The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

Up-regulation of placental leptin by human chorionic gonadotropin.

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway.

Cellular Derivatives and Efficacy in Wound and Scar Management

Biologicals have been used for decades in biopharmaceutical topical preparations. Because cellular therapies are rou-tinely used in the clinic they have gained significant attention. Different derivatives are possible from different cell and tissue sources, making the selection of cell types and establishment of consistent cell banks crucial steps in the initial whole-cell bioprocessing. Various cell and tissue types have been used in treatment of skin wounds including autolo-gous and allogenic skin cells, platelets, placenta and amniotic extracts from either human or animal sources. Experience with progenitor cells show that they may provide an interesting cell choice due to facility of out-scaling and known properties for wound healing without scar. Using defined animal cell lines to develop cell-free derivatives may provide initial starting material for pharmaceutical formulations that help in overall stability. Cell lines derived from ovine tis-sue (skin, muscle, connective tissue) can be developed in short periods of time and consistency of these cell lines was monitored by cellular life-span, protein concentrations, stability and activity. Each cell line had long culture periods up to 37 - 41 passages and protein measures for each cell line at passages 2 - 15 had only 1.4-fold maximal difference. Growth stimulation activity towards two target skin cell lines (GM01717 and CRL-1221; 40 year old human males) at concentrations ranging up to 6 μg/ml showed 2-3-fold (single extracts) and 3-7-fold (co-cultured extracts) increase. Proteins from co-culture remained stable up to 1 year in pharmaceutical preparations shown by separation on SDS- PAGE gels. Pharmaceutical cell-free preparations were used for veterinary and human wounds and burns. Cell lines and cell-free extracts can show remarkable consistency and stability for preparation of biopharmaceutical creams, moreover when cells are co-cultured, and have positive effects for tissue repair.

Human Exposure to Endocrine-Disrupting Chemicals and Prenatal Risk Factors for Cryptorchidism and Hypospadias: A Nested Case-Control Study

BACKGROUND. Exposure to xenoestrogens during pregnancy may disturb the development and function of male sexual organs. OBJECTIVE. In this study we aimed to determine whether the combined effect of environmental estrogens measured as total effective xenoestrogen burden (TEXB) is a risk factor for male urogenital malformations. METHODS. In a case-control study, nested in a mother-child cohort (n = 702) established at Granada University Hospital, we compared 50 newborns with diagnosis of cryptorchidism and/or hypospadias with 114 boys without malformations matched by gestational age, date of birth, and parity. Controls did not differ from the total cohort in confounding variables. TEXB and levels of 16 organochlorine pesticides were measured in placenta tissues. Characteristics of parents, pregnancy, and birth were gathered by questionnaire. We used conditional and unconditional regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs). RESULTS. TEXB from organohalogenated compounds was detectable in 72% and 54% of case and control placentas, respectively. Compared with controls, cases had an OR for detectable versus non-detectable TEXB of 2.82 (95% CI, 1.10-7.24). More pesticides were detected in cases than in controls (9.34 +/- 3.19 vs. 6.97 +/- 3.93). ORs for cases with detectable levels of pesticides, after adjusting for potential confounders in the conditional regression analysis, were o,p'-DDT (OR = 2.25; 95% CI, 1.03-4.89), p,p'-DDT (OR = 2.63; 95% CI, 1.21-5.72), lindane (OR = 3.38; 95% CI, 1.36-8.38), mirex (OR = 2.85; 95% CI, 1.22-6.66), and endosulfan alpha (OR = 2.19; 95% CI, 0.99-4.82). Engagement of mothers in agriculture (OR = 3.47; 95% CI, 1.33-9.03), fathers' occupational exposure to xenoestrogens (OR = 2.98; 95% CI, 1.11-8.01), and history of previous stillbirths (OR = 4.20; 95% CI, 1.11-16.66) were also associated with risk of malformations. CONCLUSIONS We found an increased risk for male urogenital malformations related to the combined effect of environmental estrogens in placenta.

Vertical Transmission of Pneumocystis jirovecii in Humans

This study is part of the project “Pneumocystis Pathogenomics: Unravelling the Colonization-to-Disease Shift,” a Coordination Action supported by the European Commission (ERANET PathoGenoMics). This study was partially supported by the Spanish Ministry of Health (FIS 03/1743). M.A.M.-C. and C.d.l.H. were supported by the Spanish Ministry of Health (FIS CP-04/217 and FIS CM-04/146).

Detection of suspected placental invasion by MRI: Do the results depend on observer' experience?

PURPOSE: To evaluate the diagnostic value of previously described MR features used for detecting suspected placental invasion according to observers' experience. MATERIALS AND METHODS: Our population included 25 pregnant women (mean age 35.16) investigated by prenatal MRI (1.5T, T1- and T2-weighted MR-sequences without i.v. contrast), among them 12 with histopathologically proven placental invasion and 13 women (52%) without placental invasion used as control group. Two senior and two junior radiologists blindly and independently reviewed MR-examinations in view of 6 previously defined MR-features indicating presence and degree of placental invasion (placenta increta, accreta or percreta). For each reader the sensibility, specificity, and receiver operating curve (ROC) were calculated. Interobserver agreements between senior and junior readers were determined. Stepwise logistic regression was performed including the 6 MR-features predictive of placental invasion. RESULTS: Demographics between both groups were statistically equivalent. Overall sensitivity and specificity for placental invasion was 90.9% and 75.0% for seniors and 81.8% and 61.8% for juniors, respectively. The best single MR-feature indicating placental invasion was T2-hypointense placental bands (r(2)=0.28), followed by focally interrupted myometrial border, infiltration of pelvic organs and tenting of the bladder (r(2)=0.36). Interobserver agreement for detecting placental invasion was 0.64 for seniors and 0.41 for juniors, thus substantial and moderate, respectively. Seniors detected placental invasion and depth of infiltration with significantly higher diagnostic certitude than juniors (p=0.0002 and p=0.0282, respectively). CONCLUSION: MRI can be a reliable and reproducible tool for the detection of suspected placental invasion, but the diagnostic value significantly depends on observers' experience.

High mobility group box 1 protein (HMGB-1): a pathogenic role in preeclampsia?

We evaluated whether preeclampsia is associated with elevated circulating levels of High mobility group box 1 protein (HMGB-1), a nuclear protein with proinflammatory effects when released extracellularly. We enrolled 48 women, 32 in third trimester pregnancy (16 with, 16 without preeclampsia), and 16 healthy non pregnant. In the peripheral blood of pregnant women, HMGB-1 concentration was assessed serially, before and after delivery. With or without preeclampsia, third trimester pregnancy was associated with elevated levels of HMGB-1. This elevation is exaggerated in preeclampsia. The source of HMGB-1 observed in these conditions is likely to involve tissues other than the placenta itself.

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)(2)cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway.

Pregnancy malaria: cryptic disease, apparent solution

Malaria during pregnancy can be severe in non-immune women, but in areas of stable transmission, where women are semi-immune and often asymptomatic during infection, malaria is an insidious cause of disease and death for mothers and their offspring. Sequelae, such as severe anaemia and hypertension in the mother and low birth weight and infant mortality in the offspring, are often not recognised as consequences of infection. Pregnancy malaria, caused by Plasmodium falciparum, is mediated by infected erythrocytes (IEs) that bind to chondroitin sulphate A and are sequestered in the placenta. These parasites have a unique adhesion phenotype and distinct antigenicity, which indicates that novel targets may be required for development of an effective vaccine. Women become resistant to malaria as they acquire antibodies against placental IE, which leads to higher haemoglobin levels and heavier babies. Proteins exported from the placental parasites have been identified, including both variant and conserved antigens, and some of these are in preclinical development for vaccines. A vaccine that prevents P. falciparum malaria in pregnant mothers is feasible and would potentially save hundreds of thousands of lives each year.

Role of Waddlia chondrophila placental infection in miscarriage.

Waddlia chondrophila is an intracellular bacterium suspected to cause human and bovine abortion. We confirmed an association between antibodies against W. chondrophila and human miscarriage and identified this organism in placenta or genital tract of women who had had miscarriages. These results suggest a possible role of W. chondrophila infection in miscarriage.

Roles of PPARβ and PPARγ in mouse placental development

RESUME Les gènes des PPARs jouent des rôles importants dans la régulation du métabolisme énergétique, lipidique et glucidique. Le présent travail, caractérise et analyse les défauts placentaires responsables de la mort embryonnaire des souris mutantes pour PPARβ et pour PPARγ, entre le jour E9.5 et E10.5. Les placentas issus d'embryons PPARP présentent un sévère retard de croissance, alors que les placentas mutants PPARγ montrent de graves défauts vasculaires. Nous montrons que les placentas issus d'embryons PPARβ-/-, au jour E9.5 présentent une réduction prononcée de la couche de cellules géantes, associée à une diminution des niveaux de protéines exprimées par les cellules géantes, tel que le placenta lactogène-I et la « proliferin ». Par ailleurs, nous montrons que le traitement d'un lignée trophoblastique par un ligand spécifique de PPARP augmente considérablement leur différentiation en cellules géantes. Cette différentiation dépendante de la voie de signalisation P13-kinase, s'accompagne d'une élévation de l'expression de l'ADRP, une protéine de structure associée aux vésicules lipidiques. Ainsi nous démontrons que PPAR5 est un régulateur majeur de la différentiation des cellules géantes, lesquelles sont primordiales aussi bien pour l'établissement de la structure placentaire, que pour la fonction endocrine. Par contre, les placentas PPARγ-/- présentent un défaut de vascularisation. Le niveau d'une protéine anti-angiogénique, la « proliferin-related protein », est très basse et ne peut pas contre-balancer l'élévation normale de la protéine pro-angiogénique « proliferin ». La formation des vaisseaux se trouve alors altérée. Ainsi, PPARγ constitue un régulateur majeur de l'activité anti-angiogénique. En conclusion, ce travail fournit de nouveaux éléments sur le rôle complémentaires de PPARβet PPARγ dans les événements complexes qui régissent le développement placentaire. SUMMARY Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors involved in energy homeostasis and growth. Herein, we characterize the placental defects that cause embryonic lethality around E9.5/E10.5 in PPARβ- and in PPARγ-deficient mouse lines. Most but not all PPARβ-null mutants die around E9.5/E10.5 with severe growth retardation. The placentas from PPARβ-/- embryos at E9.5 exhibit a strongly reduced giant cell layer, associated with reduced levels of proteins expressed by giant cells such as Placental lactogen-I and Proliferin. Ectopic treatment of a rat trophoblast cell line with PPARβ ligand markedly accelerated PI3 kinase-dependent giant cell differentiation. In addition, we demonstrate that ADRP, a pen-related lipid droplet-bound protein, is up-regulated by PPARβ in differentiated Rcho-1 cells. These results indicate that PPARβ is a crucial regulator of the differentiation secondary giant cells, which play a major role in the establishment of the placental structure as well as an important endocrine function. In contrast, the main alteration of the PPARγ-null placentas concerns the vasculogenesis. We show that in these placentas, the level of the anti-angiogenic proliferin-related protein is very low, and cannot balance the normal elevation of the pro-angiogenic proliferin expression, leading to the defective placental vessel formation. Consistently, the dramatic increase of PPARγ expression in late stage of gestation in wild-type mice is likely a major regulator of the anti-angiogenic activity, particularly important at the end of the pregnancy. This work emphasizes the important and complementary roles of PPARβ and PPARγ in mouse placental development and provides new tools for understanding the complex regulatory events that governs placental development and function. Understanding the function of PPARβ and PPARγ are of crucial interest with respect to human placental development and associated pathologies.

Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.

Dual control of hydrogen cyanide biosynthesis by the global activator GacA in Pseudomonas aeruginosa PAO1.

The global response regulator GacA of Pseudomonas aeruginosa PAO1 positively controls the production of the quorum sensing signal molecule N-butanoyl-homoserine-lactone (C4-HSL) and hence the synthesis of several C4-HSL-dependent virulence factors, including hydrogen cyanide (HCN). This study presents evidence that GacA positively influences the transcription of the rhlI gene, specifying C4-HSL synthase, explaining the quorum sensing-dependent transcriptional control of the HCN biosynthetic genes (hcnABC). In addition, GacA was found to modulate hcn gene expression positively at a post-transcriptional level involving the hcnA ribosome-binding site. Thus, the activating effect of GacA on cyanogenesis results from both transcriptional and post-transcriptional mechanisms.

An integrated genetic and functional analysis of the role of type II transmembrane serine proteases (TMPRSSs) in hearing loss.

Building on our discovery that mutations in the transmembrane serine protease, TMPRSS3, cause nonsyndromic deafness, we have investigated the contribution of other TMPRSS family members to the auditory function. To identify which of the 16 known TMPRSS genes had a strong likelihood of involvement in hearing function, three types of biological evidence were examined: 1) expression in inner ear tissues; 2) location in a genomic interval that contains a yet unidentified gene for deafness; and 3) evaluation of hearing status of any available Tmprss knockout mouse strains. This analysis demonstrated that, besides TMPRSS3, another TMPRSS gene was essential for hearing and, indeed, mice deficient for Hepsin (Hpn) also known as Tmprss1 exhibited profound hearing loss. In addition, TMPRSS2, TMPRSS5, and CORIN, also named TMPRSS10, showed strong likelihood of involvement based on their inner ear expression and mapping position within deafness loci PKSR7, DFNB24, and DFNB25, respectively. These four TMPRSS genes were then screened for mutations in affected members of the DFNB24 and DFNB25 deafness families, and in a cohort of 362 sporadic deaf cases. This large mutation screen revealed numerous novel sequence variations including three potential pathogenic mutations in the TMPRSS5 gene. The mutant forms of TMPRSS5 showed reduced or absent proteolytic activity. Subsequently, TMPRSS genes with evidence of involvement in deafness were further characterized, and their sites of expression were determined. Tmprss1, 3, and 5 proteins were detected in spiral ganglion neurons. Tmprss3 was also present in the organ of Corti. TMPRSS1 and 3 proteins appeared stably anchored to the endoplasmic reticulum membranes, whereas TMPRSS5 was also detected at the plasma membrane. Collectively, these results provide evidence that TMPRSS1 and TMPRSS3 play and TMPRSS5 may play important and specific roles in hearing.

A novel neuro-muscular marker for the heavy subunit of neurofilament proteins, NF-H, and striated muscle myosin of Xenopus laevis.

A novel monoclonal antibody, M7, is described, that reacts on Western blots with the large subunit of the neurofilament triplet proteins (NF-H) and with striated muscle myosin of Xenopus laevis. Enzymatically digested neurofilament and myosin proteins revealed different immunoreactive peptide fragments on Western blots. Therefore, the antibody must react with immunologically related epitopes common to both proteins. Immunohistochemistry showed staining of large and small axons in CNS and PNS, and nerves could be followed into endplate regions of skeletal muscles. These muscles were characterized by a striated immunostaining of the M-lines. Despite the crossreactivity of M7 with NF-H and muscle myosin, this antibody may be a tool to study innervation of muscle fibers, and to define changes in the neuromuscular organization during early development and metamorphosis of tadpoles.