Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells.

STAT transcription factors are induced by a number of growth factors and cytokines. Within minutes of induction, the STAT proteins are phosphorylated on tyrosine and serine residues and translocated to the nucleus, where they bind to their DNA targets. The leukemia inhibitory factor (LIF) mediates pleiotropic and sometimes opposite effects both in vivo and in cultured cells. It is known, for example, to prevent differentiation of embryonic stem (ES) cells in vitro. To get insights into LIF-regulated signaling in ES cells, we have analyzed protein-binding and transcriptional properties of STAT recognition sites in ES cells cultivated in the presence and in the absence of LIF. We have detected a specific LIF-regulated DNA-binding activity implicating the STAT3 protein. We show that STAT3 phosphorylation is essential for this LIF-dependent DNA-binding activity. The possibility that ERK2 or a closely related protein kinase, whose activity is modulated in a LIF-dependent manner, contributes to this phosphorylation is discussed. Finally, we show that the multimerized STAT3-binding DNA element confers LIF responsiveness to a minimal thymidine kinase promoter. This, together with our observation that overexpression of STAT3 dominant-negative mutants abrogates this LIF responsiveness, clearly indicates that STAT3 is involved in LIF-regulated transcriptional events in ES cells. Finally, stable expression of such a dominant negative mutant of STAT3 induces morphological differentiation of ES cells despite continuous LIF supply. Our results suggest that STAT3 is a critical target of the LIF signaling pathway, which maintains pluripotent cell proliferation.

Unusual sequence element found at the end of an amplicon.

In a polyomavirus-transformed rat cell line, designated LPT, the polyomavirus DNA is integrated into a single chromosomal site. Treatment of LPT cells with carcinogens induces amplification of the integrated virus DNA and flanking cellular sequences. We show that the amplification is arrested within a specific cell DNA segment that maps 1.3 to 1.85 kilobases beyond one virus-cell DNA junction, defined as the left junction. We also present the sequence of an 897-base-pair fragment spanning the arrest site. This fragment contains an unusual sequence element, which consists of two contiguous components, a potential cruciform with stems of 6 base pairs and a d(G-A)27 X d(T-C)27 tract, and maps 1,497 to 1,564 nucleotides beyond the left junction. The possibility that this unusual sequence plays a role in the arrest of the amplification process is discussed.

Interference footprinting analysis of telomerase elongation complexes.

Telomerase is a reverse transcriptase that adds single-stranded telomeric repeats to the ends of linear eukaryotic chromosomes. It consists of an RNA molecule including a template sequence, a protein subunit containing reverse transcriptase motifs, and auxiliary proteins. We have carried out an interference footprinting analysis of the Tetrahymena telomerase elongation complexes. In this study, single-stranded oligonucleotide primers containing telomeric sequences were modified with base-specific chemical reagents and extended with the telomerase by a single (32)P-labeled dGMP or dTMP. Base modifications that interfered with the primer extension reactions were mapped by footprinting. Major functional interactions were detected between the telomerase and the six or seven 3'-terminal residues of the primers. These interactions occurred not only with the RNA template region, but also with another region in the enzyme ribonucleoprotein complex designated the telomerase DNA interacting surface (TDIS). This was indicated by footprints generated with dimethyl sulfate (that did not affect Watson-Crick hydrogen bonding) and by footprinting assays performed with mutant primers. In primers aligned at a distance of 2 nucleotides along the RNA template region, the footprints of the six or seven 3'-terminal residues were shifted by 2 nucleotides. This shift indicated that during the elongation reaction, TDIS moved in concert with the 3' ends of the primers relative to the template region. Weak interactions occurred between the telomerase and residues located upstream of the seventh nucleotide. These interactions were stronger in primers that were impaired in the ability to align with the template.

Hydrodynamic and geochemical constraints on pesticide concentrations in the groundwater of an agricultural catchment (Brévilles, France).

The monitoring of a spring and seven piezometers in the 3km(2) Brévilles agricultural catchment (France) over five and a half years revealed considerable spatial and temporal variability in the concentrations of atrazine and its metabolite deethylatrazine (both systematically quantified at the outlet spring): maximum 0.97 and 2.72microgL(-1), mean 0.19 and 0.59microgL(-1), respectively. Isoproturon, the pesticide applied in the greatest amount, was detected in only 10 of the 133 samples. These observations can only partly be explained by land use and intrinsic pesticide properties. Geochemical measurements and tritium dating showed the importance of the stratification of the sandy saturated zone and the buffer function of the unsaturated limestone. Principal component analysis on 39 monthly data series of atrazine, deethylatrazine, nitrate, chloride and piezometric levels revealed a temporal structuring of the data possibly reflecting the existence within the aquifer of two different reservoirs with time-variable contributions.

Separation of the contributions from gamma- and UV-radiation to the EPR spectra of tooth enamel plates.

Anisotropy of EPR spectra of tooth enamel plates irradiated with gamma-rays and UV light has been studied. UV-irradiated enamel plates exhibit a stronger anisotropy than gamma-irradiated plates. Investigation of samples cut out of different teeth and irradiated to different doses showed that the value of anisotropy is characteristic of each type of irradiation; it equals to approximately 0.35 for gamma- rays and 0.50 for UV light. It is suggested that the difference in the anisotropy values can be attributed to different relative amounts of oriented and disordered radicals produced by the two types of radiation. This can be used for separating gamma- and UV-contributions to the total EPR signal of a tooth exposed to both types of radiation.

Pesticides in the groundwater of a spring draining a sandy aquifer: temporal variability of concentrations and fluxes.

A 250 ha agricultural catchment has been characterized with respect to its hydrogeology and groundwater contamination by pesticides from October 1999 to August 2004. Five years after the ending of atrazine (At) application, used since the sixties, At and deethylatrazine (DEA) are still systematically quantified at the outlet of the watershed with concentrations from 0.07 to 0.43 microg l(-1) for At, and between 0.14 and 1.16 microg l(-1) for DEA. Isoproturon and chlortoluron are detected in only one (0.3 microg l(-1)) and two (0.7 and 2.0 microg l(-1)) of the 124 semi-monthly samples, respectively. DEA concentrations can be very different between two samples with a 15-day time step. The annual mean exported fluxes of cumulated At and DEA are stable, which indicates a long time transfer in the unsaturated or saturated zone with a progressive leaching of the stock of At and DEA probably accumulated in the soil and the vadose zone. These fluxes, between 0.90% and 2.82% of the annual mean dose of At applied before 1999, similar to those calculated in several studies at the bottom of the root zone, could be explained by low adsorption and degradation properties of At and DEA in the unsaturated and saturated zone.

Individual and joint toxicity of the herbicide S-metolachlor and a metabolite, deethylatrazine on aquatic crustaceans: Difference between ecological groups.

We studied the individual and joint acute toxicity of S-metolachlor (SMOC) and deethylatrazine (DEA - a metabolite of atrazine) on different non-target freshwater crustaceans. We used animals from different ecological groups: two amphipods from surface running water (Gammarus pulex and Gammarus cf. orinos), an isopod from surface stagnant water (Asellus aquaticus) and an amphipod living in groundwater (Niphargus rhenorhodanensis). Organisms were exposed to different levels of SMOC and DEA, alone or in binary mixture. Temperature effect on SMOC toxicity was assessed by exposing G. pulex and N. rhenorhodanensis to SMOC at 11 °C and 15 °C. Studying mortality as the biological endpoint, N. rhenorhodanensis was more resistant than surface water species towards SMOC and DEA. Among surface water species, G. pulex was the most sensitive while Gammarus cf. orinos and A. aquaticus showed similar responses to both compounds. Temperature increase did not change SMOC toxicity but modify the shape and steepness of the dose-response curve. We used a Model Deviation Ratio (MDR) approach to evaluate the predictability of Concentration Addition (CA) and Independent Action (IA) models to mixture toxicity. Results indicated either an additive or an antagonistic or a synergistic interaction depending on the concentrations combination and the test species. Our finding conclusively show the suitability of CA and IA in predicting mixture toxicities but results should be interpreted with caution according to ecological group of exposed species in risk assessment procedures.

DNA molecules can drive the assembly of other DNA molecules into specific four-stranded structures.

Single-stranded DNA molecules containing clustered G-repeats can be assembled into various four-stranded structures linked by G-quartets. Here, we report that such molecules can also drive the assembly of other DNA molecules containing G-repeats into specific four-stranded structures. In these assays, the oligonucleotides 5'-CAGGCTGAGCAGGTACGGGGGAGCTGGGGTAGATTGGAATGTAG-3' (oligo D) and 5'-CGGGGGAGCTGGGGT-3' (oligo B), consisting of sequences found in immunoglobulin switch regions, were annealed in a buffer containing K+ and the annealing products were analyzed by polyacrylamide gel electrophoresis. This analysis revealed that whereas annealing of each oligo alone produced four-stranded structures designated D2 and B2, annealing of mixtures containing both oligos produced additional complexes designated D2* and B2*. D2* and B2* were found to contain only D molecules and only B molecules, respectively. The yield of D2* increased and the yield of B2* decreased, as the concentration ratio oligo B/oligo D was increased. These results indicated that B can drive the assembly of D into D2* and D can drive the assembly of B into B2*. Further studies revealed that while the assembly of D2 followed a second order kinetics, the B-driven assembly of D2* followed a first order kinetics. Dimethyl sulfate footprinting indicated that both D2 and D2* are four-stranded structures containing two parallel and two antiparallel chains. In addition, annealing of D mixed with various B mutants showed that only mutants containing two G-clusters can drive the assembly of D2*. Based on these data, we propose that in the process of D2* assembly, a four-stranded intermediate containing B and D is formed and then dissociates into D2* and B in a rate-limiting first order reaction. Driver mechanisms of this type may cause formation of specific four-stranded structures at G-rich chromosomal sites, thereby regulating processes such as recombination and telomere synthesis.

Determination of chloroacetanilides, triazines and phenylureas and some of their metabolites in soils by pressurised liquid extraction, GC-MS/MS, LC-MS and LC-MS/MS.

Pressurised liquid extraction (PLE) technique was used for the simultaneous extraction of phenylureas, triazines and chloroacetanilides and some of their metabolites from soils. Extractions were performed by mixing 15 g of dried soil with 30 mL of acetone under 100 atm at 50 degrees C, during 3 min and with three PLE cycles. Prior to the analysis of naturally contaminated soils, each of the five representative soil matrices used as blanks (of different depths) was spiked in triplicate with standards of each parent and degradation compound at about 10, 30 and 120 microg/kg. For each experiment, isoproturon-D6 and atrazine-D5 were used as surrogates. Analysis of phenylureas and metabolites of triazines and phenylureas was carried out by reversed phase liquid chromatography/mass spectrometry (LC-MS) and LC-MS/MS in the positive mode. Gas chromatography (GC)/ion trap mass spectrometry was used in the MS/MS mode for the parent triazines and chloroacetanilides. The average extraction recoveries were above 85%, except for didesmethyl-isoproturon, and quantification limits were between 0.5 and 5 microg/kg. The optimised multi-residue method was applied to soils and solids below the root zone, sampled from agricultural plots of a small French hydrogeological basin.

Persistent and emerging micro-organic contaminants in Chalk groundwater of England and France.

The Chalk aquifer of Northern Europe is an internationally important source of drinking water and sustains baseflow for surface water ecosystems. The areal distribution of microorganic (MO) contaminants, particularly non-regulated emerging MOs, in this aquifer is poorly understood. This study presents results from a reconnaissance survey of MOs in Chalk groundwater, including pharmaceuticals, personal care products and pesticides and their transformation products, conducted across the major Chalk aquifers of England and France. Data from a total of 345 sites collected during 2011 were included in this study to provide a representative baseline assessment of MO occurrence in groundwater. A suite of 42 MOs were analysed for at each site including industrial compounds (n=16), pesticides (n=14) and pharmaceuticals, personal care and lifestyle products (n=12). Occurrence data is evaluated in relation to land use, aquifer exposure, well depth and depth to groundwater to provide an understanding of vulnerable groundwater settings.

Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration.

PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.

'Illegitimate' recombination events in polyoma-transformed rat cells.

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.

Determination of oxanilic and sulfonic acid metabolites of acetochlor in soils by liquid chromatography-electrospray ionisation mass spectrometry.

An analytical method is presented that describes the extraction and quantification of oxanilic and sulfonic acid metabolites of the herbicide acetochlor in soil samples. Experiments were performed on 50 g of soil using a solvent extraction technique with an acetonitrile-water (60:40) mixture in an acidic medium. Analysis was carried out by reversed-phase liquid chromatography and detection by electrospray ionisation mass spectrometry in single ion monitoring and negative modes. Four different soil matrices were spiked in triplicate with standards of each degradation compound at three concentration levels between 2 and 80 microg/kg. The average recoveries range from 90 to 120% for both the metabolites, with relative standard deviations lower than 15%. The limits of quantification are about 1 and 2 microg/kg for the ethanesulfonic acid and the oxanilic acid metabolites, respectively. The method has been applied to soils and solids recovered from the deeper unsaturated zone of a small French catchment closely monitored as part of the European project "Pesticides in European Groundwaters: detailed study of Aquifers and Simulation of possible Evolution scenarios (PEGASE)".

A ten-year experience with reduction mammoplasty and abdominoplasty. What is happening in our part of the world?

The authors update their experience with 124 reduction mammoplasties (including mastopexy) and 67 abdominoplasties and discuss their applications of these procedures in Turkey. In almost all cases the Strömbeck procedure for mammoplasty and the Grazer and Pitanguy procedures for abdominoplasty were used. They present problems encountered in performing such operations and their solutions.

Urethral advancement for distal hypospadias repair in circumcised patients.

A new hypospadias repair technique is presented for distal cases. It is a modified form of the old Beck-Hacker technique and applicable only for distal hypospadias patients with minimal or no chordee. It consists of bringing the abnormally localized meatus to the tip of the glans penis by freeing and advancing the distal urethra. Thirty cases were operated on with this technique in the past 2 1/2 years with better results. The advantages of this simple technique can be summarized as follows: (1) it is easy to apply; (2) it can be applied to circumcised patients; (3) it can be done under local anesthesia even in the office; (4) the total length of the urethra is normal in its mucous membrane; (5) stricture formation is minimized by this solo glandular triangular flap; (6) ther is no need for a catheter; and (7) patients urinate freely during the healing process.

Delayed effect of denervation on wound contraction in rat skin.

Neuronal supply in soft tissues may be an important part of cutaneous wound healing. In order to observe the effect of denervation on wound contraction, rectangular full-thickness skin defects were created on the dorsum of two groups of Wistar rats. In the experimental group (n = 20), spinal nerves corresponding to the area of the open wound (T11 to L2) were isolated and divided bilaterally. In the control group (n = 20), the same pairs of spinal nerves were dissected but left intact. Limits of denervation were verified by the pinprick test. Wound healing, which is primarily in the form of wound contraction in this model, was evaluated by tracing wound margins onto millimetric paper weekly. Wound contraction was delayed significantly in the experimental group (p < 0.05) at all follow-up periods when compared with the controls. Loss of neuropeptide secretion from the nerve endings in denervated tissues may be responsible for the retarded wound contraction, since neuropeptides are thought to exert trophic effects on skin wound healing.

Facial resurfacing in xeroderma pigmentosum with monoblock full-thickness skin graft.

A case of xeroderma pigmentosum with multiple skin tumors on the face that was treated with total excision and replacement of face skin except the eyelids with a monoblock full-thickness abdominal skin graft is reported. The quality and tumor-free features of the monoblock full-thickness skin graft in xeroderma pigmentosum are indicated. Despite the increased morbidity of the donor region, the radical surgical approach advocated here has improved the prognosis in the case reported.

Vascularized double-sided preputial island flap with W flap glanuloplasty for hypospadias repair.

Hypospadias surgery is one of the most challenging surgical interventions that still need further refinements for increased success rates. During the last 5 years, we operated on 26 mid-penile or proximal hypospadias cases by using vascularized double-sided preputial island flap and W flap glanuloplasty to achieve superior functional and aesthetic results. Follow-up period of the patients revealed a 92.3 percent success rate with a single operation. Two cases developed fistula, which was located at the proximal anastomosis site. However, they were repaired in a second sitting without any additional problem. The basic aim in hypospadias surgery is the correction of chordee, reconstruction of urethra, and sufficient ventral penile skin coverage in one stage with minimal complication. The use of vascularized tissue for urethral reconstruction and ventral coverage is believed to have a superior healing capacity with better functional and cosmetic outcome. On the other hand, adding W flap glanuloplasty to this technique avoids the risk of meatal stenosis. We conclude that by combining the two above-mentioned techniques, it is possible to cope better with this devastating congenital deformity.

Vulva reconstruction with a tissue expander.

A patient with exstrophy of the bladder in whom we performed vulva reconstruction using a tissue-expansion technique and local flaps was presented. The ultimate result was in concert with the anatomic characteristics of normal external genitalia with regard to type of skin, pigmentation, hair pattern, quality of circulation, and sensory supply.

Evaluation of ELISA microtiter plate-based assays for the direct determination of isoproturon in water samples and soil extracts.

Trials were carried out on the commercially available Millipore isoproturon ELISA microtiter plate tests and on laboratory assays developed by Hirst as part of a Joint European Union research project (BIOPTICAS). The lowest detectable dose (LDD) was determined as three times the standard deviation of the blanks. Depending on the calibration curves obtained on different days with different plates, the LDD varied from 0.020 to 0.064 microgram/L for the Millipore test and from 0.080 to 0.329 microgram/L for the Hirst test. The mean coefficients of variation within a single plate for triplicate determinations of standard solutions in the 0.05 to 0.5 microgram/L range were 5.5 and 3.6% for Millipore and Hirst respectively. Cross-reactivity was studied for mono- and di-demethylated isoproturon, chlortoluron, diuron and linuron. The highest cross-reactivity with both tests was that of mono-demethylated isoproturon (22% for Millipore, 4% for Hirst). This molecule was the only one to show significant cross-reactivity in the Hirst test, whereas in the Millipore test, the di-demethylated isoproturon also cross-reacted (4%). Natural water samples, 19 ground-, 53 lysimetric plate and 47 suction cup water samples, and 32 soil samples were also analysed with the ELISA tests. HPLC with a diode array detector was used as a validated control technique for the natural samples. For each water type, ELISA concentrations of both tests were significantly correlated with the HPLC values (r > or = 0.937; p < 0.001). For the soil extracts, the correlations were also significant (p < 0.001), but the scatter in the data was greater. Overall, the Millipore correlation coefficients were higher than those of Hirst.