Cytochrome P450 17alpha hydroxylase/17,20 lyase (CYP17) function in cholesterol biosynthesis: identification of squalene monooxygenase (epoxidase) activity associated with CYP17 in Leydig cells

Cytochrome P450 17alpha-hydroxylase/17,20-lyase (CYP17) is a microsomal enzyme catalyzing two distinct activities, 17alpha-hydroxylase and 17,20-lyase, essential for the biosynthesis of adrenal and gonadal steroids. CYP17 is a potent oxidant, it is present in liver and nonsteroidogenic tissues, and it has been suggested to have catalytic properties distinct to its function in steroid metabolism. To identify CYP17 functions distinct of its 17alpha-hydroxylase/17,20-lyase activity, we used MA-10 mouse tumor Leydig cells known to be defective in 17alpha-hydroxylase/17,20-lyase activity. A CYP17 knocked down MA-10 clone (MA-10(CYP17KD)) was generated by homologous recombination and its steroidogenic capacity was compared with wild-type cells (MA-10(wt)). Although no differences in cell morphology and proliferation rates were observed between these cells, the human chorionic gonadotropin-induced progesterone formation and de novo synthesis of steroids were dramatically reduced in MA-10(CYP17KD) cells; their steroidogenic ability could be rescued in part by transfecting CYP17 DNA into the cells. Knocking down CYP17 mRNA by RNA interference yielded similar results. However, no significant difference was observed in the steroidogenic ability of cells treated with 22R-hydroxycholesterol, which suggested a defect in cholesterol biosynthesis. Incubation of MA-10(CYP17KD) cells with (14)C-labeled squalene resulted in the formation of reduced amounts of radiolabeled cholesterol compared with MA-10(wt) cells. In addition, treatment of MA-10(CYP17KD) cells with various cholesterol substrates indicated that unlike squalene, addition of squalene epoxide, lanosterol, zymosterol, and desmosterol could rescue the hormone-induced progesterone formation. Further in vitro studies demonstrated that expression of mouse CYP17 in bacteria resulted in the expression of squalene monooxygenase activity. In conclusion, these studies suggest that CYP17, in addition to its 17alpha-hydroxylase/17,20-lyase activity, critical in androgen formation, also expresses a secondary activity, squalene monooxygenase (epoxidase), of a well-established enzyme involved in cholesterol biosynthesis, which may become critical under certain conditions.     

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-dependent processes in the full-length protein

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH.The sorcin calcium binding domain (SCBD, residues 33-198) expressed in Escherichia coli was crystallized in the Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers.The structure of the SCBD leads to a model that explains the solution properties and describes the Ca2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.     

Chemical defense and antifouling activity of three Mediterranean sponges of the genus Ircinia

The defense roles and the antifouling activity of the organic extracts and the major metabolites of the sponges Ircinia oros, I. variabilis and I. spinosula were investigated. The antifeedant activity was tested in experimental aquaria on the generalist predator fish Thalassoma pavo as well as in coastal ecosystems rich in fishes. Some of the major metabolites exhibited high levels of antifeedant activity. The antifouling activity was tested in laboratory assays, against representatives of the major groups of fouling organisms (marine bacteria, marine fungi, diatoms, macroalgae and mussels). All extracts showed promising levels of activity. As was expected, no single extract was active in all tests and some fractions that were effective against one organism showed little or no activity against the others. The high but variable level of antifouling activity in combination with the absence of toxicity (tested on the development of oyster and sea urchin larvae) shows the potential of these metabolites to become ingredients in environmentally friendly antifouling preparations.     

Recovery Kinetics of Knee Flexor and Extensor Strength after a Football Match

We examined the temporal changes of isokinetic strength performance of knee flexor (KF) and extensor (KE) strength after a football match. Players were randomly assigned to a control (N = 14, participated only in measurements and practices) or an experimental group (N = 20, participated also in a football match). Participants trained daily during the two days after the match. Match and training overload was monitored with GPS devices. Venous blood was sampled and muscle damage was assessed pre-match, post-match and at 12h, 36h and 60h post-match. Isometric strength as well as eccentric and concentric peak torque of knee flexors and extensors in both limbs (dominant and non-dominant) were measured on an isokinetic dynamometer at baseline and at 12h, 36h and 60h after the match. Functional (KF_ecc/KE_con) and conventional (KF_con/KE_con) ratios were then calculated. Only eccentric peak torque of knee flexors declined at 60h after the match in the control group. In the experimental group: a) isometric strength of knee extensors and knee flexors declined (P<0.05) at 12h (both limbs) and 36h (dominant limb only), b) eccentric and concentric peak torque of knee extensors and flexors declined (P<0.05) in both limbs for 36h at 60°/s and for 60h at 180°/s with eccentric peak torque of knee flexors demonstrating a greater (P<0.05) reduction than concentric peak torque, c) strength deterioration was greater (P<0.05) at 180°/s and in dominant limb, d) the functional ratio was more sensitive to match-induced fatigue demonstrating a more prolonged decline. Discriminant and regression analysis revealed that strength deterioration and recovery may be related to the amount of eccentric actions performed during the match and athletes'' football-specific conditioning. Our data suggest that recovery kinetics of knee flexor and extensor strength after a football match demonstrate strength, limb and velocity specificity and may depend on match physical overload and players'' physical conditioning level.     

Let-7, Mir-98 and Mir-181 as Biomarkers for Cancer and Schizophrenia

Recent evidence supports a role of microRNAs in cancer and psychiatric disorders such as schizophrenia and bipolar disorder, through their regulatory role on the expression of multiple genes. The rather rare co-morbidity of cancer and schizophrenia is an old hypothesis which needs further research on microRNAs as molecules that might exert their oncosuppressive or oncogenic activity in the context of their role in psychiatric disorders. The expression pattern of a variety of different microRNAs was investigated in patients (N = 6) suffering from schizophrenia termed control, patients with a solid tumor (N = 10) and patients with both schizophrenia and tumor (N = 8). miRNA profiling was performed on whole blood samples using the miRCURY LNA microRNA Array technology (6th & 7th generation). A subset of 3 microRNAs showed a statistically significant differential expression between the control and the study groups. Specifically, significant down-regulation of the let-7p-5p, miR-98-5p and of miR-183-5p in the study groups (tumor alone and tumorand schizophrenia) was observed (p<0.05). The results of the present study showed that let-7, miR-98 and miR-183 may play an important oncosuppressive role through their regulatory impact in gene expression irrespective of the presence of schizophrenia, although a larger sample size is required to validate these results. Nevertheless, further studies are warranted in order to highlight a possible role of these and other micro-RNAs in the molecular pathways of schizophrenia.     

Synergistic Combinations of Nucleoside Analog Drugs with Other Drugs Induce Greater Apoptosis in Human Leukemic T-Cells

Abstract

Combinations of nucleoside analog drugs such as 6-MP. ara-C. or F-araA are synergistic against human leukemic T-cells and induce apoptotic cell death. Addition of Taxotere or PEG-ASNase to the synergistic combination of nucleoside analog drugs augments the synergism several fold by enhancing cellular apoptosis.     

Population-based trends of pregnancy outcome in obese mothers: what has changed over 15 years

Maternal obesity is a growing concern worldwide. We aimed to determine trends of obesity in women of reproductive age over a 15-year period, the relationship between prepregnancy weight status and pregnancy-related outcomes, and possible changes over a 15-year period. Data was derived from two identical, cross-sectional National Perinatal Surveys (NPSs). Analysis was restricted to the mothers of healthy, full-term, singleton infants. Overall, the study population consisted of 18,752 mother-infant pairs (7,208 from the 1st NPS and 11,544 from the 2nd NPS). The prevalence of overweight and obese women prior to pregnancy increased significantly between the two surveys, from 12 to 15.3% and 2.1 to 4.7%, respectively. In both study periods, prepregnancy weight status was positively associated with pre-eclampsia (P < 0.001) and method of delivery (P < 0.001). After adjusting for potential confounders, overweight and obese women had higher odds for caesarian section (adjusted (odd ratio) OR = 1.87) and pre-eclampsia (adjusted OR = 2.59) than normal-weight women. These associations did not change between the study periods. In conclusion, prepregnancy maternal obesity is steadily increasing over time. The magnitude of the problem underlies the need for public health programs to focus on obesity prevention, particularly among women of reproductive age, and re-evaluate the guidelines for various perinatal practices.     

Control of hypercholesterolemia and atherosclerosis using the cholesterol recognition/interaction amino acid sequence of the translocator protein TSPO

The translocator protein (18-kDa) TSPO is an ubiquitous high affinity cholesterol-binding protein reported to be present in the endothelial and smooth muscle cells of the blood vessels; its expression dramatically increased in macrophages found in atherosclerotic plaques. A domain in the carboxy-terminus of TSPO was identified and characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide could be used as an hypocholesterolemic, with potential anti-atherogenic properties, agent. We report herein the therapeutic benefit that resulted for the administration of the VLNYYVWR human CRAC sequence to guinea pigs fed with a high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. CRAC treatment (3 and 30 mg/kg once daily for 6 weeks) resulted in reduced circulating cholesterol levels in guinea pigs fed with 2% high cholesterol diet and ApoE knock-out B6.129P2-Apoetm1Unc/J mice. In high cholesterol fed guinea pigs, CRAC treatment administered once daily induced an increase in circulating HDL, decreased total, free and LDL cholesterol, and removed atheroma deposits in the aorta in a dose-dependent manner. The treatment also prevented the high cholesterol diet-induced increase in serum creatine kinase, total and isoforms, markers of neurological, cardiac and muscular damage. No toxicity was observed. Taken together these results support a role of TSPO in lipid homeostasis and atherosclerosis and indicate that CRAC may constitute a novel and safe treatment of hypercholesterolemia and atherosclerosis.