Indication of selective growth of human endometrial epithelial cells on extracellular matrix

Summary The culturing of human endometrium in conventional plastic dishes and media is only partially successful, mainly because a growth of a heterogeneous population of cells is achieved. Naturally produced extracellular matrix closely resembles the subepithelial basement membrane and seems to affect both growth and differentiation of cells. These qualities of the extracellular matrix (ECM) were applied for obtaining endometrial epithelial cultures. Endometrial tissue specimens were plated after slicing on ECM-coated dishes and kept for up to 8 d. The growth of a confluent homogeneous tissue composed of polygonal epithelial-like cells was demonstrated. To further characterize these cells, cultures were examined by scanning electron microscopy and transmission electron microscopy. Scanning electron microscopy revealed flattened polygonal cells covered with microvilli, among which ciliated cells were observed. By transmission electron microscopy the cells were seen as a monolayer, with some cells overlapping, closely adherent to the matrix. Microvilli, as well as intracellular vacuoles and glycogen granules were observed. Cell type specific cytoskeletal markers were demonstrated by antibodies to intermediate filament proteins (keratin and epithelial membrane antigen). Taken together, the morphologic and immunohistochemical studies indicate that a selective growth of the epithelial component of endometrial tissue was obtained after plating unprocessed endometrial tissue fragments on ECM-coated culture dishes. This work was supported by PHS grant no. CA 30289 to J.V.     

On the Relation between Effector Concentration and the Rate of Induced Enzyme Synthesis

The Jacob and Monod scheme for the regulation of enzyme formation leads to the following relation between the relative rate of enzyme synthesis α and cellular effector concentration E (the lower sign is for repressible systems): log (α/1 - α - α_b) = ± n log [E] + log α_b ± log K₁. This equation permits linear plotting of experimental data and the evaluation of three quantities: n, the number of effector molecules combining with a repressor molecule, K₁, the dissociation constant of this interaction and K₂/R_t, the ratio of repressor-operator dissociation constant to total repressor concentration. Measurements on the repression of alkaline phosphatase in Escherichia coli as a function of phosphate concentration are reported and fit the proposed equation with n = 1, indicating that the binding of a single phosphate to the repressor species may be sufficient to cause repression. K₁ of this interaction was found to be 0.58 ±0.11 × 10^-3 M. The available data regarding the enzymes of the lac operon in a variety of E. coli strains, and several other enzymes are analyzed. It is confirmed that the lac repressor interacts with 2 isopropyl thiogalactoside (IPTG) molecules to relieve repression with a K₁ = 50 ±20 × 10^-12 M². In some strains, separate binding constants for the first and second IPTG molecules can be evaluated.     

Loss of Paneth Cell Autophagy Causes Acute Susceptibility to Toxoplasma gondii-Mediated Inflammation

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Efficient Flp-Int HK022 dual RMCE in mammalian cells

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp–Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp–Int (HK022) pair depends on the variable input of the recombinases—the amount of the recombinase expression vectors added at transfection—and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of ‘simultaneous’ dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.     

Development of methods for detection and Agrobacterium-mediated transformation of the sterile,endophytic fungus Muscodor albus

Symbiotic endophytes, unlike plant pathogens, do not usually induce visible host response. This may constraint the researcher's decision whether a plant has been successfully infected by the endophyte. In order to properly study the establishment, development and progress of an endophyte in the host plant and host-endophyte interactions, methods for the identification and localization of endophytic microorganisms are needed. Towards this aim, we focused at two levels: (A) We constructed M. albus-specific primers for polymerase chain reaction (PCR). In vitro, these primers specifically detected only M. albus strains and not isolates of related fungi (such as Daldinia sp. and a Xylariaceae sp.). (B) For direct visualization of the fungi, we inserted a reporter gene (gfp) into M. albus hyphae using Agrobacterium-mediated transformation. Since M. albus is a sterile fungus (i.e., without spores or fungal fruiting bodies), we used chopped fungal mycelium for the transformation procedure. We transformed three different isolates of M. albus using Agrobacterium-mediated transformation. Fifty-nine different transformants were collected with a transformation efficacy of 0.0004–0.0026%. Although PCR-based detection and direct visualization of the transformants in planta were unsuccessful, all tested transformants (with one exception) exhibited similar biological activity to their cognate wild type. This work provides a significant step forward in molecular research of the relationships between this endophytic genus and their hosts.     

X-ray crystal structure of the hypothetical phosphotyrosine phosphatase MDP-1 of the haloacid dehalogenase superfamily

The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 A X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.     

Excess mortality in persons with severe mental disorders: a multilevel intervention framework and priorities for clinical practice,policy and research agendas

Excess mortality in persons with severe mental disorders (SMD) is a major public health challenge that warrants action. The number and scope of truly tested interventions in this area remain limited, and strategies for implementation and scaling up of programmes with a strong evidence base are scarce. Furthermore, the majority of available interventions focus on a single or an otherwise limited number of risk factors. Here we present a multilevel model highlighting risk factors for excess mortality in persons with SMD at the individual, health system and socio‐environmental levels. Informed by that model, we describe a comprehensive framework that may be useful for designing, implementing and evaluating interventions and programmes to reduce excess mortality in persons with SMD. This framework includes individual‐focused, health system‐focused, and community level and policy‐focused interventions. Incorporating lessons learned from the multilevel model of risk and the comprehensive intervention framework, we identify priorities for clinical practice, policy and research agendas.