Carcinogen and microsomal membrane interactions: changes in membrane density and ability to bind nucleic acids.

DNA and synthetic copolymer polyribocytidylic-polyriboguanylic acid bind to microsomal membrane. The nucleic acid-membrane complex may be detected by centrifugation in cesium chloride density gradients. The density of the nucleic acid-membrane complex and, in certain cases, the amount of nucleic acid associated with the membrane was changed in the presence of various carcinogenic chemicals.

Activity pattern of medium and large sized mammals and density estimates of Cuniculus paca (Rodentia: Cuniculidae) in the Brazilian Pampa.

Between July 2014 and April 2015, we conducted weekly inventories of the circadian activity patterns of mammals in Passo Novo locality, municipality of Alegrete, southern Brazil. The vegetation is comprised by a grassy-woody steppe (grassland). We used two camera traps alternately located on one of four 1 km transects, each separated by 1 km. We classified the activity pattern of species by the percentage of photographic records taken in each daily period. We identify Cuniculus paca individuals by differences in the patterns of flank spots. We then estimate the density 1) considering the area of riparian forest present in the sampling area, and 2) through capture/recapture analysis. Cuniculus paca, Conepatus chinga and Hydrochoerus hydrochaeris were nocturnal, Cerdocyon thous had a crepuscular/nocturnal pattern, while Mazama gouazoubira was cathemeral. The patterns of circadian activity observed for medium and large mammals in this Pampa region (southern grasslands) may reflect not only evolutionary, biological and ecological affects, but also human impacts not assessed in this study. We identified ten individuals of C. paca through skin spot patterns during the study period, which were recorded in different transects and months. The minimum population density of C. paca was 3.5 individuals per km2 (resident animals only) and the total density estimates varied from 7.1 to 11.8 individuals per km2, when considering all individuals recorded or the result of the capture/recapture analysis, respectively.

Activation of mitomycin C by NADPH:cytochrome P-450 reductase.

Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway.

Home range and density of three sympatric felids in the Southern Atlantic Forest, Brazil.

Home range and minimal population densities of Southern tiger cat (Leopardus guttulus), margay (Lepardus wiedii) and jaguarundi (Puma yagouaroundi) were estimated between 2005 and 2006 in Taquari Valley, near the southern edge of the Atlantic Rainforest in Brazil. Home range data were collected by conventional radio telemetry (VHF) locations in a highly fragmented landscape. The average home range size, calculated using 95% kernel density estimates, was 16.01 km2 for Southern tiger cat, 21.85 km2 for margay and 51.45 km2 for jaguarundi. Telemetry data were used to obtain minimal density estimates of 0.08 Southern tiger cats / km2, and 0.04 jaguarundi / km2. The density estimates arise from areas where ocelot (Leopardus pardalis) and other larger-bodied carnivores were locally extinct, and they suggest a specific type of mesopredator release known as the ocelot effect, which is likely enabling the increase in smaller felid populations in this area.

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes.

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. 97 +/- 2% of the total membrane phosphorus is accounted for as phospholipid phosphorus. Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been indentified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present in the 49 000-60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences. Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight. Membrane phosphorus was distributed between two chromatographic fractions: one containing membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.

Mixed-function amine oxidase of the rat hepatocyte nuclear envelope. Demonstration and effects of phenobarbital and 3-methylcholanthrene.

Mixed-function amine oxidase (EC 1.14.13.8) has been demonstrated in highly purified rat hepatocyte nuclear envelope . The enzyme was present in the nuclear envelope at a level 20 percent of that observed in microsomes. Induction studies indicated that nuclear envelope amine oxidase as well as its microsomal counterpart were refractory to the effects of phenobarbital and 3-methylcholanthrene. Phenobarbital administration increased the specific activity of the microsomal N, N-dimethylaniline N-demethylase and benzo[a]pyrene hydroxylase by 600 and 190 percent, respectively, but decreased the specific activity of the nuclear enzymes by 30-50 percent. In contrast, 3-methylcholanthrene increased the specific activity of benzo[a]pyrene hydroxylase in nuclear envelope and microsomes by 42- and 11-fold, respectively. The hydrocarbon also increased the microsomal and nuclear N, N-dimethylaniline N-demethylase by 40 and 60 percent, respectively, but the specific activity of microsomal and nuclear aniline 4-hydroxylase was decreased by 50 percent. Demonstration of amine oxidase in rat hepatocyte nuclear envelope implicates this enzyme in the toxicity and carcinogenicity of certain drugs and chemicals.

Activity pattern of medium and large sized mammals and density estimates of Cuniculus paca (Rodentia: Cuniculidae) in the Brazilian Pampa / Padrão de atividade de mamíferos de médio e grande porte e estimativa da densidade de Cuniculus paca (Rodentia: Cuniculidae) no Pampa brasileiro

Abstract Between July 2014 and April 2015, we conducted weekly inventories of the circadian activity patterns of mammals in Passo Novo locality, municipality of Alegrete, southern Brazil. The vegetation is comprised by a grassy-woody steppe (grassland). We used two camera traps alternately located on one of four 1 km transects, each separated by 1 km. We classified the activity pattern of species by the percentage of photographic records taken in each daily period. We identify Cuniculus paca individuals by differences in the patterns of flank spots. We then estimate the density 1) considering the area of riparian forest present in the sampling area, and 2) through capture/recapture analysis. Cuniculus paca, Conepatus chinga and Hydrochoerus hydrochaeris were nocturnal, Cerdocyon thous had a crepuscular/nocturnal pattern, while Mazama gouazoubira was cathemeral. The patterns of circadian activity observed for medium and large mammals in this Pampa region (southern grasslands) may reflect not only evolutionary, biological and ecological affects, but also human impacts not assessed in this study. We identified ten individuals of C. paca through skin spot patterns during the study period, which were recorded in different transects and months. The minimum population density of C. paca was 3.5 individuals per km2 (resident animals only) and the total density estimates varied from 7.1 to 11.8 individuals per km2, when considering all individuals recorded or the result of the capture/recapture analysis, respectively.

Resumo De julho de 2014 a abril de 2015, realizamos levantamentos semanais para estudar padrões de atividade circadiana da mastofauna na localidade de Passo Novo, Alegrete, sul do Brasil. A vegetação é compreendida por savana estépica (campo). Utilizamos duas armadilhas fotográficas distribuídas alternadamente ao longo de quatro transectos, com extensão de 1 km e distantes cerca de 1 km entre si. Nós classificamos o padrão de atividade das espécies através da percentagem de fotos registradas em cada período diário. Nós identificamos indivíduos de Cuniculus paca através dos diferentes padrões de manchas nos flancos dos animais. Nós então estimamos a densidade 1) considerando a área de floresta ripária presente na área amostrada, e 2) através da análise de captura/recaptura. As espécies Cuniculus paca, Conepatus chinga e Hydrochoerus hydrochaeris foram classificadas como noturnas, Cerdocyon thous apresentou um padrão crepuscular/noturno, enquanto Mazama gouazoubira foi classificada como catemeral. O padrão de atividade circadiana observado para os mamíferos de médio e grande porte nessa região do Pampa (campos sulinos) pode refletir não só aspectos evolutivos, biológicos e ecológicos, mas também impactos humanos não avaliados nesse estudo. Através do padrão de manchas da pelagem de C. paca nós identificamos dez indivíduos durante o período de estudo, que foram registrados em diferentes transectos e meses. A densidade populacional mínima de C. paca foi de 3,5 ind/km2 (apenas indivíduos residentes) e a densidade total variou de 7,1 a 11,8 ind/km2, quando consideramos todos os indivíduos registrados ou com base em análises de captura e recaptura, respectivamente.

Home range and density of three sympatric felids in the Southern Atlantic Forest, Brazil / Área de vida e densidade de três felídeos simpátricos na Mata Atlântica do Brasil

Abstract Home range and minimal population densities of Southern tiger cat (Leopardus guttulus), margay (Lepardus wiedii) and jaguarundi (Puma yagouaroundi) were estimated between 2005 and 2006 in Taquari Valley, near the southern edge of the Atlantic Rainforest in Brazil. Home range data were collected by conventional radio telemetry (VHF) locations in a highly fragmented landscape. The average home range size, calculated using 95% kernel density estimates, was 16.01 km2 for Southern tiger cat, 21.85 km2 for margay and 51.45 km2 for jaguarundi. Telemetry data were used to obtain minimal density estimates of 0.08 Southern tiger cats / km2, and 0.04 jaguarundi / km2. The density estimates arise from areas where ocelot (Leopardus pardalis) and other larger-bodied carnivores were locally extinct, and they suggest a specific type of mesopredator release known as the ocelot effect, which is likely enabling the increase in smaller felid populations in this area.

Resumo Neste estudo são apresentadas áreas de vida e estimativas mínimas de densidade populacional do gato-do-mato-pequeno (Leopardus guttulus), gato-maracajá (Leopardus wiedii) e gato-mourisco (Puma yagouaroundi) obtidas entre 2005 e 2006, no Vale do Taquari, próximo ao limite sul da Mata Atlântica no Brasil. Os dados sobre área de vida foram coletados com a utilização de telemetria convencional (VHF) em uma paisagem altamente fragmentada. A área de vida média, calculada por Kernel 95%, foi de 16,01 km2 para o gato- do-mato-pequeno, 21,85 km2 para o gato-maracajá e 51,45 km2 para o gato-mourisco. Os dados de telemetria foram utilizados para obter uma estimativa de densidade mínima de 0,08 gatos-do-mato-pequenos por km2, e 0,04 gatos-mourisco por km2. As estimativas de densidade são oriundas de áreas sem a presença de jaguatiricas (Leopardus pardalis) ou outros predadores de maior porte, todos localmente extintos, com possíveis efeitos de um tipo específico de relaxamento de mesopredadores, conhecido como “Efeito Pardalis” que podem permitir o aumento do tamanho das populações de gatos menores.

Pyrophosphate:protein phosphotransferase: a membrane-bound enzyme of endoplasmic reticulum.

Recently, we demonstrated that highly purified rat liver microsomal membrane was capable of selectively phosphorylating two intrinsic membrane polypeptides (M(r) 145,000 and M(r) 130,000) and that the course of the reaction was kinetically divided into two distinct stages [Lam, K. S. & Kasper, C. B. (1980) J. Biol. Chem. 255, 259-266]. Evidence was also presented that strongly suggested that a phosphoryl donor other than ATP was involved in the second stage of phosphorylation. In the present study, we demonstrate that incubation of microsomal membrane with [gamma-(32)P]ATP produces a prominent (32)P-labeled compound detectable by thin-layer chromatography on polyethyleneimine-impregnated cellulose. DEAE-cellulose fractionation of detergent-solubilized microsomal membrane generated a protein fraction that could convert in excess of 90% of the [gamma-(32)P]ATP into this newly (32)P-labeled unknown compound (I approximately P) without the formation of significant levels of (32)P(i). When [alpha-(32)P]ATP was used, I approximately P was unlabeled. Enzymically synthesized I approximately P was purified and determined to be pyrophosphate by using (31)P NMR spectroscopy. [(32)P]Pyrophosphate, synthesized chemically or enzymically, was capable of selectively phosphorylating the M(r) 145,000 and M(r) 130,000 polypeptides. Time course studies utilizing pyrophosphate as the phosphate source showed only one phase of phosphorylation that was strongly inhibited by micromolar levels of ATP as well as by NaF (5 mM). These studies further establish that pyrophosphate is the phosphoryl donor involved in the second stage of phosphorylation.

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development.

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.

Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains.

The coding nucleotide sequence of the mRNA for NADPH-cytochrome P-450 oxidoreductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complementary to rat liver oxidoreductase mRNA. The single open reading frame of 2034 nucleotides spanning these cDNAs codes for a 678 amino acid polypeptide with a molecular weight of 76,962. The deduced amino acid composition is in excellent agreement with that determined by direct amino acid analysis of purified rat liver P-450 oxidoreductase, and the amino-terminal region (residues 1-80) largely coincides with the amino-terminal sequence of the oxidoreductase isolated from rabbit liver. Comparison of the amino acid sequence to those of other flavoproteins revealed two separate domains that are likely to be involved in flavin binding: a long segment (residues 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an FMN-containing protein, and a shorter segment (residues 452-477) homologous with the FAD-binding segment of fumarate reductase from Escherichia coli.

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins.

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.

Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis.

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

Enzyme-substrate binding interactions of NADPH-cytochrome P-450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies.

The pH dependence of the kinetic parameters for the reaction catalyzed by NADPH-cytochrome P-450 oxidoreductase (P-450R) has been determined, using various substrates and inhibitors. All Vmax and (V/K) profiles show pKas of 6.2-7.3, for an acidic group that is preferentially unprotonated for catalysis, and of 8.1-9.6, for a basic group that is preferentially protonated for catalysis. The presence of the wrong ionization state for both of these groups is tolerated more at lower ionic strength (300 mM) than at higher ionic strength (850 mM). Ionization of the basic group has a more pronounced effect on binding of substrate (cytochrome c or dichloroindophenol) than on catalysis, since ionization has only a 2-fold effect on Vmax with cytochrome c, and only a 5-fold effect on Vmax with dichloroindophenol, while (V/K) for both substrates continues to drop at high pH with no sign of reaching a plateau. Therefore, this basic group affects predominantly substrate binding and, to a lesser extent, catalysis. It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol binding site, near the FMN prosthetic group. The NADP+ pKi profile shows a pKa of 5.95 for the 2'-phosphate of NADP+, which is bound to P-450R as the dianion, and a pKa of 9.53 for an enzyme group that must be protonated in order to bind NADP+.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH.

Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)