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.     

Bioconversion of substituted naphthalenes to the corresponding salicylic acids

The Pseudomonas fluorescens N3 was isolated from soil for its ability to utilize naphthalene as a carbon source. The strain transforms 2,3-dimethyl-, 2-methoxy-, 1- and 2-ethylnaphthalenes to the corresponding salicylic acids competitively with chemical synthesis. The identification of 2-hydroxy-2-carboxy-7-ethylchromane by biotransformation of 2-ethylnaphthalene, contributes to elucidating the steps involved in the catabolic pathways of naphthalenes to salicylaldehydes. Correspondence to: F. Pelizzoni     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Signal transduction during liver regeneration: role of insulin and vasopressin.

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of 11, 4, 5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane-bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol-derived signal molecules are associated with hepatic regeneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle.     

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     

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.     

The primary structure of the Fab fragment of protein KAU, a monoclonal immunoglobulin M cold agglutinin

The complete amino acid sequence of the Fab fragment of protein KAU, a human monoclonal cold agglutinin (IgMk) with anti-I activity, was determined. The light chain (L-chain) consists of 215 residues; the variable (V)L region belongs to the Hum/Kv325/kIIIb sub-subgroup that is preferentially selected in human IgM autoimmune response. The joining (J) region is encoded by the Jk4 gene, and the constant region (C)L domain expresses the km3 allotypic marker. The Fd fragment contains 232 amino acids, and 120 of them comprise the variable domain. The VH region corresponds to the VHIV subgroup and is closely related to the VHIV 2.1 gene isolated from genomic DNA expressed in peripheral blood of a healthy Caucasian. The complementary-determining region 1 has a unique amino acid (Asp) at position 31, and the complementary-determining region 3 codified by the diversity segment (D) gene, shows poor homology with other known D sequences. The joining segment with two unusual substitutions at the D-J junction is encoded by the JH4 gene. Thus, cold agglutinin KAU is an IgM, VkIIIb-Jk4-km3; VHIV-JH4-C mu.     

Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA.

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant density, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhis, tRNAval, tRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.