Glucocorticoid Modulates Angiotensin II Receptor Expression Patterns and Protects the Heart from Ischemia and Reperfusion Injury

Glucocorticoid regulates angiotensin II receptor (ATR) expression via activating glucocorticoid receptors and binding to glucocorticoid response elements. The regulation of ATR by glucocorticoids in the context of myocardial injury from ischemia/reperfusion (I/R) is yet to be elucidated. The present study determined the role of ATR in glucocorticoid-induced cardiac protection. Adult male rats were administered once a day i.p. 1 mg/kg/day dexamethasone or dexamethasone plus 10 mg/kg/day RU486 for 5 days. Hearts were then isolated and subjected to I/R injury in a Langendorff preparation. Dexamethasone treatment significantly decreased I/R injury and improved post-ischemic recovery of cardiac function. Dexamethasone increased glucocorticoid receptor binding to glucocorticoid response elements at AT_1aR and AT₂R promoters, resulting in a significant increase in expression of AT₁R protein but a decrease in AT₂R expression in the heart. In addition, dexamethasone treatment significantly increased PKCε expression and p-PKCε protein abundance. These dexamethasone-mediated effects were blocked by RU486. More importantly, blockade of AT₁R and AT₂R with losartan and PD123319 abrogated dexamethasone-induced protection of the heart from I/R injury. The results indicate that glucocorticoid promotes a cardioprotective phenotype associated with the upregulation of AT₁R and PKCε and downregulation of AT₂R in the heart.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.     

Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli

Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b₅ (Cyt-b₅) was further coexpressed with CPR, indicating that Cyt-b₅ supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b₅ but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an “alternative” electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.     

The effects of lycopene on hepatic ischemia/reperfusion injury in rats

There is a very little information about the protective effect of lycopene (LYC) against hepatic ischemia–reperfusion injury. The present study was designed to examine the possible protective effect of the strong antioxidant and anti-inflammatory agent, LYC, on hepatic ischemia/reperfusion injury. For this purpose, rats were subjected to 45 min of hepatic ischemia followed by 60 min of reperfusion period. LYC at the doses of 2.5 and 5 mg/kg body weight (bw) were injected intraperitoneally, 60 min prior to ischemia. Upon sacrification, hepatic tissue samples were used for the measurement of catalase (CAT) activity and malondialdehyde (MDA) levels. Also, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) were assayed in serum samples. As a result of the use of LYC at the doses of 2.5 and 5 mg/kg bw; while improvements of the ALT, AST, LDH and MDA values were partial and dose-dependent, the improvement of CAT activity was total and dose-independent (p < 0.05). Our findings suggest that LYC has a protective effect against ischemia/reperfusion injury on the liver.     

Lycopene bioavailability is associated with a combination of genetic variants

The intake of tomatoes and tomato products, which constitute the main dietary source of the red pigment lycopene (LYC), has been associated with a reduced risk of prostate cancer and cardiovascular disease, suggesting a protective role of this carotenoid. However, LYC bioavailability displays high interindividual variability. This variability may lead to varying biological effects following LYC consumption. Based on recent results obtained with two other carotenoids, we assumed that this variability was due, at least in part, to several single nucleotide polymorphisms (SNPs) in genes involved in LYC and lipid metabolism. Thus, we aimed at identifying a combination of SNPs significantly associated with the variability in LYC bioavailability. In a postprandial study, 33 healthy male volunteers consumed a test meal containing 100 g tomato puree, which provided 9.7 mg all-trans LYC. LYC concentrations were measured in plasma chylomicrons (CM) isolated at regular time intervals over 8 h postprandially. For the study 1885 SNPs in 49 candidate genes, i.e., genes assumed to play a role in LYC bioavailability, were selected. Multivariate statistical analysis (partial least squares regression) was used to identify and validate the combination of SNPs most closely associated with postprandial CM LYC response. The postprandial CM LYC response to the meal was notably variable with a CV of 70%. A significant (P=0.037) and validated partial least squares regression model, which included 28 SNPs in 16 genes, explained 72% of the variance in the postprandial CM LYC response. The postprandial CM LYC response was also positively correlated to fasting plasma LYC concentrations (r=0.37, P<0.05). The ability to respond to LYC is explained, at least partly, by a combination of 28 SNPs in 16 genes. Interindividual variability in bioavailability apparently affects the long-term blood LYC status, which could ultimately modulate the biological response following LYC supplementation.     

Differential sensitivity of CYP1A to 3,3′,4′,4-tetrachlorobiphenyl and benzo(a)pyrene in two Lepomis species

Although Lepomis species are abundant in a wide variety of habitats throughout North America and could serve as potentially valuable biomonitoring tools, few studies have examined the induction of pollutant biomarkers in this genus. We hypothesized that the induction of cytochrome P-450 1A (CYP1A), a sensitive and widely used indicator of response to aquatic contaminants, would serve as an effective biomarker of organic pollutant exposure in Lepomis species. We examined the response of CYP1A and two of the major pollutant-responsive phase II enzymes, glutathione S-transferase (GST), and uridine diphosphate glucuronyltransferase (UDPGT), in Lepomis exposed to organic pollutants under laboratory and field conditions. Two Lepomis species (longear sunfish, Lepomis megalottis and bluegill, Lepomis macrochirus) were exposed in the laboratory via intraperitoneal injection to corn oil (vehicle), benzo(a)pyrene (BaP) (10 and 50 mg/kg), a polynuclear aromatic hydrocarbon (PAH) or 3,4,3′,4′-tetrachlorobiphenyl (PCB 77) (0.1 and 1.0 mg/kg), a dioxin-like planar halogenated aromatic hydrocarbon (HAH), and sacrificed 2 (BaP) or 7 (corn oil, PCB77) days later. Lepomis hepatic CYP1A exhibited differential sensitivity to these two classes of environmental contaminants. CYP1A activity was weakly induced in bluegill exposed to 1.0 mg/kg PCB 77 (3 fold induction over controls) but strongly induced in both bluegill and longear sunfish exposed to 50 mg/kg BaP (37 and 15 fold induction over controls, respectively). In contrast, hepatic GST activity in both species remained unchanged following the treatment with either compound and hepatic UDPGT activity, which was assessed only in BaP-treated longear sunfish, was unaffected by that chemical, indicating these phase II enzymes may not be sensitive pollutant biomarkers in this genus. Further, longear sunfish collected from a PCB contaminated site displayed relatively low levels of CYP1A activity despite PCB body burdens associated with strong induction of CYP1A activity in other fish species. The strong induction of CYP1A by BaP with much weaker CYP1A response to PCB indicates that CYP1A in Lepomis sp. could be an excellent biomarker for PAH pollution, but may not be a reliable indicator of site contamination by halogenated hydrocarbons. We conclude that Lepomis species provide a useful model for examining the regulation and potential consequences of differential pollutant sensitivity, but that CYP1A in these species should be used with caution as an indicator of halogenated contaminants.     

The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells

Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Maintaining blood retinal barrier homeostasis to attenuate retinal ischemia-reperfusion injury by targeting the KEAP1/NRF2/ARE pathway with lycopene

Retinal ischemia-reperfusion (I/R) often results in intractable visual impairments, where blood retinal barrier (BRB) homeostasis mediated by retinal pigment epithelium (RPE) and retinal microvascular endothelium (RME) is crucial. However, strategies targeting the BRB are limited. Thus, we investigated the inconclusive effect of lycopene (LYC) in retinal protection under I/R. LYC elevated cellular viability and reversed oxidative stress in aRPE-19 cells/hRME cells under I/R conditions based on oxygen-glucose deprivation (OGD) in vitro. Molecular analysis showed that LYC promoted NRF2 expression and enhanced the downstream factors of the KEAP1/NRF2/ARE pathway: LYC increased the activities of antioxidants, including SOD and CAT, whereas it enhanced the mRNA expression of HO-1 (ho-1) and NQO-1 (nqo-1). The activation resulted in restrained ROS and MDA. On the other hand, LYC ameliorated the damage to retinal function and morphology in a mouse I/R model, which was established by unilateral ligation of the left pterygopalatine artery/external carotid artery and reperfusion. LYC promoted the expression of NRF2 in both the neural retina and the RPE choroid in vivo. This evidence revealed the potential of LYC in retinal protection under I/R, uncovering the pharmacological effect of the KEAP1/NRF2/ARE pathway in BRB targeting. The study generates new insights into scientific practices in retinal research.     

Melatonin Reverses Fas,E2F-1 and Endoplasmic Reticulum Stress Mediated Apoptosis and Dysregulation of Autophagy Induced by the Herbicide Atrazine in Murine Splenocytes

Exposure to the herbicide Atrazine (ATR) can cause immunotoxicity, apart from other adverse consequences for animal and human health. We aimed at elucidating the apoptotic mechanisms involved in immunotoxicity of ATR and their attenuation by Melatonin (MEL). Young Swiss mice were divided into control, ATR and MEL+ATR groups based on daily (x14) intraperitoneal administration of the vehicle (normal saline), ATR (100 mg/kg body weight) and MEL (20 mg/kg body weight) with ATR. Isolated splenocytes were processed for detection of apoptosis by Annexin V-FITC and TUNEL assays, and endoplasmic reticulum (ER) stress by immunostaining. Key proteins involved in apoptosis, ER stress and autophagy were quantified by immunoblotting. ATR treatment resulted in Fas-mediated activation of caspases 8 and 3 and inactivation of PARP1 which were inhibited significantly by co-treatment with MEL. MEL also attenuated the ATR-induced, p53 independent mitochondrial apoptosis through upregulation of E2F-1 and PUMA and suppression of their downstream target Bax. An excessive ER stress triggered by ATR through overexpression of ATF-6α, spliced XBP-1, CREB-2 and GADD153 signals was reversed by MEL. MEL also reversed the ATR-induced impairment of autophagy which was indicated by a decline in BECN-1, along with significant enhancement in LC3B-II and p62 expressions. Induction of mitochondrial apoptosis, ER stress and autophagy dysregulation provide a new insight into the mechanism of ATR immunotoxicity. The cytoprotective role of MEL, on the other hand, was defined by attenuation of ER stress, Fas-mediated and p53 independent mitochondria-mediated apoptosis as well as autophagy signals.     

The effect of a natural molecule in ovary ischemia reperfusion damage: does lycopene protect ovary?

Ovarian ischemia is a gynecological emergency case that occurs as a result of ovarian torsion. Oxidative stress plays a central role in the development of ischemia/reperfusion (IR) injuries. Lycopene (LYC) is a lipophilic, natural carotenoid well known for its antioxidant properties. This study provides information on the potential applications of lycopene. The Wistar Albino rats were distributed into six groups: Sham group (only a laparotomy was performed), Control group [laparotomy and intraperitoneal dissolvent (olive oil)], IR group, IR+olive oil group, IR+LYC 2.5 mg/kg/dose, intraperitoneal group, IR+LYC 5 mg/kg/dose intraperitoneal group. Evaluated in terms of histopathological changes, tissue malondialdehyde levels (MDA), ovarian expressions of phosphorylated nuclear factor-kappa B (p-NF-κB) and the TUNEL method was utilized to show apoptosis of ovarian tissue. There was a significant decrease in MDA, p-NF-κB values and the proportion of apoptotic cells assessed by TUNEL compared to the group that did not receive intraperitoneal LYC in rat injury with IR damage (P<0.05). In histopathological damage scoring, it was observed that the cell damage was significantly reduced in LYC-administered groups. LYC showed significant ameliorative effects on ovary injury caused by IR through acting as an antioxidant, antiinflammatory, and antiapoptotic agent.     

CYP5122A1, a novel cytochrome P450 is essential for survival of Leishmania donovani

Background

Cytochrome P450s (CYP450s) are hemoproteins catalysing diverse biochemical reactions important for metabolism of xenobiotics and synthesis of physiologically important compounds such as sterols. Therefore, they are functionally important for survival of invading pathogens. One such opportunistic pathogen Leishmania donovani causes visceral leishmaniasis worldwide, which is an important public health problem due to significant disease burden. The parasite genome database, Gene DB, annotates 3 CYP450s in Leishmania, however, the functional role of cytochrome P450 enzymes in Leishmania spp. remains elusive.

Methodology/Principal Findings

A CYP450-like gene cloned from Leishmania donovani was identified as a novel CYP450, the CYP5122A1. Upon co-localization with organelle specific markers, CYP5122A1 distribution was shown to be localized in the promastigote ER, mitochondria and the glycosomes. Replacement of one allele of CYP5122A1 with either neomycin or hygromycin gene by homologous recombination in Leishmania promastigotes induced substantial reduction of CYP5122A1 expression. These parasites showed impaired growth, lower mitochondrial Ca²⁺ and membrane potential resulting in low ATP generation. Also, these parasites were less infective in vitro and in vivo than their wild-type counterparts as assessed by incubation of Leishmania promastigotes with macrophages in vitro as well as through administration of parasites into hamsters. The HKOs were more susceptible to drugs like miltefosine and antimony, but showed reduced sensitivity to amphotericin B. Removal of two alleles of CYP5122A1 did not allow the parasites to survive. The mutant parasites showed 3.5 times lower ergosterol level as compared to the wild-type parasites when estimated by Gas chromatography/mass spectrometry. Complementation of CYP5122A1 through episomal expression of protein by using pXG-GFP+2 vector partially rescued CYP5122A1 expression and restored ergosterol levels by 1.8 times. Phenotype reversal included restored growth pattern and lesser drug susceptibility.

Conclusions/Significance

In summary, this study establishes CYP5122A1 as an important molecule linked to processes like cell growth, infection and ergosterol biosynthesis in Leishmania donovani.     

Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens.     

Anti-obesity potential of Moringa olifera seed extract and lycopene on high fat diet induced obesity in male Sprauge Dawely rats

Present research explored the anti-obesity effect of Moringa olifera seed oil extract and lycopene (LYC). Forty eight male Sprauge Dawely rats were divided equally into 6 groups. Group Ι (C) served as control, group ΙΙ (MC) was given Moringa olifera seed oil extract (800 mg/kg b.wt) for 8 weeks, group ΙΙΙ (LC) was given (20 mg/kg b.wt) LYC for 8 weeks, group ΙV (O) received high fat diet (HFD) for 20 weeks, group Ѵ (MO), was given HFD for 20 weeks and received (800 mg/kg b.wt) Moringa olifera seed oil extract for last 8 weeks and group ѴΙ (LO), received HFD for 20 weeks and was given (20 mg/kg b.wt) LYC for last 8 weeks. Hematology, lipid peroxidation and antioxidants, non-esterified fatty acids (NEFA), glucose, lipid profile, serum liver and kidney biomarkers, inflammatory markers, leptin, resistin and heart fatty acid binding protein (HFABP) were determined. Also histopathology for liver, kidney and aorta were performed besides immunohistochemistry (IHC) for aortic inducible nitric oxide synthase (iNOS). Administration of Moringa olifera seed oil extract and LYC significantly ameliorated the HFD induced hematological and metabolic perturbations as well as reduced leptin and resistin. Both treatments exerted these effects through promotion of antioxidant enzymes and reducing lipid peroxidation as well as inflammatory cytokines along with reduced iNOS protein expression. Administration of Moringa olifera seed oil extract and LYC have anti-obesity potential in HFD induced obesity in male Sprauge Dawely rats.     

In silico analysis of cytochrome P450 monooxygenases in chronic granulomatous infectious fungus Sporothrix schenckii: Special focus on CYP51

Sporotrichosis is an emerging chronic, granulomatous, subcutaneous, mycotic infection caused by Sporothrix species. Sporotrichosis is treated with the azole drug itraconazole as ketoconazole is ineffective. It is a well-known fact that azole drugs act by inhibiting cytochrome P450 monooxygenases (P450s), heme-thiolate proteins. To date, nothing is known about P450s in Sporothrix schenckii and the molecular basis of its resistance to ketoconazole. Here we present genome-wide identification, annotation, phylogenetic analysis and comprehensive P450 family-level comparative analysis of S. schenckii P450s with pathogenic fungi P450s, along with a rationale for ketoconazole resistance by S. schenckii based on in silico structural analysis of CYP51. Genome data-mining of S. schenckii revealed 40 P450s in its genome that can be grouped into 32 P450 families and 39 P450 subfamilies. Comprehensive comparative analysis of P450s revealed that S. schenckii shares 11 P450 families with plant pathogenic fungi and has three unique P450 families: CYP5077, CYP5386 and CYP5696 (novel family). Among P450s, CYP51, the main target of azole drugs was also found in S. schenckii. 3D modeling of S. schenckii CYP51 revealed the presence of characteristic P450 motifs with exceptionally large reductase interaction site 2. In silico analysis revealed number of mutations that can be associated with ketoconazole resistance, especially at the channel entrance to the active site. One of possible reason for better stabilization of itraconazole, compared to ketoconazole, is that the more extended molecule of itraconazole may form a hydrogen bond with ASN-230. This in turn may explain its effectiveness against S. schenckii vis-a-vis resistant to ketoconazole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.