The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

DRUG METABOLISM IN LIVER TUMORS: PURIFICATION AND CHARACTERIZATION OF NADPH-CYTOCHROME-P450 REDUCTASE AND THE RESOLUTION OF MULTIPLE FORMS OF CYTOCHROME P-450

The hydroxylation of N- and O-methyl drugs and a polycyclic hydrocarbon has been demonstrated in microsomes prepared from two transplantable Morris hepatomas (i.e., 7288C. t.c. and 5123 t.c.(H). The hydroxylation rates of the drug benzphetamine and the polycyclic hydrocarbon benzo {(alpha)} pyrene by tumor microsomes were inducible 2 to 3-fold and 2-fold, respectively by pretreatment of rats with phenobarbital/hydrocortisone. Hepatoma 5123t.c.(h) microsomal hydroxylation activities were more inducible after these pretreatments than hepatoma 7288C.t.c. Two chemotherapeutic drugs (cyclophosphamide and isophosphamide) were shown to be mutagenic after activation by the tumor hemogenate with the TA100 strain of Salmonella typhimurium bacteria. NADPH-cytochrome P-450 was purified from phenobarbital/hydrocortisone treated rat hepatoma 5123t.c.(H) microsomes 353-fold with a specific activity 63.6 nmol of cytochrome c reduced per min per mg of protein. The purified enzyme, has an apparent molecular weight of 79,500 daltons, and contained an equal molar ratio of FMN and FAD, with a total flavin content of 16.4 nmol per mg of protein. The purified enzyme also catalyzed electron transfer to artificial electron acceptors with the K(,m) values of the hepatoma reductase similar to those of purified liver reductase. The K(,m) value of the hepatoma reductase (13 uM) for NADPH was similar to that of purified liver reductase (5.0 uM). In addition the purified hepatoma reductase was immunochemically similar to the liver reductase.^ Hepatoma cytochrome P-450, the hemeprotein component of the hepatoma microsomes of rats pretreated with phenobarbital/hydrocortisone. The resolution of the six forms was achieved by the DE-53 ion-exchange chromatography, and further purified by hydroxyapatite. The six different fractions that contained P-450 activity, had specific contents from 0.47 to 1.75 nmol of cytochrome P-450 per mg of protein, and indicated a 2 to 9-fold purification as compared to the original microsomes. In addition, difference spectra, molecular weights and immunological results suggest there are at least six different forms of cytochrome P-450 in hepatoma 5123 t.c.(H). ^

Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-fos, c-jun, JunB, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome c (Cyt c), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25–3 hr) and followed sequentially by c-jun (0.5–3 hr), JunB (0.5–6 hr), NRF-1 (1–12 hr), Cyt c (12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-fos, β-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt c expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.

Analysis of Respiratory Chain Regulation in Roots of Soybean Seedlings1

Changes in the respiratory rate and the contribution of the cytochrome (Cyt) c oxidase and alternative oxidase (COX and AOX, respectively) were investigated in soybean (Glycine max L. cv Stevens) root seedlings using the 18O-discrimination method. In 4-d-old roots respiration proceeded almost entirely via COX, but by d 17 more than 50% of the flux occurred via AOX. During this period the capacity of COX, the theoretical yield of ATP synthesis, and the root relative growth rate all decreased substantially. In extracts from whole roots of different ages, the ubiquinone pool was maintained at 50% to 60% reduction, whereas pyruvate content fluctuated without a consistent trend. In whole-root immunoblots, AOX protein was largely in the reduced, active form at 7 and 17 d but was partially oxidized at 4 d. In isolated mitochondria, Cyt pathway and succinate dehydrogenase capacities and COX I protein abundance decreased with root age, whereas both AOX capacity and protein abundance remained unchanged. The amount of mitochondrial protein on a dry-mass basis did not vary significantly with root age. It is concluded that decreases in whole-root respiration during growth of soybean seedlings can be largely explained by decreases in maximal rates of electron transport via COX. Flux via AOX is increased so that the ubiquinone pool is maintained in a moderately reduced state.

Crystal structure of the membrane-exposed domain from a respiratory quinol oxidase complex with an engineered dinuclear copper center.

Cytochrome oxidase is a membrane protein complex that catalyzes reduction of molecular oxygen to water and utilizes the free energy of this reaction to generate a transmembrane proton gradient during respiration. The electron entry site in subunit II is a mixed-valence dinuclear copper center in enzymes that oxidize cytochrome c. This center has been lost during the evolution of the quinoloxidizing branch of cytochrome oxidases but can be restored by engineering. Herein we describe the crystal structures of the periplasmic fragment from the wild-type subunit II (CyoA) of Escherichia coli quinol oxidase at 2.5-A resolution and of the mutant with the engineered dinuclear copper center (purple CyoA) at 2.3-A resolution. CyoA is folded as an 11-stranded mostly antiparallel beta-sandwich followed by three alpha-helices. The dinuclear copper center is located at the loops between strands beta 5-beta 6 and beta 9-beta 10. The two coppers are at a 2.5-A distance and symmetrically coordinated to the main ligands that are two bridging cysteines and two terminal histidines. The residues that are distinct in cytochrome c and quinol oxidases are around the dinuclear copper center. Structural comparison suggests a common ancestry for subunit II of cytochrome oxidase and blue copper-binding proteins.

Molecules, wing pattern and distribution: an approach to species delimitation in the "loxurina group" (Lepidoptera: Lycaenidae: Penaincisalia)

The wide range of morphological variations in the “loxurina group” makes taxa identification difficult, and despite several reviews, serious taxonomical confusion remains. We make use of DNA data in conjunction with morphological appearance and available information on species distribution to delimit the boundaries of the “loxurina” group species previously established based on morphology. A fragment of 635 base pairs within the mtDNA gene cytochrome oxidase I (COI) was analysed for seven species of the “loxurina group”. Phylogenetic relationships among the included taxa were inferred using maximum parsimony and maximum likelihood methods. Penaincisalia sigsiga (Bálint et al), P. cillutincarae (Draudt), P. atymna (Hewitson) and P. loxurina (C. Felder & R. Felder) were easily delimited as the morphological, geographic and molecular data were congruent. Penaincisalia ludovica (Bálint & Wojtusiak) and P. loxurina astillero (Johnson) represent the same entity and constitute a sub-species of P. loxurina. However, incongruence among morphological, genetic, and geographic data is shown in P. chachapoya (Bálint & Wojtusiak) and P. tegulina (Bálint et al). Our results highlight that an integrative approach is needed to clarify the taxonomy of these neotropical taxa, but more genetic and geographical studies are still required.

Determining the age of adult wild dogs (Canis lupus dingo, C-l. domesticus and their hybrids). I. Pulp cavity: tooth width ratios

In order to determine the age of adult wild dogs, we compared two methods ( that of Thomson and Rose (TR method) and that of Knowlton and Whittemore (KW method)) of measuring and calculating pulp cavity : tooth width ratios on upper and lower canine teeth from 68 mixed-sex, known-age wild dogs of 9 months to 13 years of age reared at two localities. Although significant relationships ( P = 0.0001) were found between age and pulp cavity ratios by both methods, the TR ratio calculation and measurement showed heteroscedasity in error variance whereas the KW ratios had a more stable error variance and were normally distributed. The KW method also found significant differences between pulp cavity ratios between teeth of the upper and lower jaws ( P < 0.0001) and sex ( P = 0.01) but not geographic origin ( P = 0.1). Regressions and formulae for fitted curves are presented separately for male and female wild dogs. Males show greater variability in pulp cavity decrements with age than do females, suggesting a physiological difference between the sexes. We conclude that the KW method of using pulp cavity as a proportion of tooth width, measured 15 mm from the root tip and averaged over both upper canines, is the more accurate method of estimating the age of adult wild dogs.

Studies on the anti-inflammatory effect of S-Adenosyl-L-methionine

1. S-adenosyl-L-methionine (SAMe) had no effect on cytochrome C reduction by superoxide generated from xanthine oxidase except at high concentrations. This was due to direct inhibition of the enzyme. 2. SAMe inhibited the neutrophil respiratory burst , measured by luminol enhanced chemiluminescence, to FMLP and zymosan A but not to PMA. 3. Adenosine and methylthioadenosine (MTA) inhibited the respiratory burst elicited by FMLP. 4. SAMe inhibited the phagocytosis of latex particles by neutrophils at high concentrations but methionine and S-adenosyl L-homocysteine had no effect. 5. Treatment with SAMe had no effect on cell infiltration or PGE2 production in 6-day air pouches. 6. Treatment with SAMe at the optimum dose of 50mg/kg inhibited the early phases of carrageenan induced rat hind paw inflammation but had a lesser effect on the secondary response. The antiinflammatory effect was sustained after inhibiton of polyamine synthesis. 7. SAMe increased liver putrescine levels in the presence and absence of inflammation Spermidine levels were increased in the presence of inflammation but spermine levels were unaffected by any of the treatments. 8. MT A and adenosine increased liver putrescine and spermidine levels 9. Treatment with SAMe had no effect on the polyamine status of blood. lO.Treatment with SAMe had no effect on the levels of glutathione in liver or blood. 11.SAMe and MTA inhibited histamine and platelet-activating factor (PAF) induced hind paw inflammation but had no effect on inflammation induced by dextran, zymosan, compound 48/80, 5-hydroxytryptamine, arachidonic acid or glucose oxidase. MTA was more effective than SAMe. 12. PAP-induced rat hind paw inflammation was inhibited by isoprenaline and verapamil. Combinations of these drugs with SAMe or MT A had no further enhancement of effect. 13. Incubation of rat PMNLs with [14c ] SAMe increased the intracellular levels of S-adenosyl-L-homocysteine in a dose dependent manner, but had no effect on the intracellular levels of SAMe, adenosine or MT A. 14. Pharmacokinetic studies of plasma SAMe following a single dose of the drug (50mg/kg) i.p. demonstrated that SAMe is rapidly absorbed and metabolised

Sampling information, length measurements, DNA alignments, models and phylogenetics trees for Oithona similis s.l.

Traditionally, many small-sized copepod species are considered to be widespread, bipolar or cosmopolitan. However, these large-scale distribution patterns need to be re-examined in view of increasing evidence of cryptic and pseudo-cryptic speciation in pelagic copepods. Here, we present a phylogeographic study of Oithona similis s.l. populations from the Arctic Ocean, the Southern Ocean and its northern boundaries, the North Atlantic and the Mediterrranean Sea. O. similis s.l. is considered as one of the most abundant species in temperate to polar oceans and acts as an important link in the trophic network between the microbial loop and higher trophic levels such as fish larvae. Two gene fragments were analysed: the mitochondrial cytochrome oxidase c subunit I (COI), and the nuclear ribosomal 28S genetic marker. Seven distinct, geographically delimitated, mitochondrial lineages could be identified, with divergences among the lineages ranging from 8 to 24 %, thus representing most likely cryptic or pseudocryptic species within O. similis s.l. Four lineages were identified within or close to the borders of the Southern Ocean, one lineage in the Arctic Ocean and two lineages in the temperate Northern hemisphere. Surprisingly the Arctic lineage was more closely related to lineages from the Southern hemisphere than to the other lineages from the Northern hemisphere, suggesting that geographic proximity is a rather poor predictor of how closely related the clades are on a genetic level. Molecular clock application revealed that the evolutionary history of O. similis s.l. is possibly closely associated with the reorganization of the ocean circulation in the mid Miocene and may be an example of allopatric speciation in the pelagic zone.

Soybean Stem Fly, Melanagromyza sojae (Diptera: Agromyzidae), in the New World: detection of high genetic diversity from soybean fields in Brazil

Soybean Stem Fly (SSF), Melanagromyza sojae (Zehntner), belongs to the family Agromyzidae and is highly polyphagous, attacking many plant species of the family Fabaceae, including soybean and other beans. SSF is regarded as one of the most important pests in soybean fields of Asia (e.g., China, India), North East Africa (e.g., Egypt), parts of Russia, and South East Asia. Despite reports of Agromyzidae flies infesting soybean fields in Rio Grande do Sul State (Brazil) in 1983 and 2009 and periodic interceptions of SSF since the 1940s by the USA quarantine authorities, SSF has not been officially reported to have successfully established in the North and South Americas. In South America, M. sojae was recently confirmed using morphology and its complete mitochondrial DNA (mtDNA) was characterized. In the present study, we surveyed the genetic diversity of M. sojae, collected directly from soybean host plants, using partial mtDNA cytochrome oxidase I (COI) gene, and provide evidence of multiple (>10) maternal lineages in SSF populations in South America, potentially representing multiple incursion events. However, a single incursion involving multiple-female founders could not be ruled out. We identified a haplotype that was common in the fields of two Brazilian states and the individuals collected from Australia in 2013. The implications of SSF incursions in southern Brazil are discussed in relation to the current soybean agricultural practices, highlighting an urgent need for better understanding of SSF population movements in the New World, which is necessary for developing effective management options for this significant soybean pest. © FUNPEC-RP.

Genetic differentiation among Parastichopus regalis populations in the Western Mediterranean Sea: potential effects from its fishery and current connectivity

Parastichopus regalis (Cuvier, 1817) is the most expensive seafood product on the Catalonian market (NE Spain), with prices at approximately 130 €/Kg (fresh weight). Despite its ecological and economic importance, biological and genetic information on this sea cucumber species is scarce. Here, we provide both the first insight on the genetic structure of P. regalis using sequences of cytochrome oxidase I (COI) and 16S genes and a morphological description of its population. Individual sea cucumbers were collected in six locations along the Spanish Mediterranean coast, including an area under fishery pressure (Catalonia). We found high haplotype diversity and low nucleotide diversity for both genes, with higher levels of genetic diversity observed in the COI gene. The population pairwise fixation index (FST), AMOVA and correspondence analysis (CA) based on the COI gene revealed significant genetic differentiation among some locations. However, further analysis using nuclear markers (e.g., microsatellites) is necessary to corroborate these results. Moreover, the genetic and morphological data may indicate fishery effects on the Catalonian population with a decrease in the size and weight averages and lower genetic diversity compared with locations that lack fishery pressure. For the appropriate management of this species, we suggest the following: 1) accurately assessing the stock status along the Spanish coasts; 2) studying the reproductive cycle of this target species and the establishment of a closed fishery season according to the reproductive cycle; and 3) establishing protected areas (i.e., not take zones) to conserve healthy populations and favour recruitment in the nearby areas.

Population genetics of Cerastoderma edule in Ria Formosa (southern Portugal): the challenge of understanding an intraspecific hotspot of genetic diversity

Coastal lagoons are highly variable environments that may act as hotspots of genetic diversity as a consequence of their ecological role as nursery habitats of marine species with both ecological and fisheries importance. The edible cockle (Cerastoderma edule) is a commercially important shellfish resource inhabiting coastal lagoons in Europe and their fisheries management urgently needs genetic studies to design appropriate strategies to promote the recovery of exploited populations. The aim of this study was to assess the C. edule genetic diversity and population structure at a small geographic scale, inside Ria Formosa coastal lagoon (southern Portugal) using mitochondrial cytochrome oxidase I sequences in six locations. Outcomes pointed to a common pattern of high haplotype diversity and non-significant genetic structuring inside the Ria Formosa lagoon. A high level of gene flow was detected between all localities and the presence of a single stock from a genetic point of view may be considered for fisheries management purposes. The existence of a high number of haplotypes and high values of haplotype diversity of C. edule in Ria Formosa lagoon could be consistent with the hypothesis that higher genetic diversity is expected in populations occurring in coastal lagoons, suggesting that lagoons could increase standing genetic variation and an adaptive potential of lagoon populations as an ecological response to a highly variable environment.

DNA markers to disentangle complexes of cryptic taxa in mealybugs (Hemiptera: Pseudococcidae).

Mealybugs (Hemiptera: Pseudococcidae) are major pests of a wide range of crops and ornamental plants worldwide. Their high degree of morphological similarity makes them difficult to identify and limits their study and management. We aimed to identify a set of markers for the genetic characterization and identification of complexes of taxa in the Pseudococcidae. We surveyed and tested the genetic markers used in previous studies and then identified new markers for particularly relevant genomic regions for which no satisfactory markers were available. We tested all markers on a subset of four taxa distributed worldwide. Five markers were retained after this first screening: two regions of the mitochondrial cytochrome oxidase I gene, 28S-D2, the entire internal transcriber space 2 locus and the rpS15-16S region of the primary mealybug endosymbiont Tremblaya princeps. We then assessed the utility of these markers for the characterization and identification of 239 samples from 43 sites in France and Brazil. The five markers studied (i) successfully distinguished all species identified by morphological examination, (ii) disentangled complexes of species by revealing intraspecific genetic variation and identified a set of closely related taxa for which taxonomic status requires clarification through further studies, and (iii) facilitated the inference of phylogenetic relationships between the characterized taxa.

Protostrongylids nematodes as a tool to study the biogeography of wild mammals

The European brown hare (Lepus europaeus Pallas, 1778) is an important game species, distributed across Europe and introduced in other regions. Recently, a geographically isolated population, closely related to an ancestral lineage of Lepus europaeus meridiei, was found on Pianosa Island, off the coast of Tuscany, Italy (Mengoni et al., 2018). Thus, the unique opportunity to explore the evolution and genetic structure of its helminth parasites was added to its exceptional isolation condition. Various lungworm species within the genus Protostrongylus (Nematoda: Protostrongylidae) are described in European brown hares. Our aim was to analyze the parasite population through morphological and molecular approaches in order to study the biogeography of the European brown hares (L. e. meridiei) population from Pianosa Island. Moreover, we investigated the morphology of a monospecific genus, i.e. Orthostrongylus, considering its quite intrigant descriptive history and its still unclear and debated classification. Nuclear and mitochondrial markers were used based on their resolution power and expected polymorphism; the whole Internal Transcribed Spacer 1 and 2 (ITS), including the 5.8S rRNA sequence and the Large Subunit (28S) were used, as nuclear genes, for confirmation of the species identification. Conversely, the cytochrome oxidase c subunit I (COI) was used, as mithocondrial genes, to assess interspecific genetic relationships. Molecular analysis corroborated the morphological identification since all the generated ITS and LSU sequences were 100% consistent with the species Protostrongylus oryctolagi and Orthostrongylus macrotis. The paucity of molecular data existent about this genus of parasites underlines the need for more insight’s studies. An in-depth analysis of broncho-pulmonary parasites and the host-parasites relationships along with the improvement of the use of mitochondrial genes, as well as the assessment of new polymorphic markers could contribute to an extensive understanding of parasites fauna and taxonomy, as well as their relationship with wild mammals’ hosts.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data