Role of human hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) in steroid-dependent diseases in females –novel indications for HSD17B1 inhibitors. Phenotypic analysis of transgenic mice overexpressing human HSD17B1.

Hormone-dependent diseases, e.g. cancers, rank high in mortality in the modern world, and thus, there is an urgent need for new drugs to treat these diseases. Although the diseases are clearly hormone-dependent, changes in circulating hormone concentrations do not explain all the pathological processes observed in the diseased tissues. A more inclusive explanation is provided by intracrinology – a regulation of hormone concentrations at the target tissue level. This is mediated by the expression of a pattern of steroid-activating and -inactivating enzymes in steroid target tissues, thus enabling a concentration gradient between the blood circulation and the tissue. Hydroxysteroid (17beta) dehydrogenases (HSD17Bs) form a family of enzymes that catalyze the conversion between low active 17-ketosteroids and highly active 17beta-hydroxysteroids. HSD17B1 converts low active estrogen (E1) to highly active estradiol (E2) with high catalytic efficiency, and altered HSD17B1 expression has been associated with several hormone-dependent diseases, including breast cancer, endometriosis, endometrial hyperplasia and cancer, and ovarian epithelial cancer. Because of its putative role in E2 biosynthesis in ovaries and peripheral target tissues, HSD17B1 is considered to be a promising drug target for estrogen-dependent diseases. A few studies have indicated that the enzyme also has androgenic activity, but they have been ignored. In the present study, transgenic mice overexpressing human HSD17B1 (HSD17B1TG mice) were used to study the effects of the enzyme in vivo. Firstly, the substrate specificity of human HSD17B1 was determined in vivo. The results indicated that human HSD17B1 has significant androgenic activity in female mice in vivo, which resulted in increased fetal testosterone concentration and female disorder of sexual development appearing as masculinized phenotype (increased anogenital distance, lack of nipples, lack of vaginal opening, combination of vagina with urethra, enlarged Wolffian duct remnants in the mesovarium and enlarged female prostate). Fetal androgen exposure has been linked to polycystic ovary syndrome (PCOS) and metabolic syndrome during adulthood in experimental animals and humans, but the genes involved in PCOS are largely unknown. A putative mechanism to accumulate androgens during fetal life by HSD17B1 overexpression was shown in the present study. Furthermore, as a result of prenatal androgen exposure locally in the ovaries, HSD17B1TG females developed ovarian benign serous cystadenomas in adulthood. These benign lesions are precursors of low-grade ovarian serous tumors. Ovarian cancer ranks fifth in mortality of all female cancers in Finland, and most of the ovarian cancers arise from the surface epithelium. The formation of the lesions was prevented by prenatal antiandrogen treatment and by transplanting wild type (WT) ovaries prepubertally into HSD17B1TG females. The results obtained in our non-clinical TG mouse model, together with a literature analysis, suggest that HSD17B1 has a role in ovarian epithelial carcinogenesis, and especially in the development of serous tumors. The role of androgens in ovarian carcinogenesis is considered controversial, but the present study provides further evidence for the androgen hypothesis. Moreover, it directly links HSD17B1-induced prenatal androgen exposure to ovarian epithelial carcinogenesis in mice. As expected, significant estrogenic activity was also detected for human HSD17B1. HSD17B1TG mice had enhanced peripheral conversion of E1 to E2 in a variety of target tissues, including the uterus. Furthermore, this activity was significantly decreased by treatments with specific HSD17B1 inhibitors. As a result, several estrogen-dependent disorders were found in HSD17B1TG females. Here we report that HSD17B1TG mice invariably developed endometrial hyperplasia and failed to ovulate in adulthood. As in humans, endometrial hyperplasia in HSD17B1TG females was reversible upon ovulation induction, triggering a rise in circulating progesterone levels, and in response to exogenous progestins. Remarkably, treatment with a HSD17B1 inhibitor failed to restore ovulation, yet completely reversed the hyperplastic morphology of epithelial cells in the glandular compartment. We also demonstrate that HSD17B1 is expressed in normal human endometrium, hyperplasia, and cancer. Collectively, our non-clinical data and literature analysis suggest that HSD17B1 inhibition could be one of several possible approaches to decrease endometrial estrogen production in endometrial hyperplasia and cancer. HSD17B1 expression has been found in bones of humans and rats. The non-clinical data in the present study suggest that human HSD17B1 is likely to have an important role in the regulation of bone formation, strength and length during reproductive years in female mice. Bone density in HSD17B1TG females was highly increased in femurs, but in lesser amounts also in tibias. Especially the tibia growth plate, but not other regions of bone, was susceptible to respond to HSD17B1 inhibition by increasing bone length, whereas the inhibitors did not affect bone density. Therefore, HSD17B1 inhibitors could be safer than aromatase inhibitors in regard to bone in the treatment of breast cancer and endometriosis. Furthermore, diseases related to improper growth, are a promising new indication for HSD17B1 inhibitors.

Characterization of nontypable haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Genetic and phenotypic characterization of isolates of Pyricularia grisea from the rice cultivars epagri 108 and 109 in the State of Tocantins

An epidemic of rice (Oryza sativa) blast occurred on cultivars Epagri 108 and 109 in the municipalities of Lagoa da Confusão and Duerê in the State of Tocantins, during the rice-growing season 1998-99. DNA fingerprinting and virulence phenotype analysis were utilized to determine the diversity of Pyricularia grisea isolates collected from these cultivars in one epidemic year. Rep-PCR analysis of isolates was done by using two primer sequences from Pot2. Two distinct fingerprint groups or lineages were identified among 53 isolates collected from nine different commercial fields. The virulence pattern of isolates retrieved from these two cultivars was analyzed in artificial inoculation tests utilizing 32 genotypes in the greenhouse. A dendrogram constructed from virulence phenotype data showed a single group considering 77% similarity level. The predominant pathotype IB-45 was represented by 47 of the 53 isolates corresponding to 83%. Four other pathotypes (IB-1, IB-9, IB-13 and IB-41) were identified at random among the isolates from these cultivars. There was no relation between rep-PCR grouping and pathotypes. The results showed that the isolates of P. grisea recovered from cultivars Epagri108 and 109 in farmers' fields had narrow phenotypic and genetic diversity. The blast outbreak on these two cultivars one year after their introduction could be attributed to the new pathotype IB-45 or its increase, which was hitherto existing in low frequency.

Estimation of phenotypic diversity in field populations of Magnaporthe grisea from two upland rice cultivars

The phenotypic diversity of Magnaporthe grisea was evaluated based on leaf samples with blast lesions collected from eight commercial fields of the upland rice cultivars 'BRS Primavera' and 'BRS Bonança', during the growing seasons of 2001/2002 and 2002/2003, in Goias State. The number of M. grisea isolates from each field utilized for virulence testing varied from 28 to 47. Three different indices were used based on reaction type in the eight standard international differentials and eight Brazilian differentials. The M. grisea subpopulations of ´Primavera' and 'Bonança', as measured by Simpson, Shannon and Gleason indices, showed similar phenotypic diversities. The Simpson index was more sensitive relation than those of Shannon and Gleason for pathotype number and standard deviation utilizing Brazilian differentials. However, the Gleason index was sensitive to standard deviation for international differentials. The sample size did not significantly influence the diversity index. The two sets of differential cultivars used in this study distinguished phenotypic diversity in different ways in all of the eight subpopulations analyzed. The phenotypic diversity determined based on eight differential Brazilian cultivars was lower in commercial rice fields of 'Primavera' than in the fields of 'Bonança,' independent of the diversity index utilized, year and location. Considering the Brazilian differentials, the four subpopulations of 'BRS Primavera' did not show evenness in distribution and only one pathotype dominated in the populations. The even distribution of pathotype was observed in three subpopulations of 'BRS Bonança'. The pathotype diversity of M. grisea was determined with more precision using Brazilian differentials and Simpson index.

Evaluation of phenotypic stability of resistance to Phytophthora spp. in cacao clones

Resistance of fourteen Theobroma cacao clones to Phytophthora spp. was evaluated using stem inoculations on grafted seedlings. Concepts of phenotypic stability were used to interpret the results and to express horizontality of the resistance. The linear regression coefficient 'b', the determination coefficient (R²) and average lesion size were used to determine the level of horizontal resistance, the phenotypic stability and the predictability of all clones. The results indicated that clones P 7 and MA 15 present highest levels of horizontal resistance and stability, but with moderate predictability. Clones CAS 1 and CEPEC 13 were classified as those with high horizontal resistance, stability and predictability, while clones PA 30, UF 650 and SIAL 88 and EET 59 showed intermediate resistance and stability and high predictability. Clones SPA 17, OC 61, PA 150, SIAL 505, ICS 1 and R 41 presented high susceptibility and intermediate or low stability and moderate or high predictability.

The spindle assembly checkpoint as a drug target - Novel small-molecule inhibitors of Aurora kinases

Cell division (mitosis) is a fundamental process in the life cycle of a cell. Equal distribution of chromosomes between the daughter cells is essential for the viability and well-being of an organism: loss of fidelity of cell division is a contributing factor in human cancer and also gives rise to miscarriages and genetic birth defects. For maintaining the proper chromosome number, a cell must carefully monitor cell division in order to detect and correct mistakes before they are translated into chromosomal imbalance. For this purpose an evolutionarily conserved mechanism termed the spindle assembly checkpoint (SAC) has evolved. The SAC comprises a complex network of proteins that relay and amplify mitosis-regulating signals created by assemblages called kinetochores (KTs). Importantly, minor defects in SAC signaling can cause loss or gain of individual chromosomes (aneuploidy) which promotes tumorigenesis while complete failure of SAC results in cell death. The latter event has raised interest in discovery of low molecular weight (LMW) compounds targeting the SAC that could be developed into new anti-cancer therapeutics. In this study, we performed a cell-based, phenotypic high-throughput screen (HTS) to identify novel LMW compounds that inhibit SAC function and result in loss of cancer cell viability. Altogether, we screened 65 000 compounds and identified eight that forced the cells prematurely out of mitosis. The flavonoids fisetin and eupatorin, as well as the synthetic compounds termed SACi2 and SACi4, were characterized in more detail utilizing versatile cell-based and biochemical assays. To identify the molecular targets of these SAC-suppressing compounds, we investigated the conditions in which SAC activity became abrogated. Eupatorin, SACi2 and SACi4 preferentially abolished the tensionsensitive arm of the SAC, whereas fisetin lowered also the SAC activity evoked by lack of attachments between microtubules (MTs) and KTs. Consistent with the abrogation of SAC in response to low tension, our data indicate that all four compounds inhibited the activity of Aurora B kinase. This essential mitotic protein is required for correction of erratic MT-KT attachments, normal SAC signaling and execution of cytokinesis. Furthermore, eupatorin, SACi2 and SACi4 also inhibited Aurora A kinase that controls the centrosome maturation and separation and formation of the mitotic spindle apparatus. In line with the established profound mitotic roles of Aurora kinases, these small compounds perturbed SAC function, caused spindle abnormalities, such as multi- and monopolarity and fragmentation of centrosomes, and resulted in polyploidy due to defects in cytokinesis. Moreover, the compounds dramatically reduced viability of cancer cells. Taken together, using a cell-based HTS we were able to identify new LMW compounds targeting the SAC. We demonstrated for the first time a novel function for flavonoids as cellular inhibitors of Aurora kinases. Collectively, our data support the concept that loss of mitotic fidelity due to a non-functional SAC can reduce the viability of cancer cells, a phenomenon that may possess therapeutic value and fuel development of new anti-cancer drugs.

Weak phenotypic reversion of ivermectin resistance in a field resistant isolate of Haemonchus contortus by verapamil

Recent advances in anthelmintic resistant phenotype reversion by Pgp modulating drugs in ruminant nematodes indicate that this can be a useful tool to helminth control. The aim of the present study was to evaluate the efficacy of ivermectin (IVM) in combination with verapamil (VRP), in oil or water-based vehicle, against an IVM-resistant field isolate of Haemonchus contortus through a larval migration assay and experimental infection trial. In the in vitro assay was observed a phenotypic reversion of H. contortus resistance to ivermectin at a high concentration of VRP, increasing IVM efficacy from 53.1% to 94.3. In the in vivo trial, IVM + VRP demonstrated 36.02% efficacy compared to the 7.75% of IVM alone. The vehicle formulation showed no influence in efficacy. These are the first results demonstrating the effect of VRP as a partial IVM-resistance phenotype reverser in a field isolate of IVM-resistant H. contortus experimentally inoculated in sheep.

Holstein white coat color and performance: phenotypic, genetic and environmental correlations

Correlations of measures of percentages of white coat color, five measures of production and two measures of reproduction were obtained from 4293 first lactation Holsteins from eight Florida dairy farms. Percentages of white coat color were analyzed as recorded and transformed by an extension of Box-Cox procedures. Statistical analyses were by derivative-free restricted maximum likelihood (DFREML) with an animal model. Phenotypic and genetic correlations of white percentage (not transformed) were with milk yield, 0.047 and 0.097; fat yield, 0.002 and 0.004; fat percentage, -0.047 and -0.090; protein yield, 0.024 and 0.048; protein percentage, -0.070 and -0.116; days open, -0.012 and -0.065; and calving interval, -0.007 and -0.029. Changes in magnitude of correlations were very small for all variables except days open. Genetic and phenotypic correlations of transformed values with days open were -0.027 and -0.140. Modest positive correlated responses would be expected for white coat color percentage following direct selection for milk, fat, and protein yields, but selection for fat and protein percentages, days open, or calving interval would lead to small decreases.

Synthesis of stocks and phenotypic effects of dwarf and bantam sex-linked major genes in egg-type chickens

The present study describes the production of stocks segregating dwarf (dw), bantam (dwB) and normal (dw+) alleles, as well as the characters, shank length, adult body weight, age at sexual maturity and egg production. Heterozygous K dw+/k dwB sires were mated to normal (dw+) dams to produce stock D6.a, and mated to dwB females to produce stock D6.b. Stock D4.a came from mating F1 heterozygous dwB dw sires to dwarf Leghorns. In a third series of matings, 7/8 Sebright and 1/8 dw-Leghorn dwB dw sires were crossed to three groups of dams of different genotypes. The progeny of the normal (dw+), dwarf (dw), and bantam (dwB) dams were designated as stocks D4.b, D4.c and D4.d, respectively. The dw+ dams were White Leghorn strain cross females. The difference between the rate of laying of normal (69.7%) and their bantam sisters (68.6%) was not statistically significant when the average 32-week body weight of the dw+ sisters was 1,897 g. However, when the 32-week body weight of the normal daughters from the same sires and smaller dams was around 1,646 g, the difference between the rate of laying of the normal (78.1%) and their bantam sisters (75.9%) was significant (P < 0.05). The dwB gene may have a similar but smaller effect on the rate of egg laying than its dwarf allele. The difference between sexual maturity of normal and bantam daughters of either the largest or the smallest dams was not statistically significant, even though the smallest dwB pullets were in average 2.9 days older at first egg. The use of shank length combined with adult body weight allowed a precise discrimination between bantams and dwarfs

Estimates of genetic and phenotypic parameters in a segregant population of rice irrigated by continuous flooding

Estimates of genetic and phenotypic parameters were obtained by using data from families of a recurrent selection program in rice. An experiment using population CNA-IRAT 4ME/1/1 was conducted at two locations (Lambari and Cambuquira) in the State of Minas Gerais, Brazil. At Lambari, families S0:2 and S0:3 were assessed during crop seasons 1992/1993 and 1993/1994, respectively. In the Cambuquira trial, only S0:3 families were tested in 1993/1994. The experimental design was a 10 x 10 lattice with three replications. The following traits were assessed: grain yield (GY), mean number of days to flowering (FL), plant height (PH), and the incidence of neck blast (NB) caused by Pyricularia grisea and grain staining (GS) caused by Drechslera oryzae. This population proved to be promising for recurrent selection, as it had high average yield and genetic variability. Heritability estimates obtained using variance components were generally greater than estimates of realized heritability, and heritability obtained by parent-offspring regression

Novel genes and regulatory systems in epididymal differentiation and sperm maturation of the mouse.

Mammalian spermatozoa gain their fertilizing ability during maturation in the epididymis. Proteins and lipids secreted into the epididymal lumen remodel the sperm membrane, thereby providing the structure necessary for progressive motility and oocyte interaction. In the current study, genetically modified mouse models were utilized to determine the role of novel genes and regulatory systems in the postnatal development and function of the epididymis. Ablation of the mouse β-defensin, Defb41, altered the flagellar movements of sperm and reduced the ability of sperm to bind to the oocyte in vitro. The Defb41-deficient iCre knock-in mouse model was furthermore utilized to generate Dicer1 conditional knock-out (cKO) mice. DICER1 is required for production of mature microRNAs in the regulation of gene expression by RNA interference. Dicer1 cKO gave rise to dedifferentiation of the epididymal epithelium and an altered expression of genes involved in lipid synthesis. As a consequence, the cholesterol:polyunsaturated fatty acid ratio of the Dicer1 cKO sperm membrane was increased, which resulted in membrane instability and infertility. In conclusion, the results of the Defb41 study further support the important role of β-defensin family members in sperm maturation. The regulatory role of Dicer1 was also shown to be required for epididymal development. In addition, the study is the first to show a clear connection between lipid homeostasis in the epididymis and sperm membrane integrity. Taken together, the results give important new evidence on the regulatory system guiding epididymal development and function

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.