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Collagenase isolated rat hepatocytes were transfected with liposome encapsulated pEJ (LE-pEJ), a plasmid carrying the human cellular activated Ha-rasEJ oncogene. A proliferative cell line was cloned from these cells transfected in vitro. It secreted per day 0.87 µg albumin and 0.32 µg transferrin per 106 cells, and 11.06 nmol free and conjugated bile acids (BA) per mg protein. Also, it metabolized 2-acetylaminoflourene (2-AFAF) into N- and ring-hydroxylated metabolites and 2-aminofluorene at rates of 1.50, 9.73, and 1.98 nmol/mg cell protein/24 hr, respectively. Rats were i.v. injected with both LE-pEJ and LE-p17hGHnneo carrying the hGH cDNA gene, and secreted hGH in the plasma which induced the synthesis of anti-hGH antibodies. A cell line was cloned from cultures of primary hepatocytes isolated from the liver of transfected rats. After 2 to 3 months in culture, this cell line secreted per day 18.9 µg albumin and 11.0 µg transferrin per 106 cells, 38.75 nmol total BA per mg cell protein, and up to 31 ng hGHper 106 cells without cloning hGH recombinant cells. A 24 hr control culture of primary hepatocytes isolated from non transfected rats secreted 25.5 µg albumin and 11.7 µg transferrin per 106 cells, and produced 21.64 nmol total BA and 2.13 nmol N-OH-2-AFAF per mg cell protien. Hence, Ha-rasEJ transfection of either hepatocytes in vitro or liver cells in vivo, initiated cell cycles leading to presumptive proliferating hepatocytes which express liver function.

• Objective:To evaluate the efficacy, tolerance and acceptability an innovative two-layer system (KTwo; Laboratoires URGO) versus an established four-layer bandage system (Profore; Smith & Nephew) in the local management of venous leg ulcers.• Method:A non-inferiority European randomised controlled trial, conducted in 37 centres, in three countries (France, UK and Germany), on patients presenting with venous leg ulcers (VLUs). Participants were adult, non-immunosuppressed patients who presented with non-infected, non-malignant leg ulcers, predominantly of venous origin (ABPI > 0.8), with a surface area of 2–50cm2and duration 1-24 months. Patients were followed-up every 2 weeks for a period of 12 weeks, or until full closure. Visits included a clinical examination, wound area tracings and photographic evidence. The primary endpoint was the percentage of leg ulcers healed after the 12 weeks, with secondary endpoints of relative wound area reduction (RWAR), absolute wound area reduction (AWAR) and the percentage of wounds with RWAR ≥ 40%.• Results:In total, 187 patients were randomised to either the two-layer bandage (2LB, n=94) or four-layer bandage (4LB; n=93) system. The two groups were comparable, with regard to wound and patient characteristics, at baseline. By week 12, 44% of VLUs in the 2LB group and 39% in the 4LB group had healed (intention-to-treat [ITT] analysis). The per-protocol (PP) analysis showed that complete wound closure was obtained in 48% and 38% of the 2LB and 4LB groups, respectively. A non-inferiority margin within -10% is considered as demonstrating a 95% and 97.5% confidence interval (p=0.001). The AWAR was 6.6cm2in the test and 4.9cm2in the control group. The percentage of wounds with a RWAR ≥ 40% was 47% and 44% for the 2LB and 4LB systems, respectively. Pain between dressing changes was reported in 27% of the test and 40% of the control group, and the incidence of adverse events was 17% and 25%, respectively. The 2LB compression system was considered to be significantly easier to apply than the 4LB (p=0.038).• Conclusion:The 2LB system (KTwo) was not seen to be any less effective than a well-known 4LB system (Profore) in the management of VLUs. Furthermore, the 2LB system was considered to be easier to apply, representing an alternative to the conventional treatment with 4LB currently available.• Declaration of interest:This study was sponsored by a grant from Laboratoires URGO, manufacturers of KTwo. S. Bohbot and A. Sauvadet are employees of Laboratoires Urgo. S. Meaume has received monetary compensation as a speaker for Laboratoires Urgo. Data management and statistical analyses were conducted by Vertical (J.C. Kerihuel; Paris, France).

The diagnosis of malignant pleural mesothelioma is difficult, with the most common differential diagnoses being benign pleural diseases and metastatic adenocarcinomas (ADCA). To identify novel markers that would be able to improve diagnostic accuracy, we performed a genome-wide gene expression analysis on tumor cell lines established from pleural effusions (malignant pleural mesothelioma and lung ADCA). This analysis led to the identification of genes encoding novel and pertinent cellular and soluble markers, for which the expression was validated by real-time RT-PCR. Immunohistochemical staining of tumor biopsy specimens with anti–type III collagen antibodies showed positive labeling for mesothelioma cells but not for ADCA cells. Using enzyme-linked immunosorbent assay, we showed that the C-C motif chemokine 2 (CCL2) concentration was significantly higher in pleural effusions from patients with mesothelioma (n= 61) than in subjects with ADCA (n= 25) or with benign pleural effusions (n= 15): median (interquartile range) = 2.99 ng/ml (1.76 to 6.01) vs 0.99 ng/ml (0.51 to 1.83) and 1.47 ng/ml (0.80 to 1.56), respectively, P< 0.0001. Conversely, the galectin-3 concentration was lower in mesothelioma: 11.50 ng/ml (6.73 to 23.53) vs 24.74 ng/ml (20.42 to 70.35) and 17.64 ng/ml (14.81 to 24.68), respectively, P< 0.0001. The areas under the curve for CCL2 were 0.8030 and 0.7716 for the differentiation of mesothelioma from ADCA or benign pleural effusions, respectively. Similarly, the areas under the curve obtained for galectin-3 were 0.7980 and 0.6923, respectively. In conclusion, type III collagen, CCL2, and galectin-3 are promising new diagnostic markers for mesothelioma.

To test the Kirsten‐Ras (Ki‐Ras) alternative prenylation hypothesis in malignant transformation, we used a novel farnesyltransferase inhibitor competitive to farnesyl‐pyrophosphate, RPR130401, and a CaaX peptidomimetic geranylgeranyltransferase‐1 inhibitor GGTI‐298. In Ki‐Ras‐overexpressing transformed adrenocortical cells, RPR130401 at 1–10 μM inhibited very efficiently the [3H]farnesyl but not [3H]geranylgeranyl transfer to Ras. However, proliferation of these cells was only slightly sensitive to RPR130401 (IC50=30 μM). GGTI‐298 inhibited the growth of these cells with an IC50of 11 μM but cell lysis was observed at 15 μM. The combination of 10 μM RPR130401 and 10 μM GGTI‐298 inhibited efficiently (80%) cell proliferation. These combined inhibitors but not each inhibitor alone blocked the cell cycle in G0/G1and disrupted MAP kinase activation. Thus, combination of two inhibitors, at non‐cytotoxic concentrations, acting on the farnesyl‐pyrophosphate binding site of the farnesyltransferase and the CaaX binding site of the geranylgeranyltransferase‐1 respectively is an efficient strategy for disrupting Ki‐Ras tumorigenic cell proliferation.

We have established cell lines that contain plasmid constructs carrying combinations of genes coding for human growth hormone (hGH) or the human hepatitis B virus surface antigen complex (HBsAg), placed under the expression control of a human heat shock promoter. In addition, neomycin resistance genes and oncogenes have been employed for selection of cell lines. These lines have been passed as tumours in rodents. After proteolytic dissociation of cells from excised tumours, up to 109tumour cells were obtained in culture, per (20 mm diameter) tumour. These cells can produce, after heat induction, approximately similar quantities of hGH or HBsAg as the parental cells prior to passage as tumours in animals. In addition, we have cultivated one of these cell lines on spherical microcarriers (cytodex 1R) in 1 liter fermentors, using media with or without serum. Under these conditions, a daily production of mg quantities of hGH per tumour, per heat shock, was obtained. This procedure is applicable to the pilot scale production of natural proteins, or their protein-engineered variants.

La st?rilit? m?le nucl?ocytoplasmique ?447? de la f?verole (Vicia faba L) manifeste de fr?quentes instabilit?s qui s'expriment par des retours ? la fertilit? parmi les lign?es en cours de conversion vers la st?rilit?. La mise en ?vidence de particules cytoplasmiques pseudovirales li?es ? la st?rilit? et la forte relation entre teneur en particules et degr? de stabilit? du maintien a permis d'envisager l'utilisation de cette teneur comme crit?re de s?lection. Un test immunoenzymatique du type Enzyme linked immunosorbant assay (ELISA) apr?s l'obtention d'un antis?rum et d'immunoglobulines de classe G (IgG) a ?t? d?velopp? pour mesurer la teneur en particules pseudovirales dans la plante. L'?tude de cette teneur dans les divers organes de la plante, de leur r?partition dans les diff?rents niveaux foliaires, selon l'?ge de la plante et selon la temp?rature, d?bouche sur une nouvelle m?thode d'?valuation de la st?rilit? m?le nucl?ocytoplasmique ?447?. Cette m?thode d'?valuation de la st?rilit? m?le est compar?e ? la m?thode directe bas?e sur l'observation pollinique. La bonne h?ritabilit? du caract?re teneur en particules permet de s?lectionner efficacement pour le maintien de la st?rilit? ?447?. Use of an immunoenzymatic assay in breeding for higher stability in cytoplasmic male sterile faba bean (Vicia faba L). The nucleocytoplasmic male sterility "447" of faba bean (Vicia faba L) shows frequent instabilities expressed by the occurrence of male fertile plants in backcross progenies. The discovery of cytoplasmic virus-like particles (VLPs) linked to male sterility and the observation of a strong relationship between particle contents and stability level led the breeder to utilise virus-like particle content as a selection criterion. An enzyme linked immunosorbant assay (ELISA) test has been developed through antiserum and IgG production for quantifying the VLPs. The evaluation of VLP contents in different plant organs (tables I and II), their repartition according to node level (fig 2), plant age (tables III and IV) and temperature have led to a new method for male sterility evaluation in breeding programs. The efficiency of this method appears higher when compared to the direct method of pollen observation (fig 5). The good heritability of VLP content character analyzed in figs 3 and 4 allows efficient selection for stabilizing male sterility ?447?.

Flavescence dorée (FD), a grapevine yellows disease, is caused by a mycoplasma-like organism (MLO). A colloidal gold indirect immunolabeling technique identified MLO in salivary glands of a vector leafhopper, Euscelidius variegatus. After aldehyde fixation, tissue samples were prepared by cryoultramicrotomy or embedding in acrylic resins. Double fixation with aldehydes and osmium retroxide, followed by embedding in epon, was also performed. Thin or semi-thin serial sections were treated with polyclonal anti-FD-MLO rabbit antibodies, then with gold-conjugated anti-rabbit IgG. Labeling was revealed using the silver enhancement technique for light microscopy. MLO in frozen thin sections of glands were efficiently labeled. Optimal results were obtained with 4% paraformaldehyde-0.1% glutaraldehyde fixation and low-temperature embedding in LR White resin. Both scattered MLO and unusual dense forms of MLO were easily detected with the electron-dense gold probe. This method distinguished MLO from other membrane-limited bodies and provided a good tool for studying infection in large regions of FD-infected tissues by light microscopy.

Flavescence doréc (FD) MLO is experimentally maintained in broadbean and transmitted by the leafhopper vector Euscelidius variegatus. Indirect ELISA allowed the study, according to time after feeding access, of the distribution of MLO in the different organs of the vector. Colloidal gold indirect immunolabeling was used to visualize the MLO cells in the FD-infected organs. The alimentary tract was the first organ to become infected. Infected cells were not numerous but were crowded with MLO particles. Infection did not seem to be localized in specific cells. MLO multiplied in the cytoplasm of midgut cells and reached the hemolymph either by crossing between two cells or through individual infected cells. By circulating in the hemolymph, MLO infected salivary glands, in which they subsequently multiplied. Abdomen fat bodies and finally brain were infected; an increase in the number of MLO was observed in these organs. Observation by electron microscopy showed that MLO infection in fat bodies was important, with no particular localization. In contrast, MLO infection in the brain was low. Mycetomes were occasionally infected. No MLO particles were detected in malpighian tubules or in male and female genital organs. Copyright 1994, 1999 Academic Press

Phytoplasmas are plant-pathogenic mollicutes restricted to phloem. They belong to several groups in a unique phylogenetic clade. Non-related phytoplasmas may infect the same plant species, often with similar symptoms. Hence methods are needed to specifically localize phytoplasmas and to study their multiplication and movement in their hosts. Conditions for post-embeddingin situhybridization (ISH) with transmission electron microscopy using oligodeoxynucleotides as probes for labelling of phytoplasmas in plant tissues have been searched. Sections of acrylic resin-embedded tissues of phytoplasma-infected periwinkle were submitted to ISH using digoxigenin or biotin-labelled oligoprobes (22 mers). These probes were the complementary sequences of primers used in group-specific polymerase chain reaction (PCR) amplification of 16S rDNA of stolbur and elm yellows phytoplasma, respectively. Together with preliminary digestion with pepsin, differentin situdenaturation conditions and formamide concentrations were tested. The grids were incubated in the hybridization mixture at 37°C overnight. Detection of hybridized material was performed with gold immunocytochemistry. Specificity of labelling was checked with appropriate controls. Stringency conditions could be found to ensure specific hybridization with such short probes. A specific labelling was obtained for stolbur phytoplasma on groups of mature as well as senescent phytoplasma cells. The results show that oligonucleotides may be used as probes for phytoplasma identification in post-embedding ISH with electron microscopy.

The steroids in urine from normal pregnant women have been studied. After extraction of conjugate steroids, solvolysis and enzymatic hydrolysis, the liberated steroids were separated by chromatography on Sephadex LH-20, and were analysed by gas-liquid chromatography and gas chromatography mass spectrometry. The following steroids were isolated and completely identified in the LH-20 fraction 7: 5ß-pregnane-3a,20a-diol, 5ß-pregnane-3a,17,20a-triol, 5a-pregnane-3a,20a,21-triol and 5a-pregnane-3ß,16a,20a-triol. In addition, two metabolites tentatively identified as 5?-pregnane-2?,3?,20?-triol and 2?,3?,16?-trihydroxy-5?-pregnan-20-one, have not been reported as occurring in urine from pregnant women. The 5ß-pregnane-3a,20a,21-triol was detected only in the third trimester of pregnancy and the urinary excretion values are between 320 and 650 µg per 24 h. With the present data, it is not possible to establish the precursor(s) of this steroid. However, these results tentatively suggest that 5ß-pregnane-3a,20a,21-triol arises from foeto-placental unit.

Flavescence dorée (FD) and other types of grapevine yellows are serious diseases of grapevines caused by phytoplasmas. FD is spread by a leafhopper species, and it has been shown to be transmitted by using infected cuttings. Data are presented for the effective use of hot‐water treatment to eliminate FD. Immersion time has to be increased logarithmically when temperature is reduced linearly. The effective treatments range from 10 h at 40°C to 10 min at 55°C. The eggs of the leafhopper vector of FD, Scaphoideus titanus, are also killed by immersion at 50°C for 45 minutes. This temperature/time combination is recommended for treating dormant cuttings of grapevines for the elimination of FD.

The direct analysis of pregnancy urinary steroids by GC-MS appears very difficult due to the numerous steroids and their great diversity of relative amounts, especially in overlapping peaks. Urinary steroids are hydrolyzed and extracted according to classical methods. Fractionation is carried out on Sephadex LH-20. On each fraction, separation and identification of steroids were done by GC-MS and then quantitated by GLC. We have positively identified and quantitated three 11-deoxy-17-oxosteroids, two 11-oxy-17-oxosteroids, five 16-OH-17-oxosteroids, one 18-OH-17-oxosteroid, 3 stereoisomers of 3-OH-5-pregnan-20-one, and three of pregnane-3,20-diol, seven hydroxylated compounds of 3-OH-pregnan-20-one, the 5α-pregnane-3α,17α,20α-triol, four isomers of pregnane-3,16,20-triol, two isomers of pregnane-3,20,21-triol, the 5-pregnene-3β,20α-diol and 5-pregnene-3β,16α,20α-triol, 17-oxo-androstane-3,17-diol, two 5-androstenetriols, two androstanetriols and four estrogens. The two main interests of this technique rely on the module liquid fractionation which gives groups of steroids having well differentiated retention time and the quantitative analysis.

A method is described for the separation, identification and quantitation of endogenous hydrogenated metabolities of corticosterone in rat liver Corticosterone, 5a- and 5ß-dihydrocorticosterone, 3a,11ß,21-trihydroxy-5a-pregnane-3,20-dione, 3ß,11ß,21-trihydroxy-5a-pregnane-3,20-dione, 3a,11ß,21-trihydroxy-5ß-pregnane-3,20-dione, 3ß,11ß,21-trihydroxy-5a-pregnane-3,20-dione, 5a-pregnane-3a,11ß,20a,21-tetrol, 5a-pregnane-3a,11ß,20ß,21-tetrol and 5a-pregnane-3ß,11ß,20ß,21-tetrol are specifically quantitated in one analysis by mass fragmentography all as their O-methyloxime pertrimethylsilyl derivatives. A pronounced difference in the amounts of these nine metabolites between male and female adult rats was found, while in newborn rats only the two tetrahydro-compounds bearing a 5a-hydrogen were detected in both sexes. On the other hand 3a,11ß,1 5a,21-tetrahydroxy-5a-pregnan-20-one was detected only in the adult female as its second prominent metabolite of corticosterone. Corticosterone metabolites being biochemical markers of the sex-linked differentiation of the hepatocytes, their mass fragmentographic assay is proposed as a tool for the quantitative analysis of this type of gene expression.