforms). Whereas TAp73 functions as a tumor suppressor with pro-apoptotic function, DeltaNp73 is likely to prevent the induction of apoptosis in tumor cells and to participate in oncogenesis. Here we used a loss-of-function strategy to assess the role of DeltaNp73 in SH-SY5Y neuroblastoma cells. An antisense oligonucleotide designed to target DeltaNp73 mRNA, but not TAp73, was used to effectively downregulate this transcript. DeltaNp73 downregulation was accompanied by increased levels of the pro-apoptotic BH3 family member PUMA at the mRNA and protein level, and by conformational activation of BAX which translocated to mitochondria. These DeltaNp73 antisense-mediated alterations led to the induction of apoptosis as detected by decreased cell viability, augmented DNA fragmentation and increased caspase-3 activity in cell lysates. Our results demonstrate the cytoprotective role of DeltaNp73 in neuroblastoma and suggest its use as a target for molecular intervention therapy.

Apoptosis↗

PubMed2005

Activation of p53 and the pro-apoptotic p53 target gene PUMA during depolarization-induced apoptosis of chromaffin cells.

The pathogenesis of non-glutamatergic, depolarization-induced cell death is still enigmatic. Recently, we have shown that veratridine induces apoptosis in chromaffin cells, and we have demonstrated protective effects of antioxidants in this system, suggesting a role for Na+ channels and oxidative stress in depolarization-induced cell death. We examined the possible contribution of p53, a transcription factor that has a major role in determining cell fate, and the mitochondrial apoptosis pathway in veratridine-induced cell death of cultured bovine chromaffin cells. Nuclear condensation and fragmentation were detected several hours after a 60-min exposure to 30 microM veratridine. Apoptosis was associated with a transitory increase in p53 protein levels. Veratridine induced transcription of the pro-apoptotic p53 target gene PUMA, but not of bax or pig3. Using transient transfection experiments, we found that wild-type p53, but not the mutant form p53-273H, was sufficient to induce cell death in the chromaffin cells, which was caspase-9 dependent. The down-regulation of either p53, by overexpressing p53-273H, or caspase-9 activity using a dominant-negative caspase-9 mutant protected chromaffin cells against veratridine-induced toxicity. Our data demonstrate the importance of p53 and the downstream activation of the mitochondrial apoptosis pathway in depolarization-induced apoptosis.

Animals↗

PubMed2005

Measurement and modelling of air pollution and atmospheric chemistry in the U.K. West Midlands conurbation: overview of the PUMA Consortium project.

The PUMA (Pollution of the Urban Midlands Atmosphere) Consortium project involved intensive measurement campaigns in the Summer of 1999 and Winter of 1999/2000, respectively, in which a wide variety of air pollutants were measured in the UK West Midlands conurbation including detailed speciation of VOCs and major component analysis of aerosol. Measurements of the OH and HO2 free radicals by the FAGE technique demonstrated that winter concentrations of OH were approximately half of those measured during the summer despite a factor of 15 reduction in production through the photolysis of ozone. Detailed box modelling of the fast reaction chemistry revealed the decomposition of Criegee intermediates formed from ozone-alkene reactions to be responsible for the majority of the formation of hydroxyl in both the summer and winter campaigns, in contrast to earlier rural measurements in which ozone photolysis was predominant. The main sinks for hydroxyl are reactions with NO2, alkenes and oxygenates. Concentrations of the more stable hydrocarbons were found to be relatively invariant across the conurbation, but the impacts of photochemistry were evident through analyses of formaldehyde which showed the majority to be photochemical in origin as opposed to emitted from road traffic. Measurements on the upwind and downwind boundaries of the conurbation revealed substantial enhancements in NOx as a result of emissions within the conurbation, especially during westerly winds which carried relatively clean air. Using calcium as a tracer for crustal particles, it proved possible to reconstruct aerosol mass from the major chemical components with a fairly high degree of success. The organic to elemental carbon ratios showed a far greater influence of photochemistry in summer than winter, presumably resulting mainly from the greater availability of biogenic precursors during the summer campaign. Two urban airshed models were developed and applied to the conurbation, one Eulerian, the other Lagrangian. Both were able to give a good simulation of concentrations of both primary and secondary pollutants at urban background locations.

Acetone↗

PubMed2002

Nonpathogenic lion and puma lentiviruses impart resistance to superinfection by virulent feline immunodeficiency virus.

Lion lentivirus (LLV) and puma lentivirus (PLV) exist as highly divergent virus clades among populations of indigenously infected nondomestic Felidae. The feline immunodeficiency virus (FIV) is highly divergent from LLV and PLV and is pathogenic for domestic cats. When domestic cats are infected with LLV or PLV, they have immunologically and clinically silent persistent infections. We examined whether LLV or PLV infection might impart resistance to FIV superinfection in vitro by infecting domestic cat lymphoid cells with PLV and assessing resistance of these cells to FIV. We found that infection with FIV was highly restricted by prior established PLV infection. To examine whether this resistance applied in vivo, domestic cats were asymptomatically infected with either LLV or PLV and then challenged with pathogenic FIV. Although all cats became infected with FIV, prior LLV or PLV exposure blunted CD4 + cell depletion and suppressed plasma and peripheral blood mononuclear cell FIV loads relative to FIV-challenged controls not infected with LLV or PLV, despite the lack of prechallenge neutralizing antibody activity against FIV. Thus, as compared with naive controls, cats previously infected with LLV or PLV were able to more effectively control FIV infection and resist its immunologic effects, despite the substantial genetic divergence between these lentiviruses-raising the possibility that superinfection may impart resistance to lentivirus infection by heightening innate immune mechanisms.

Animals↗

PubMed1997

Infectivity of lion and puma lentiviruses for domestic cats.

Infection of domestic cats with feline immunodeficiency virus (FIV) causes progressive immunological deterioration similar to that caused by human immunodeficiency virus (HIV). Lentiviruses related to but phylogenetically distinct from FIV have been detected in several non-domestic feline species. Serological cross-reactivity of these viruses raises the question as to whether inter-species transmission may occur. To address this issue, we asked whether lion lentivirus (FIV-Ple) or two strains of puma lentivirus (FIV-Pco) could replicate or cause disease in domestic cats. We found that domestic cats inoculated with FIV-Ple developed persistent cell-associated viraemia, transient cell-free viraemia and antiviral antibody. Clinical disease was not detected throughout a 6 month observation period. Two of four cats inoculated with FIV-Pco developed cell-associated viraemia, seroconverted and exhibited transient lymphadenopathy. No changes in white blood cell parameters or other haematological abnormalities were detected in any of the infected cats. Virus-specific RNA was detected in co-cultivated lymphocytes of all infected cats by RT-PCR. These findings reveal that non-domestic cat lentiviruses are infectious for domestic cats and can establish persistent infection in the absence of disease.

Animals↗

PubMed1986

Mycoplasma felifaucium, a new species isolated from the respiratory tract of pumas.

Mycoplasmas isolated from the throats of three pumas (Felis concolor) were each cloned and examined in detail. All three were serologically and biologically indistinguishable from each other, and were serologically distinct from 83 recognized Mycoplasma and Acholeplasma species. They were designated as a new species, Mycoplasma felifaucium, with strain PU (NCTC 11703) as the type strain.

Animals↗

PubMed1982

Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma).

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding beta-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of beta-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.

Journal Article↗

PubMed1983

Isolation and Identification of Plasma Membrane from Light-Grown Winter Rye Seedlings (Secale cereale L. cv Puma).

An effective method for the isolation of plasma membrane from light-grown winter rye seedlings (Secale cereale L. cv Puma) was established using a liquid two-polymer phase separation. The conditions for the specific partition of plasma membrane into the polyethylene glycol-enriched upper phase were examined, including variations in the polymer concentration, buffer system, pH, and NaCl addition in the phase partition system. The most effective phase partition system for the isolation of plasma membrane from winter rye consisted of 5.6/5.6% (w/w) polyethylene glycol 4000/dextran T500 in 0.25 molar sucrose-10 millimolar potassium phosphate-30 millimolar NaCl (pH 7.8), repeated once. When the isolated plasma membrane was centrifuged on a linear sucrose density gradient, a single band was found at the 34% (w/w) sucrose layer (1.141 grams per cubic centimeter) which co-fractionated with the pH 6.5-ATPase.Identification of plasma membrane was performed by the combination of phosphotungstic acid-chromic acid stain and specific binding of N-1-naphthylphthalamic acid. Based on morphometrical observations after phosphotungstic acid-chromic acid stain, the isolated plasma membrane consisted mostly of vesicles of high purity. The isolated plasma membrane also showed extremely high specificity for N-1-naphthylphthalamic acidbinding, 10-fold higher than other membranes. It was also confirmed that there is a distinct difference in properties between plasma membrane and other membranes. The endomembranes such as from chloroplasts, mitochondria, and endoplasmic reticulum were observed to be highly sensitive to Zn(2+) ion and lower pH, which resulted in an abrupt aggregation of membranes. On the contrary, plasma membrane was very stable to these treatments and no aggregation was observed. These unique properties of isolated plasma membrane are generally observed in a wide variety of plant species and can be utilized for the assessment of the purity of preparations of isolated plasma membranes and for their identification.

Journal Article↗

PubMed1984

Involvement of Plasma Membrane Alterations in Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma).

Using plasma membranes with high purity isolated from winter rye seedlings (Secale cereale L. cv Puma) by an aqueous two-polymer phase partition technique, lipid and protein changes were determined during the development of cold hardiness.The degree of fatty acid unsaturation and proportions of phospholipid classes changed only slightly during cold acclimation. A small change, however, was observed in sterol composition; beta-sitosterol increased gradually with a corresponding decrease in campesterol plus stigmasterol during cold acclimation. The phospholipid to protein ratio increased proportionally to the increase in cold hardiness. During the period of acclimation, the sterol to phospholipid ratio declined and was inversely related to the development of cold hardiness.A distinct difference between polypeptide components of nonacclimated and acclimated plasma membrane was observed on two-dimensional sodium dodecyl sulfate slab polyacrylamide gels. A change was also detected in glycopeptides. These results suggest that the plasma membrane is in a dynamic state during cold acclimation, changing in response to the development of cold hardiness.

Journal Article↗

PubMed1987

Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma).

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free beta-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing beta-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.

Journal Article↗