Functional analysis of gap junctions in ovarian granulosa cells: distinct role for connexin43 in early stages of folliculogenesis

Ovarian granulosa cells arecoupled via gap junctions containing connexin43 (Cx43 or -1connexin). In the absence of Cx43, granulosa cells stop growing in anearly preantral stage. However, the fact that granulosa cells of maturefollicles express multiple connexins complicated interpretation of thisfinding. The present experiments were designed to clarify the role ofCx43 vs. these other connexins in the earliest stages offolliculogenesis. Dye injection experiments revealed that granulosacells from Cx43 knockout follicles are not coupled, and this wasconfirmed by ionic current injections. Furthermore, electron microscopyrevealed that gap junctions are extremely rare in mutant granulosacells. In contrast, mutant granulosa cells were able to form gapjunctions with wild-type granulosa cells in a dye preloading assay. Itwas concluded that mutant granulosa cells contain a population of connexons, composed of an unidentified connexin, that do not normally contribute to gap junctions. Therefore, although Cx43 is not the onlygap junction protein present in granulosa cells of early preantralfollicles, it is the only one that makes a significant contribution tointercellular coupling.

     

Solid state coordination chemistry of the oxovanadium-diphosphonate system: Structural consequences of aryl tethers on materials of the type cation/VxOy/diphos, where cation = organoammonium and diphos = 1,4-phenyldiphosphonic acid

Hydrothermal reactions of V₂O₅, 1,4-phenyldiphosphonic acid and an appropriate organoamine, in the presence of HF as solubilizer, were exploited to prepare a series of materials of the general type [organoammonium cation]_n[V_xO_y(H_mO₃PC₆H₄PO₃H_p)₃]. Compound 1, [H₃N(CH₂)₄NH₃][V₂O₄(O₃PC₆H₄PO₃)], exhibits a one-dimensional V-P-O substructure, linked through the phenyl tethers of the ligand into a layer. Compound 1 is a unique example of a V(V)-diphosphonate phase. Compounds 2 and 3, [H₃N(CH₂)₂NH₃][V₂O₂(O₃PC₆H₄PO₃H)₂] and [H₂pip][V₂O₂(O₃PC₆H₄PO₃H)₂] (H₂pip = piperazinium), exhibit identical two dimensional substructures, constructed from ribbons connected through the phenyl tethers of the ligands. The three-dimensional framework of [H₃N(CH₂)₇NH₃]₂[V₃O₄(O₃PC₆H₄PO₃)₂] (4) consists of V-P-O layers characterized by trinuclear V(IV)-oxide subunits and 9 and 12 polyhedral connect rings; the layers are buttressed by the phenyl spacers to provide the typical “pillared” layer structure common to metal diphosphonate materials. Compound 5, [H₂dabco][V₂F₃O₂(O₃PC₆H₄PO₃H)]·H₂O, is also three-dimensional with oxyfluoro-vanadium(IV) chains linked through the diphosphonate ligands into a framework structure with void spacers to accommodate the {H₂dabco}²⁺ cations (dabco = diamino bicyclo octane). The magnetic properties of 2-5 reflect the structural characteristics of the materials.     

A modular treatment of molecular traffic through the active site of cholinesterase

We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.     

Age-dependent alterations of c-fos and growth regulation in human fibroblasts expressing the HPV16 E6 protein.

Normal human cells in culture become senescent after a limited number of population doublings. Senescent cells display characteristic changes in gene expression, among which is a repression of the ability to induce the c-fos gene. We have proposed a two-stage model for cellular senescence in which the mortality stage 1 (M1) mechanism can be overcome by agents that bind both the product of the retinoblastoma susceptibility gene (pRB)-like pocket proteins and p53. In this study we determined whether the repression of c-fos at M1 was downstream of the p53 or pRB-like "arms" of the M1 mechanism. We examined c-fos expression during the entire lifespan of normal human fibroblasts carrying E6 (which binds p53), E7 (which binds pRB), or both E6 and E7 of human papilloma virus type 16. The results indicate a dramatic change in cellular physiology at M1. Before M1, c-fos inducibility is controlled by an E6-independent mechanism that is blocked by E7. After M1, c-fos inducibility becomes dependent on E6 whereas E7 has no effect. In addition, a novel oscillation of c-fos expression with an approximately 2-h periodicity appears in E6-expressing fibroblasts post-M1. Accompanying this shift at M1 is a dramatic change in the ability to divide in low serum. Before M1, E6-expressing fibroblasts growth arrest in 0.3% serum, although they continue dividing under those conditions post-M1. These results demonstrate the unique physiology of fibroblasts during the extended lifespan between M1 and M2 and suggest that p53 might participate in the process that represses the c-fos gene at the onset of cellular senescence.     

Fast deprotection of synthetic oligodeoxyribonucleotides using standard reagents under microwave irradiation

Fast methods for the removal of permanent amide exo-cyclic protective groups widely used in phosphoramidite-method DNA synthesis are desirable for many genomics and proteomics applications. In this communication, we present a method for the deprotection of a range of N-acyl deoxyribonucleosides (T, dA Bz, dC Bz, dC Ac, dG ibu, dG PAC) and synthetic oligodeoxyribonucleotides, ranging in length from 5-mer to 50-mer. Oligodeoxyribonucleotides were synthesized using standard amide protecting groups (dA Bz, dC Bz, dG ibu) and phosphoramidite chemistry on cis-diol solid phase support. This deprotection method utilizes 29% aqueous ammonia solution at 170 degrees C for 5 minutes under monomode microwave irradiation at a 20-nmole reaction scale. Reaction products were analyzed by TLC, RP-HPLC, CE, ESI-MS, real-time PCR, agarose gel electrophoresis, and by DNA uracil glycosylase (UDG) and phosphodiesterase I (PDE) enzymatic digestions.     

Molecular systematics of basal subfamilies of ants using 28S rRNA (Hymenoptera: Formicidae)

For many years, the ant subfamily Ponerinae was hypothesized to contain the basal (early branching) lineages of ants. Recently the Ponerinae were reclassified into six poneromorph subfamilies based on morphological analysis. We evaluate this new poneromorph classification using 1240 base pairs of DNA sequence data obtained from 28S rRNA gene sequences of 68 terminal taxa. The molecular tree supported the monophyly of the ant family Formicidae, with 100% parsimony bootstrap (PB) support and posterior probabilities (PP) of 1.00, with the ant subfamily Leptanillinae as a sister group to all other ants (PB=62, PP=93). However, our analyses strongly support the polyphyly of the Poneromorph subfamilies (sensu Bolton). The Ectatomminae and Heteroponerinae are more closely related to the Formicoid subfamilies than to the rest of the poneromophs (PB=96, PP=100). The Amblyoponinae (PB=52, PP=96), Paraponerinae (PB=100, PP=100), Ponerinae (PB<50, PP=71), and Proceratiinae (PB=98, PP=100) appear as distinct lineages at the base of the tree and are identified as a poneroid grade. Monophyletic origins for the poneroid subfamilies Amblyoponinae, Paraponerinae, Ponerinae and Proceratiinae are supported in our analysis. However, the genus Platythyrea forms a distinct sister group to the Ponerini within the Ponerinae. The Heteroponerinae, based on our sample of Heteroponera, are associated with the subfamily Ectatomminae (PB=98, PP=100). Furthermore, our data indicate the genus Probolomyrmex belongs to the Proceratiinae as suggested by recent morphological analysis (PB=98, PP=100).     

Gene expression profiling and molecular characterization of antimony resistance in Leishmania amazonensis

Background

Drug resistance is a major problem in leishmaniasis chemotherapy. RNA expression profiling using DNA microarrays is a suitable approach to study simultaneous events leading to a drug-resistance phenotype. Genomic analysis has been performed primarily with Old World Leishmania species and here we investigate molecular alterations in antimony resistance in the New World species L. amazonensis.

Methods/Principal Findings

We selected populations of L. amazonensis promastigotes for resistance to antimony by step-wise drug pressure. Gene expression of highly resistant mutants was studied using DNA microarrays. RNA expression profiling of antimony-resistant L. amazonensis revealed the overexpression of genes involved in drug resistance including the ABC transporter MRPA and several genes related to thiol metabolism. The MRPA overexpression was validated by quantitative real-time RT-PCR and further analysis revealed that this increased expression was correlated to gene amplification as part of extrachromosomal linear amplicons in some mutants and as part of supernumerary chromosomes in other mutants. The expression of several other genes encoding hypothetical proteins but also nucleobase and glucose transporter encoding genes were found to be modulated.

Conclusions/Significance

Mechanisms classically found in Old World antimony resistant Leishmania were also highlighted in New World antimony-resistant L. amazonensis. These studies were useful to the identification of resistance molecular markers.     

HistoChoice® as an alternative to formalin fixation of undecalcified bone specimens

We compared histochemical and immunohistochemical staining as well as fluorochrome labeling in murine bone specimens that were fixed with 10% neutral buffered formalin to those fixed with HistoChoice®. We showed that sections from undecalcified tibiae fixed for 4 h in HistoChoice® resulted in enhanced toluidine blue and Von Kossa histochemical staining compared to formalin fixation. HistoChoice® produced comparable or improved staining for alkaline phosphatase. Acid phosphatase localization was better in formalin fixed specimens, but osteoclasts were visuralized more easily in HistoChoice® fixed specimens. As expected, immunohistochemical labeling was antibody dependent; some antibodies labeled better in HistoChoice® fixed specimens while others were better in formalin fixed specimens. Toluidine blue, Von Kossa, and alkaline phosphatase staining of sections fixed for 12 h produced sections that were similar to 4 h fixed sections. Fixation for 12 h preserved acid phosphatase activity better. Increasing fixation to 12 h affected immunolocalization differentially. Bone sialoprotein labeling in HistoChoice® fixed specimens was comparable to formalin fixed samples. On the other hand, after 12 h formalin fixation, osteocalcin labeling was comparable to HistoChoice®. For most histochemical applications, fixing murine bone specimens for 4 h with HistoChoice® yielded superior staining compared to formalin fixation. If immunohistochemical localization is desired, however, individual antibodies must be tested to determine which fixation process retains antigenicity better. In addition, there was no detectable difference in the intensity of fluorochrome labeling using either fixative. Finally, fixation duration did not alter the intensity of labeling.