Ovarian mesothelial and extramesothelial cells in interactive culture

Summary The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14±4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93±0.40 × 10⁶ cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%–300 U/ml) under periodical resuspension and gentle scraping of SC (1.40±0.25 × 10⁶/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4μg/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2′-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.     

Electrotransformation of Streptococcus agalactiae with plasmid DNA

Abstract A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli - Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-μl aliquot containing about 5×10⁹ colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 μg. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2×10⁴ cfu μg⁻¹. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae .     

Presence of telomeric and subtelomeric sequences at the fusion points of ring chromosomes indicates that the ring syndrome is caused by ring instability

In situ hybridization of a telomeric (TTA-GGG) _n sequence to metaphases from three cases of ring chromosome, involving respectively chromosomes 4, 16, and 20, showed the presence of the cognate sequences in all three rings. To investigate whether these ring chromosomes originated by telomere-telomere fusion, we determined, by in situ hybridization, whether telomere-associated sequences and/or specific distal sequences were still present in the ring chromosomes. The finding that these sequences were preserved in all the ring chromosomes strongly indicates that they originated by telomere-telomere fusion. All three subjects carrying the ring chromosomes are affected by the so-called ring syndrome, with failure to thrive, minor dysmorphic signs and no major anomalies. The r(4) patient has the ring in mosaic form with a normal cell line and has normal intelligence. The r(16) and the r(20) patients have moderate mental retardation and suffer from seizures. We conclude that the ring syndrome, even in its more severe manifestation, is caused by ring chromosome instability.     

Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine     

The effect of membrane preparation and cellular maturation on human erythrocyte adenylate cyclase

We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5′-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg²⁺ or Mn²⁺. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5′-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.     

Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, -methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.     

Enhanced erythroid burst formation in mice after testosterone treatment

A single administration of testosterone propionate (TP) in ex-hypoxic polycythemic mice induces, at 24 hr after androgen, an amplification of the erythroid burst-forming unit (BFU-E or B) pool in marrow. This phenomenon is not associated with an amplification of the erythroid colony-forming unit (CFU-E or E) compartment and is followed by its depletion. In the other hand, the 36–49 hr rise of erythropoietin (Ep) levels in serum is followed by a 60-hr amplification of the E pool. It is suggested that the latter phenomenon is mediated by enhanced Ep production, whereas the early amplification of the B compartment may derive from a direct influence of TP at the stem cell level.