Phylogeography, more than elevation, accounts for sex chromosome differentiation in Swiss populations of the common frog (Rana temporaria).

Sex chromosomes in vertebrates range from highly heteromorphic (as in most birds and mammals) to strictly homomorphic (as in many fishes, amphibians, and nonavian reptiles). Reasons for these contrasted evolutionary trajectories remain unclear, but species such as common frogs with polymorphism in the extent of sex chromosome differentiation may potentially deliver important clues. By investigating 92 common frog populations from a wide range of elevations throughout Switzerland, we show that sex chromosome differentiation strongly correlates with alleles at the candidate sex-determining gene Dmrt1. Y-specific Dmrt1 haplotypes cluster into two main haplogroups, YA and YB , with a phylogeographic signal that parallels mtDNA haplotypes: YA populations, with mostly well-differentiated sex chromosomes, occur primarily south of the main alpine ridge that bisects Switzerland, whereas YB populations, with mostly undifferentiated (proto-)sex chromosomes, occur north of this ridge. Elevation has only a marginal effect, opposing previous suggestions of a major role for climate on sex chromosome differentiation. The Y-haplotype effect might result from differences in the penetrance of alleles at the sex-determining locus (such that sex reversal and ensuing X-Y recombination are more frequent in YB populations), and/or fixation of an inversion on YA (as supported by the empirical observation that YA haplotypes might not recombine in XYA females).

An organelle gatekeeper function for Caenorhabditis elegans UNC-16 (JIP3) at the axon initial segment.

Neurons must cope with extreme membrane trafficking demands to produce axons with organelle compositions that differ dramatically from those of the cell soma and dendrites; however, the mechanism by which they accomplish this is not understood. Here we use electron microscopy and quantitative imaging of tagged organelles to show that Caenorhabditis elegans axons lacking UNC-16 (JIP3/Sunday Driver) accumulate Golgi, endosomes, and lysosomes at levels up to 10-fold higher than wild type, while ER membranes are largely unaffected. Time lapse microscopy of tagged lysosomes in living animals and an analysis of lysosome distributions in various regions of unc-16 mutant axons revealed that UNC-16 inhibits organelles from escaping the axon initial segment (AIS) and moving to the distal synaptic part of the axon. Immunostaining of native UNC-16 in C. elegans neurons revealed a localized concentration of UNC-16 at the initial segment, although UNC-16 is also sparsely distributed in distal regions of axons, including the synaptic region. Organelles that escape the AIS in unc-16 mutants show bidirectional active transport within the axon commissure that occasionally deposits them in the synaptic region, where their mobility decreases and they accumulate. These results argue against the long-standing, untested hypothesis that JIP3/Sunday Driver promotes anterograde organelle transport in axons and instead suggest an organelle gatekeeper model in which UNC-16 (JIP3/Sunday Driver) selectively inhibits the escape of Golgi and endosomal organelles from the AIS. This is the first evidence for an organelle gatekeeper function at the AIS, which could provide a regulatory node for controlling axon organelle composition.

Does the speciation clock tick more slowly in the absence of heteromorphic sex chromosomes?

Squamates may be an attractive group in which to study the influence of sex chromosomes on speciation rates because of the repeated evolution of heterogamety (both XY and ZW), as well as an apparently large number of taxa with environmental sex-determination.

Mutagenesis of the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding site in the NH(2)-terminal domain of ezrin correlates with its altered cellular distribution.

The cytoskeleton-membrane linker protein ezrin has been shown to associate with phosphatidyl-inositol 4,5-bisphosphate (PIP(2))-containing liposomes via its NH(2)-terminal domain. Using internal deletions and COOH-terminal truncations, determinants of PIP(2) binding were located to amino acids 12-115 and 233-310. Both regions contain a KK(X)(n)K/RK motif conserved in the ezrin/radixin/moesin family. K/N mutations of residues 253 and 254 or 262 and 263 did not affect cosedimentation of ezrin 1-333 with PIP(2)-containing liposomes, but their combination almost completely abolished the capacity for interaction. Similarly, double mutation of Lys 63, 64 to Asn only partially reduced lipid interaction, but combined with the double mutation K253N, K254N, the interaction of PIP(2) with ezrin 1-333 was strongly inhibited. Similar data were obtained with full-length ezrin. When residues 253, 254, 262, and 263 were mutated in full-length ezrin, the in vitro interaction with the cytoplasmic tail of CD44 was not impaired but was no longer PIP(2) dependent. This construct was also expressed in COS1 and A431 cells. Unlike wild-type ezrin, it was not any more localized to dorsal actin-rich structures, but redistributed to the cytoplasm without strongly affecting the actin-rich structures. We have thus identified determinants of the PIP(2) binding site in ezrin whose mutagenesis correlates with an altered cellular localization.

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures.

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.

Approximation techniques for neuromimetic calculus.

Approximation Theory plays a central part in modern statistical methods, in particular in Neural Network modeling. These models are able to approximate a large amount of metric data structures in their entire range of definition or at least piecewise. We survey most of the known results for networks of neurone-like units. The connections to classical statistical ideas such as ordinary least squares (LS) are emphasized.

CAFASP-1: critical assessment of fully automated structure prediction methods.

The results of the first Critical Assessment of Fully Automated Structure Prediction (CAFASP-1) are presented. The objective was to evaluate the success rates of fully automatic web servers for fold recognition which are available to the community. This study was based on the targets used in the third meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP-3). However, unlike CASP-3, the study was not a blind trial, as it was held after the structures of the targets were known. The aim was to assess the performance of methods without the user intervention that several groups used in their CASP-3 submissions. Although it is clear that "human plus machine" predictions are superior to automated ones, this CAFASP-1 experiment is extremely valuable for users of our methods; it provides an indication of the performance of the methods alone, and not of the "human plus machine" performance assessed in CASP. This information may aid users in choosing which programs they wish to use and in evaluating the reliability of the programs when applied to their specific prediction targets. In addition, evaluation of fully automated methods is particularly important to assess their applicability at genomic scales. For each target, groups submitted the top-ranking folds generated from their servers. In CAFASP-1 we concentrated on fold-recognition web servers only and evaluated only recognition of the correct fold, and not, as in CASP-3, alignment accuracy. Although some performance differences appeared within each of the four target categories used here, overall, no single server has proved markedly superior to the others. The results showed that current fully automated fold recognition servers can often identify remote similarities when pairwise sequence search methods fail. Nevertheless, in only a few cases outside the family-level targets has the score of the top-ranking fold been significant enough to allow for a confident fully automated prediction. Because the goals, rules, and procedures of CAFASP-1 were different from those used at CASP-3, the results reported here are not comparable with those reported in CASP-3. Nevertheless, it is clear that current automated fold recognition methods can not yet compete with "human-expert plus machine" predictions. Finally, CAFASP-1 has been useful in identifying the requirements for a future blind trial of automated served-based protein structure prediction.

Plasma total and glycosylated corticosteroid-binding globulin levels are associated with insulin secretion.

In humans, steroid hormones circulate in the blood mainly bound to specific steroid transport proteins, namely corticosteroid-binding globulin (CBG) for cortisol and sex hormone-binding globulin (SHBG) for testosterone and estradiol. The binding activities of these proteins are believed to modulate the biodisposal of steroids to target cells. It has been shown in vitro that insulin is a potent inhibitor of both CBG and SHBG secretion by a human hepatoblastoma cell (HepG2) line. To further investigate this potential effect of insulin in vivo, we prospectively studied three groups of lean subjects, obese subjects, and obese subjects with glucose intolerance, all of whom were otherwise healthy. The three groups were comparable in sex and age, and in the two obese groups, body mass index, waist to hip ratio, and blood pressure were similar. Plasma total CBG concentrations (38.2 +/- 5.4 vs. 31.7 +/- 4.05 mg/L; P = 0.016) and glycosylated CBG levels (37.3 +/- 5.2 vs. 31 +/- 3.9 mg/L; P = 0.018) were significantly increased in obese subjects with glucose intolerance. Plasma CBG correlated positively with fasting glucose levels (r = 0.49; P = 0.002), hemoglobin A1c levels (r = 0.35; P = 0.03), and area under the curve of glucose after an oral glucose tolerance test (r = 0.45; P = 0.005) and correlated negatively with the insulin response to i.v. glucose (AIRg; -0.38, P = 0.02) as well as to oral glucose (r = -0.40; P = 0.01) challenge tests. CBG levels did not covariate with insulin sensitivity. Multiple linear regression analysis showed that only AIRg contributed to the variability of the CBG concentration (P = 0.03), explaining 41% of its variance. Morning cortisol levels did not differ between the groups and did not correlate to any of the glucose or insulin metabolism parameters. Because carbohydrate chains influence the biological activity and half-life of glycoproteins, we analyzed the migration profile of CBG by Western blot and the interaction of CBG with lectin, Con A. The results indicated that the CBG mol wt and interaction with Con A did not differ between lean and obese patients. These data favor the hypothesis that the inhibitory effect of insulin on CBG liver secretion might be relevant in vivo and therefore contribute to decrease CBG levels in obese patients with enhanced insulin secretion. In both men and women, SHBG levels correlated negatively with fasting glucose (r = -0.55; P < 0.0001) and hemoglobin A1c (r = -0.38; P = 0.02) and positively with insulin sensitivity (S(I); r = 0.65; P = 0.003 and r = 0.63; P = 0.007 in men and women, respectively), but not with insulin secretion. The disposition index (S(I) x AIRg) was significantly decreased in the obese, glucose-intolerant subjects, suggesting that AIRg was inadequate for their degree of insulin resistance. The disposition index correlated positively with plasma SHBG levels (r = 0.52; P = 0.001) and negatively with plasma CBG levels (r = -0.54; P = 0.001). Our data suggest that CBG is a marker of insulin secretion in a similar way as SHBG is a marker of insulin sensitivity. As high plasma CBG levels have been associated with increased incidence of type 2 diabetes, this important issue merits further investigations.

A peptide derived from a beta2-adrenergic receptor transmembrane domain inhibits both receptor dimerization and activation.

One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F.(1990) Nature 348, 125-132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that beta2-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits beta-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.

Improved binding of cytochrome P450cam substrate analogues designed to fill extra space in the substrate binding pocket.

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties.

[Iridium 192 brachytherapy of supra-tentorial high grade glioma recurring in irradiated areas: technique and preliminary results of the Pitié-Salpêtrière hospital group]. / Curiethérapie par iridium 192 des gliomes sus-tentoriels de haut grade récidivant en territoire irradié: technique du groupe hospitalier Pitié-Salpêtrière et résultats préliminaires.

UNLABELLED: Our aim was to analyse feasibility and preliminary results obtained with iridium 192 re-irradiation of recurrent high grade gliomas. MATERIAL AND METHODS: a technique for implanting rigid plastic tubes afterloaded with iridium 192 wires was developed that utilised a stereotactic Leksell frame. Nineteen glioblastomas and one anaplastic glioma (12 males and 8 females: age: 20-69 years, median: 50) were implanted between January 1993 and December 1994. Previous treatments included surgery (18/20). 55-60 Gy external beam radiotherapy (20/20), and chemotherapy (16/20); interval between initial treatment and retreatment with iridium 192 was 6 to 39 months (median: 10). Maximum diameter of the tumour at the moment of implantation was 2.1-10.1 cm (median: 6.4 cm) and tumour volume 2.122 cm3 (median: 22 cm3). All tumours were supra-tentorial (right hemisphere: 9; left hemisphere: 11). Karnofsky index was 60-100 (median: 80). Implantation was carried out under local anaesthesia; tumour contours were visualised using either a CT-scan (16/20) or a MRI (4/20). Dosimetry was carried out using two orthogonal films and CT-scan images. Total dose on the reference isodose was 40-60 Gy (60 Gy: 9; 50 Gy: 7; 40 Gy: 4); dose-rate was 0.24-0.73 Gy/h (median: 0.38). RESULTS: probability of overall survival is 90% at 6 months, 55% at one year, and 26% at two years. Median survival is 56 weeks. Eleven patients died from local failure, and three from leptomeningeal metastasis. Six patients are alive, 15-30 months after the implantation. Two were reoperated for brain necrosis. Three patients showed evidence of bacterial meningitis, and three others of skin necrosis. CONCLUSION: according to this preliminary analysis, results obtained after reirradiation of high grade gliomas with iridium 192 are encouraging. More patients and longer follow-up are needed to draw definitive conclusions.

Dosimetry of intraluminal brachytherapy.

Dose specification in intraluminal brachytherapy varies considerably in the literature. A terminology is proposed, inspired from the Paris System of interstitial brachytherapy, to define some dosimetric guidelines. The dose is specified in the central plane which is a plane perpendicular to the source and passing through its centre. The reference dose rate is calculated in this plane. The treated volume is the volume encompassed by the reference isodose. The hyperdose sleeve is the volume receiving a dose equal to or greater than twice the reference dose. A study of the dose distribution along a linear source of iridium-192 was undertaken. Results obtained, also valid for caesium-137 and cobalt-60, show that the radius of the hyperdose sleeve is relatively independent of the length of the source. It is approximately 0.6 times the distance between the source axis and the point of dose specification when the active length varies from 3 to 20 cm. Reporting not only the dose, but also the thickness of tissue covered by the reference isodose and the thickness of tissue included in the hyperdose sleeve, is recommended to assist in evaluation of the results of treatment and to facilitate the exchange of clinical information in intraluminal brachytherapy. Practical examples are given.

[Current indications of laparoscopy]. / Indications actuelles de la laparoscopie.

The authors illustrate the indications of laparoscopy by reporting their clinical experience in a Department of gastroenterology between 1982 and 1984. In addition to the clinical indications which remain valid, new indications of laparoscopy are described in cases of failure of ultrasonography and/or computerised tomography, and of direct opacification of the biliary and pancreatic ducts. Laparoscopy has been practically abandoned in the diagnosis of obstructive jaundice and pancreatic pathology. It is irreplaceable in peritoneal pathology. The role of laparoscopy in hepatic tumours, hepatitis, cirrhosis, certain abdominal emergencies, unexplained abdominal pain and pelvic disease is discussed. Some rare indications may be considered only when the technical possibilities of laparoscopy are understood.

The guide gutter or loop techniques of interstitial implantation and the Paris system of dosimetry.

The predictive dosimetry system for implants known as the Paris system can be used with either loops or hairpins. When using the guide gutter technique, implant geometry is predetermined by the inherent spacing and parallelism of the branches of the hairpins. When using loops, their branches should not be spaced too widely apart and should be parallel over an adequate distance to obtain a fairly regular dose distribution between them. The basic principles of implantation are the same as for rectilinear sources. Branches must be rectilinear, parallel, arranged so that their centers are located in the same plane (central plane). Adjacent branches must be equidistant from each other and the reference linear kerma rate (or the linear activity) must be uniform and identical for all sources. When these conditions are met, the dimensions of the treated volume (volume encompassed by the reference isodose surface with a value equal to 85% of the basal dose rate) can be estimated at the time of the implantation procedure. In practice, only a few relationships presented in this paper, with examples of application, must be known. Although, the Paris system permits forecasting the final dosimetry, the geometry of the implant must be verified and the dose calculated according to the implantation as actually achieved. The best method of checking the exact position of radioactive sources in an implant and determining the dose rate at any desired point is a reconstruction by computer program although alternative methods are occasionally appropriate.(ABSTRACT TRUNCATED AT 250 WORDS)