Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of different biotransformation enzymes during primary culturing of rat proximal tubular cells (PT cells). Specific marker substrates for determining cytochrome P450 (CYP450) activity of primary cultured PT cells include 7-ethoxyresorufin (CYP1A1), caffeine (CYP1A), testosterone (CY2B/C, CYP3A), tolbutamide (CYP2C) and dextromethorphan (CYP2D1). Activities of the CYP450 isoenzymes decreased considerably during culture with the greatest loss in activity within 24 h of culture. In addition, expression of CYP450 apoprotein, including CYP1A, CYP2C, CYP2D, CYP2E and CYP4A, was detected in microsomes from freshly isolated PT cells by immunoblotting using specific antibodies. CYP2B and CYP3A apoprotein could not be detected. Activity of the phase II biotransformation enzymes GST, GGT, beta-lyase and UGT was determined with 1-chloro-2,4-dinitrobenzene, L-glutamic acid gamma-(7-amido-4-methyl-coumarin), S-(1,1,2,2-tetrafluoroethyl)-L-cysteine and 1-naphthol, respectively, as marker substrates. Activity of the phase II enzymes remained more stable and, in contrast to CYP450 activity, significant activity was still expressed after 1 week of PT cell culture. Thus, despite the obvious advantages of PT cells as an in-vitro model for studies of biotransformation mediated toxicity, the strong time dependency of especially phase I and, to a lesser extent, phase II biotransformation activities confers limitations to their application.     

Conservation and non-conservation of genetic pathways in eye specification

In this review we highlight two genetic pathways important for eye morphogenesis that are partially conserved between flies and vertebrates. Initially we focus on the ey paradigm and establish which aspects of this genetic hierarchy are conserved in vertebrates. We discuss experiments that evaluate the non-linear relationship amongst the genes of the hierarchy with a concentration on vertebrate functional genetics. We specifically consider the Six genes and their relationship to sine oculis, as tremendous amounts of new data have emerged on this topic. Finally, we highlight similarities between Shh/Hh directed morphogenesis mediated by basic Helix-Loop-Helix factors in vertebrate retinal cell specification and in specification of fly photoreceptors.     

Mesomelic form of chondrodysplasia and congenital glaucoma associated with de novo translocation (13;18)(q14;q23)

Mesomelic form of chondrodysplasia and congenital glaucoma associated with de novo translocation (13;18)(q14:q23): Mesomelic dysplasias are characterized by limb shortening most prominent of the middle segment of the extremities (forearm and lower leg). In addition to several syndromic forms a few patients with sporadic or familial forms and without precise nosological classification have been reported so far. In this report we present a young female with disproportionate mesomelic dwarfism, dysmorphic facial features, bilateral glaucoma, patent ductus arteriosus, low and hoarse voice, and generalized muscular hypotonia. At the age of 2.5 years mental development is normal. High resolution G-banded chromosome studies revealed a de novo reciprocal translocation with karyotype 46,XX t (13;18)(q14;q23). The concurrence of this de novo autosomal translocation with this distinct phenotype supports the hypothesis that disruption of (a) gene(s) at the translocation breakpoints causes this unusual, apparently new form of skeletal chondrodysplasia.     

Cranial neural crest-derived mesenchymal proliferation is regulated by Msx1-mediated p19(INK4d) expression during odontogenesis

Neural crest cells are multipotential progenitors that contribute to various cell and tissue types during embryogenesis. Here, we have investigated the molecular and cellular mechanism by which the fate of neural crest cell is regulated during tooth development. Using a two- component genetic system for indelibly marking the progeny of neural crest cells, we provide in vivo evidence of a deficiency of CNC-derived dental mesenchyme in Msx1 null mutant mouse embryos. The deficiency of the CNC results from an elevated CDK inhibitor p19(INK4d) activity and the disruption of cell proliferation. Interestingly, in the absence of Msx1, the CNC-derived dental mesenchyme misdifferentiates and possesses properties consistent with a neuronal fate, possibly through a default mechanism. Attenuation of p19(INK4d) in Msx1 null mutant mandibular explants restores mitotic activity in the dental mesenchyme, demonstrating the functional significance of Msx1-mediated p19(INK4d) expression in regulating CNC cell proliferation during odontogenesis. Collectively, our results demonstrate that homeobox gene Msx1 regulates the fate of CNC cells by controlling the progression of the cell cycle. Genetic mutation of Msx1 may alternatively instruct the fate of these progenitor cells during craniofacial development.     

Eya1 is required for the morphogenesis of mammalian thymus,parathyroid and thyroid

Eyes absent (Eya) genes regulate organogenesis in both vertebrates and invertebrates. Mutations in human EYA1 cause congenital Branchio-Oto-Renal (BOR) syndrome, while targeted inactivation of murine Eya1 impairs early developmental processes in multiple organs, including ear, kidney and skeletal system. We have now examined the role of Eya1 during the morphogenesis of organs derived from the pharyngeal region, including thymus, parathyroid and thyroid. The thymus and parathyroid are derived from 3rd pharyngeal pouches and their development is initiated via inductive interactions between neural crest-derived arch mesenchyme, pouch endoderm, and possibly the surface ectoderm of 3rd pharyngeal clefts. Eya1 is expressed in all three cell types during thymus and parathyroid development from E9.5 and the organ primordia for both of these structures failed to form in Eya1(-/-) embryos. These results indicate that Eya1 is required for the initiation of thymus and parathyroid gland formation. Eya1 is also expressed in the 4th pharyngeal region and ultimobranchial bodies. Eya1(-/-) mice show thyroid hypoplasia, with severe reduction in the number of parafollicular cells and the size of the thyroid lobes and lack of fusion between the ultimobranchial bodies and the thyroid lobe. These data indicate that Eya1 also regulates mature thyroid gland formation. Furthermore, we show that Six1 expression is markedly reduced in the arch mesenchyme, pouch endoderm and surface ectoderm in the pharyngeal region of Eya1(-/-) embryos, indicating that Six1 expression in those structures is Eya1 dependent. In addition, we show that in Eya1(-/-) embryos, the expression of Gcm2 in the 3rd pouch endoderm is undetectable at E10.5, however, the expression of Hox and Pax genes in the pouch endoderm is preserved at E9.5-10.5. Finally, we found that the surface ectoderm of the 3rd and 4th pharyngeal region show increased cell death at E10.5 in Eya1(-/-) embryos. Our results indicate that Eya1 controls critical early inductive events involved in the morphogenesis of thymus, parathyroid and thyroid.     

Formin-2, polyploidy,hypofertility and positioning of the meiotic spindle in mouse oocytes

Successful reproduction in mammals requires a competent egg, which is formed during meiosis through two assymetrical cell divisions. Here, we show that a recently identified formin homology (FH) gene, formin-2 (Fmn2), is a maternal-effect gene that is expressed in oocytes and is required for progression through metaphase of meiosis I. Fmn2(-/-) oocytes cannot correctly position the metaphase spindle during meiosis I and form the first polar body. We demonstrate that Fmn2 is required for microtubule-independent chromatin positioning during metaphase I. Fertilization of Fmn2(-/-) oocytes results in polyploid embryo formation, recurrent pregnancy loss and sub-fertility in Fmn2(-/-) females. Injection of Fmn2 mRNA into Fmn2-deficient oocytes rescues the metaphase I block. Given that errors in meiotic maturation result in severe birth defects and are the most common cause of chromosomal aneuploidy and pregnancy loss in humans, studies of Fmn2 may provide a better understanding of infertility and birth defects.     

Ultrastructure of a haplosporidian containing Rickettsiae,associated with mortalities among cultured paua Haliotis iris

Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.