Mixed spermatogenic germ cell nuclear extracts exhibit high base excision repair activity

Spermatogenic cells exhibit a lower spontaneous mutation frequency than somatic tissues in a lacI transgene and many base excision repair (BER) genes display the highest observed level of expression in the testis. In this study, uracil-DNA glycosylase-initiated BER activity was measured in nuclear extracts prepared from tissues obtained from each of three mouse strains. Extracts from mixed spermatogenic germ cells displayed the greatest activity followed by liver then brain for all three strains, and the activity for a given tissue was consistent among the three strains. Levels of various BER proteins were examined by western blot analyses and found to be consistent with activity levels. Nuclear extracts prepared from purified Sertoli cells, a somatic component of the seminiferous epithelium, exhibited significantly lower activity than mixed spermatogenic cell-type nuclear extracts, thereby suggesting that the high BER activity observed in mixed germ cell nuclear extracts was not a characteristic of all testicular cell types. Nuclear extracts from thymocytes and small intestines were assayed to assess activity in a mitotically active cell type and tissue. Overall, the order of tissues/cells exhibiting the greatest to lowest activity was mixed germ cells > Sertoli cells > thymocytes > small intestine > liver > brain.

Peropsin, a novel visual pigment-like protein located in the apical microvilli of the retinal pigment epithelium

A visual pigment-like protein, referred to as peropsin, has been identified by large-scale sequencing of cDNAs derived from human ocular tissues. The corresponding mRNA was found only in the eye, where it is localized to the retinal pigment epithelium (RPE). Peropsin immunoreactivity, visualized by light and electron microscopy, localizes the protein to the apical face of the RPE, and most prominently to the microvilli that surround the photoreceptor outer segments. These observations suggest that peropsin may play a role in RPE physiology either by detecting light directly or by monitoring the concentration of retinoids or other photoreceptor-derived compounds.

Nested expression domains for odorant receptors in zebrafish olfactory epithelium

The mapping of high-dimensional olfactory stimuli onto the two-dimensional surface of the nasal sensory epithelium constitutes the first step in the neuronal encoding of olfactory input. We have used zebrafish as a model system to analyze the spatial distribution of odorant receptor molecules in the olfactory epithelium by quantitative in situ hybridization. To this end, we have cloned 10 very divergent zebrafish odorant receptor molecules by PCR. Individual genes are expressed in sparse olfactory receptor neurons. Analysis of the position of labeled cells in a simplified coordinate system revealed three concentric, albeit overlapping, expression domains for the four odorant receptors analyzed in detail. Such regionalized expression should result in a corresponding segregation of functional response properties. This might represent the first step of spatial encoding of olfactory input or be essential for the development of the olfactory system.

Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium

Age-related macular degeneration, a major cause of blindness for which no satisfactory treatments exist, leads to a gradual decrease in central high acuity vision. The accumulation of fluorescent materials, called lipofuscin, in retinal pigment epithelial cells of the aging retina is most pronounced in the macula. One of the fluorophores of retinal pigment epithelial lipofuscin has been characterized as A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine. An investigation aimed at optimizing the in vitro synthesis of A2E has resulted in the one-step biomimetic preparation of this pigment in 49% yield, readily producing more than 50 mg in one step. These results have allowed for the optimization of HPLC conditions so that nanogram quantities of A2E can be detected from extracts of tissue samples. By using 5% of the extract from individual aged human eyes, this protocol has led to the quantification of A2E and the characterization of iso-A2E, a new A2E double bond isomer; all-trans-retinol and 13-cis-retinol also have been identified in these HPLC chromatograms. Exposure of either A2E or iso-A2E to light gives rise to 4:1 A2E:iso-A2E equilibrium mixtures, similar to the composition of these two pigments in eye extracts. A2E and iso-A2E may exhibit surfactant properties arising from their unique wedge-shaped structures.

Functional domains are specified to single-cell resolution in a Drosophila epithelium

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P{GAL4} enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary (“stellate”) cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an “identified cell” system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Development of an epithelium-specific expression cassette with human DNA regulatory elements for transgene expression in lung airways

The efficient expression of therapeutic genes in target cells or tissues is an important component of efficient and safe gene therapy. Utilizing regulatory elements from the human cytokeratin 18 (K18) gene, including 5′ genomic sequences and one of its introns, we have developed a novel expression cassette that can efficiently express reporter genes, as well as the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, in cultured lung epithelial cells. CFTR transcripts expressed from the native K18 enhancer/promoter include two alternative splicing products, due to the activation of two cryptic splice sites in the CFTR coding region. Modification of the K18 intron and CFTR cDNA sequences eliminated the cryptic splice sites without changing the CFTR amino acid sequence, and led to enhanced CFTR mRNA and protein expression as well as biological function. Transgenic expression analysis in mice showed that the modified expression cassette can direct efficient and epithelium-specific expression of the Escherichia coli LacZ gene in the airways of fetal lungs, with no detectable expression in lung fibroblasts or endothelial cells. This is the first expression cassette which selectively directs lung transgene expression for CFTR gene therapy to airway epithelia.

E-cadherin induces mesenchymal-to-epithelial transition in human ovarian surface epithelium

Ovarian carcinomas are thought to arise in the ovarian surface epithelium (OSE). Although this tissue forms a simple epithelial covering on the ovarian surface, OSE cells exhibit some mesenchymal characteristics and contain little or no E-cadherin. However, E-cadherin is present in metaplastic OSE cells that resemble the more complex epithelia of the oviduct, endometrium and endocervix, and in primary epithelial ovarian carcinomas. To determine whether E-cadherin was a cause or consequence of OSE metaplasia, we expressed this cell-adhesion molecule in simian virus 40-immortalized OSE cells. In these cells the exogenous E-cadherin, all three catenins, and F-actin localized at sites of cell–cell contact, indicating the formation of functional adherens junctions. Unlike the parent OSE cell line, which had undergone a typical mesenchymal transformation in culture, E-cadherin-expressing cells contained cytokeratins and the tight-junction protein occludin. They also formed cobblestone monolayers in two-dimensional culture and simple epithelia in three-dimensional culture that produced CA125 and shed it into the culture medium. CA125 is a normal epithelial-differentiation product of the oviduct, endometrium, and endocervix, but not of normal OSE. It is also a tumor antigen that is produced by ovarian neoplasms and by metaplastic OSE. Thus, E-cadherin restored some normal characteristics of OSE, such as keratin, and it also induced epithelial-differentiation markers associated with weakly preneoplastic, metaplastic OSE and OSE-derived primary carcinomas. The results suggest an unexpected role for E-cadherin in ovarian neoplastic progression.

Water does not flow across the tight junctions of MDCK cell epithelium

Although it has been known for decades that the tight junctions of fluid-transporting epithelia are leaky to ions, it has not been possible to determine directly whether significant transjunctional water movement also occurs. An optical microscopic technique was developed for the direct visualization of the flow velocity profiles within the lateral intercellular spaces of a fluid-absorptive, cultured renal epithelium (MDCK) and used to determine the velocity of the fluid flow across the tight junction. The flow velocity within the lateral intercellular spaces fell to near zero adjacent to the tight junction, showing that significant transjunctional flow did not occur, even when transepithelial fluid movement was augmented by imposition of osmotic gradients.

Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Nuclear Factor-κB (NF-κB) Regulates Proliferation and Branching in Mouse Mammary Epithelium

The nuclear factor-κB (NF-κB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-κB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-κB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-κB during murine early postnatal mammary gland development by observing the consequences of increased NF-κB activity in mouse mammary epithelium lacking the gene encoding IκBα, a major inhibitor of NF-κB. Mammary tissue containing epithelium from inhibitor κBα (IκBα)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IκBα-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IκBα-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IκBα-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IκBα-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-κB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

Cell stress-regulated human major histocompatibility complex class I gene expressed in gastrointestinal epithelium.

Conventional major histocompatibility complex (MHC) class I genes encode molecules that present intracellular peptide antigens to T cells. They are ubiquitously expressed and regulated by interferon gamma. Two highly divergent human MHC class I genes, MICA and MICB, are regulated by promoter heat shock elements similar to those of HSP70 genes. MICA encodes a cell surface glycoprotein, which is not associated with beta 2-microglobulin, is conformationally stable independent of conventional class I peptide ligands, and almost exclusively expressed in gastrointestinal epithelium. Thus, this MHC class I molecule may function as an indicator of cell stress and may be recognized by a subset of gut mucosal T cells in an unusual interaction.

T-cell alpha beta + and gamma delta + deficient mice display abnormal but distinct phenotypes toward a natural, widespread infection of the intestinal epithelium.

Vertebrate immune systems contain T cells bearing either alpha beta or gamma delta T-cell antigen receptors (TCRs). alpha beta T cells perform all well-characterized T-cell effector functions, while the biological functions of gamma delta + cells remain unclear. Of particular interest is the role of gamma delta + cells during epithelial infections, since gamma delta + cells are commonly abundant within epithelia. Eimeria spp. are intracellular protozoa that infect epithelia of most vertebrates, causing coccidiosis. This study shows that in response to Eimeria vermiformis, mice lacking alpha beta T cells display defects in protective immunity, while mice lacking gamma delta + cells display exaggerated intestinal damage, apparently due to a failure to regulate the consequences of the alpha beta T cell response. An immuno-downregulatory role during infection, and during autoimmune disease, may be a general one for gamma delta + cells.

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.

Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.

The mammalian olfactory epithelium (OE) supports continual neurogenesis throughout life, suggesting that a neuronal stem cell exists in this system. In tissue culture, however, the capacity of the OE for neurogenesis ceases after a few days. In an attempt to identify conditions that support the survival of neuronal stem cells, a population of neuronal progenitors was isolated from embryonic mouse OE and cultured in defined serum-free medium. The vast majority of cells rapidly gave rise to neurons, which died shortly thereafter. However, when purified progenitors were co-cultured with cells derived from the stroma underlying the OE, a small subpopulation (0.07-0.1%) gave rise to proliferative colonies. A morphologically identifiable subset of these colonies generated new neurons as late as 7 days in vitro. Interestingly, development of these neuronal colonies was specifically inhibited when purified progenitors were plated onto stromal feeder cells in the presence of a large excess of differentiated OE neurons. These results indicate that a rare cell type, with the potential to undergo prolonged neurogenesis, can be isolated from mammalian OE and that stroma-derived factors are important in supporting neurogenesis by this cell. The data further suggest that differentiated neurons provide a signal that feeds back to inhibit production of new neurons by their own progenitors.