Spatial and temporal expression of histone demethylase,Kdm2a,during murine molar development

The histone demethylase, lysine (K)-specific demethylase 2A (Kdm2a), is highly conserved and expressed ubiquitously. Kdm2a can regulate cell proliferation and osteo/dentinogenic, adipogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from dental tissue. We used quantitative real-time RT-PCR analysis and immunohistochemistry to detect Kdm2a expression during development of the murine molar at embryonic days E12, E14, E16 and E17 and postnatal days P3 and P14. Immunohistochemistry results showed no positive staining of Kdm2a at E12. At E14, Kdm2a was expressed weakly in the inner enamel epithelium, stellate reticulum cells and dental sac. At E16, Kdm2a was expressed mainly in the inner and outer enamel epithelium, stratum intermedium and dental sac, but weaker staining was found in cervical loop and dental papilla cells adjacent to the basement membrane. At E17, the strongest Kdm2a staining was detected in the ameloblasts and stronger Kdm2a staining also was detected in the stratum intermedium, outer enamel epithelium and dental papilla cells compared to the expression at E16. Postnatally, we found that Kdm2a was localized in secretory and mature ameloblasts and odontoblasts, and dentin was unstained. Real-time RT-PCR showed that Kdm2a mRNA levels in murine germ cells increased from E12 to E14 and from E14 to E16; no significant change occurred at E16, E17 or P3, then the levels decreased at P14 compared to P3. Kdm2a expression may be closely related to cell proliferation, to ameloblast and odontoblast differentiation and to the secretion of extracellular enamel and dentin during murine tooth development.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

The ontogeny of mullerian inhibiting substance in granulosa cells of the bovine ovarian follicle

Mullerian Inhibiting Substance (MIS) previously detected in the Sertoli cells of the calf testis has been localized in the granulosa cells of the ovarian Graafian follicle by using an immunoperoxidase technique and a monoclonal antibody (IG8) to MIS that almost completely blocks its biological activity. The immunoperoxidase technique (avidin-biotin complex method) demonstrated specific localization of MIS in the cytoplasm of the ovarian granulosa cells in the bovine Graafian follicles over a wide age span, i.e. one day, one week, three months, two-and-a-half years and five years. The presence of MIS in the ovary implies a function that is as yet unknown.     

Cholinergic nerve development in fetal lung

Cholinergic nerve development in rat fetal lung can be monitored effectively using acetylcholine esterase histochemical staining techniques on frozen sections. Neuroblasts were detected first by these techniques on fetal Day 13 in the indifferent mesenchyme surrounding the trachea. Subsequently, they migrated to the level of the third tracheal branch and matured progressively to ganglia. Neither nerve fibers, ganglia, nor nerve endings of cholinergic variety were detected further in the periphery of the lung. Neuroepithelial cells with lightly staining cytoplasmic acetylcholine esterase-positive granules were first seen on Day 17, after which they were found along the epithelial lining of the entire tracheobronchial tree. These epithelial cells may be potential intrapulmonary chemoreceptors.     

Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants

We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.     

Requirement of mature dendritic cells for efficient activation of influenza A-specific memory CD8+ T cells

It is critical to identify the developmental stage of dendritic cells (DCs) that is most efficient at inducing CD8+ T cell responses. Immature DCs can be generated from monocytes with GM-CSF and IL-4, while maturation is accomplished by the addition of stimuli such as monocyte-conditioned medium, CD40 ligand, and LPS. We evaluated the ability of human monocytes and immature and mature DCs to induce CD8+ effector responses to influenza virus Ags from resting memory cells. We studied replicating virus, nonreplicating virus, and the HLA-A*0201-restricted influenza matrix protein peptide. Sensitive and quantitative assays were used to measure influenza A-specific immune responses, including MHC class I tetramer binding assays, enzyme-linked immunospot assays for IFN-gamma production, and generation of cytotoxic T cells. Mature DCs were demonstrated to be superior to immature DC in eliciting IFN-gamma production from CD8+ effector cells. Furthermore, only mature DCs, not immature DCs, could expand and differentiate CTL precursors into cytotoxic effector cells over 7 days. An exception to this was immature DCs infected with live influenza virus, because of the virus's known maturation effect. Finally, mature DCs pulsed with matrix peptide induced CTLs from highly purified CD8+ T cells without requiring CD4+ T cell help. These differences between DC stages were independent of Ag concentrations or the number of immature DCs. In contrast to DCs, monocytes were markedly inferior or completely ineffective stimulators of T cell immunity. Our data with several qualitatively different assays of the memory CD8+ T cell response suggest that mature cells should be considered as immunotherapeutic adjuvants for Ag delivery.     

Human immunodeficiency virus type 1- and cytomegalovirus-specific cytotoxic T lymphocytes can persist at high frequency for prolonged periods in the absence of circulating peripheral CD4(+) T cells

CD4(+) T cells are thought to be critical in the maintenance of virus-specific CD8(+) cytotoxic T-cell (CTL) responses. In human immunodeficiency virus type 1 (HIV-1) infection, a selective decline in HIV-1-specific CTL as the CD4(+) T-cell count decreases has been reported. Using HLA-peptide tetrameric complexes, we show the presence at high frequency of HIV-1- and cytomegalovirus-specific CD8(+) T cells when the peripheral CD4(+) T-cell count was low or zero in three HIV-1-infected patients. No direct virus-specific CD8(+)-mediated effector activity was seen in these subjects, suggesting antigen unresponsiveness, although tetramer-sorted cells could be expanded in vitro in the presence of interleukin-2 into responsive effector cells. Thus, virus-specific CD8(+) T cells can be maintained in the peripheral circulation at high frequency in the absence of circulating peripheral CD4(+) T cells, but these cells may lack direct effector activity. Strategies designed to overcome this antigen unresponsiveness may be of value in therapies for the treatment of AIDS.