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Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths.

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.

Crystallization↗

bloodthirsty, an RBCC/TRIM gene required for erythropoiesis in zebrafish.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Amino Acid Sequence↗

Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridisation.

For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. Following microdissection, most samples fail to produce sufficient quantities of DNA for genome-wide experiments. Various PCR-based amplification methods have been used, but these usually produce nonuniform representations of the genome. Bacteriophage Phi29 DNA polymerase random-primed DNA amplification is based on isothermal multiple displacement amplification. We sought to define the genome representation of this method in a bacterial artificial chromosome microarray comparative genomic hybridisation (aCGH) platform. Test genomic female DNA was amplified using Phi29 amplification at four different starting concentrations (0.5, 5, 10 and 50 ng). These products were combined with unamplified and amplified genomic female DNA as reference. In addition, 50 ng of DNA from five microdissected breast cancer frozen samples, were amplified using the same method. Three combinations were performed: unamplified test with unamplified reference, amplified test with unamplified reference and both amplified tumour and reference DNA. aCGH was performed with an in-house 16 K BAC platform (a resolution of approximately 100 Kb). Pearson's correlation tests and hierarchical clustering were performed to compare the profiles obtained. aCGH profiles obtained with amplified test and unamplified reference female genomic DNA showed copy number biases throughout the genome. These biases were more conspicuous with smaller amounts of starting material and mapped to regions of known copy number polymorphisms. When similar concentrations of test and reference DNA were amplified, the biases were significantly reduced, rendering accurate profiles. For the tumours, representative profiles were obtained when both test and reference DNA were amplified. Phi29 amplification induces copy number biases and unamplified material remains the gold standard for copy number analysis. For accurate results using Phi29 amplification, samples subjected to aCGH analysis should be combined with reference DNA amplified with the same method, using similar amounts of starting template.

Breast Neoplasms↗

Regulation of a distinctive set of genes in glucocorticoid-evoked apoptosis in CEM human lymphoid cells.

Gene expression was evaluated in clones of the acute lymphoblastic leukemic cell line CEM that were sensitive or resistant to apoptosis evoked by the glucocorticoid, dexamethasone (Dex). Founding clones CEM-C7 (glucocorticoid sensitive) and CEM-C1 (glucocorticoid resistant) were subcloned to maximize uniformity of each population studied. Among subclones of C1, our original pseudodiploid clone of glucocorticoid-resistant cells, we found a high proportion of hyperploid clones. Most C1 subclones were glucocorticoid resistant but two C1 subclones were found to be revertants to glucocorticoid sensitivity. Glucocorticoid receptor content of the C1 subclones varied almost 5-fold but higher quantity of receptors did not guarantee steroid sensitivity. Gene expression analysis was carried out on microchips containing representations for approximately 12,600 human genes. When a group of four subclones from C1 (three glucocorticoid-resistant and one glucocorticoid-sensitive revertant) were compared with the glucocorticoid-sensitive subclone CEM-C7-14 for basal gene expression, the four C1 subclones clustered closely and far from C7-14. Thus, basal gene expression in the C1 subclones differed for a large number of genes from that in the C7 subclone. Reversion to glucocorticoid sensitivity did not cause a major shift in basal gene expression to a more C7-like state. Three clones (one revertant glucocorticoid sensitive from C1 subclone, one C7 sensitive subclone, and one C1 glucocorticoid-resistant subclone) were compared for the genes regulated by treatment for 20 hours with 10(-6)M Dex. This interval brings the cells to a point just before the onset of apoptosis. We tested the hypothesis that a distinctive set of genes would be regulated in the glucocorticoid-sensitive clones. This proved to be so. In three experiments, at our chosen levels of discrimination, 39 genes were consistently induced > or = 2.5-fold and 21 genes were consistently reduced > or = 2-fold in glucocorticoid-sensitive clones but not in the glucocorticoid-resistant clone. The glucocorticoid-resistant clone showed induction or reduction of 88 genes different from those regulated in the glucocorticoid-sensitive clones. These data support our hypothesis and further show that the glucocorticoid-resistant clone is capable of responding to steroid but with a different set of genes. We propose that a general metabolic switch accounts for the alteration.

Apoptosis↗

Dynamic simulations of the molecular conformations of wild type and mutant xanthan polymers suggest that conformational differences may contribute to observed differences in viscosity.

Xanthan gum is an exopolysaccharide secreted by the bacterium Xanthamonas campestris whose ability to make solutions viscous at low concentrations and over a pH and temperature range have generated much interest in both academic and industrial environments. Mutant Xanthamonas strains have been derived that produce xanthan gums with an altered or variant subunit chemical structure and different measured viscosities when compared with the wild type (wt) form of the polymer. Two variant gums were targeted as potentially interesting in this study, these being the nonacetylated tetramer (natet) and the acetylated tetramer (atet), which both lack a side-chain terminal mannose residue and in one case (natet) lacks an acetate group on an internal mannose residue. Solutions of these tetrameric gums possess viscosities higher (natet) and lower (atet) than the wt gum, and therefore we have attempted to determine whether these molecules possess unique conformational preferences when compared with the wt and with each other. In this manner we can initiate an understanding of how a polysaccharide's conformation contributes to its solution properties. The GEGOP software permits a sampling of the static and dynamic equilibrium states of carbohydrate molecules, and this software was employed to calculate equilibrium states of representative oligosaccharides with chemical structures representative of xanthan-like molecules. Energy minimization techniques revealed similar local minima for all three molecules. Some of these minima are comprised of elongate backbone conformations (A type) in which side chains fold onto backbone surfaces. Other minima with A backbones possessed side chains in less intimate backbone contact especially when calculations were performed with a low dielectric constant. This phenomenon was particularly pronounced in the wt molecule where an increased number of negatively charged side-chain residues experience charge repulsion resulting in reduced side-chain-backbone contact. Metropolis Monte Carlo (MMC) dynamic simulations performed with an elevated temperature factor (1000 K) allowed a better qualitative representation of conformational space than 300 K simulations. Employing a nonhierarchical cluster analysis method (population density profile: PDP) coupled with a classification scheme, it was possible to partition resulting MMC data sets into conformational families. This analysis revealed that in simulations performed with different dielectric constant values (10, 25, and infinity) all molecules possessed primarily A-type backbones. Less elongate, more open helical backbone forms (B, C, D, J, and Flat-a) did occur during the simulations but were populated to a lesser extent. In the natet molecule significantly open helical backbones existed (E, F, G, H, and I) that did not occur in the lower viscosity wt and atet molecules. PDP clustering methods and subsequent conformational classification applied to the first residue (mannose) of the side chain permitted a determination of side-chain orientation. Comparison of all three molecules indicated a larger population of side-chain conformational families in less direct backbone contact for the wt molecule than either of the variant molecules (natet/atet) suggesting that the side chains in the wt are more flexible. Thus, a major conformational difference between the high viscosity natet and the lower viscosities of the wt/atet is the increased amount of open helical backbone in the natet. In addition, the significant difference between the higher viscosity wt and the lower viscosity atet is the increase side-chain flexibility in the wt. We hypothesize that conformational differences of this kind could form a partial explanation of the observed differences in viscosity between these xanthan-like polymers.

Carbohydrate Conformation↗

A biological question and a balanced (orthogonal) design: the ingredients to efficiently analyze two-color microarrays with Confirmatory Factor Analysis.

BACKGROUND: Factor analysis (FA) has been widely applied in microarray studies as a data-reduction-tool without any a-priori assumption regarding associations between observed data and latent structure (Exploratory Factor Analysis).A disadvantage is that the representation of data in a reduced set of dimensions can be difficult to interpret, as biological contrasts do not necessarily coincide with single dimensions. However, FA can also be applied as an instrument to confirm what is expected on the basis of pre-established hypotheses (Confirmatory Factor Analysis, CFA). We show that with a hypothesis incorporated in a balanced (orthogonal) design, including 'SelfSelf' hybridizations, dye swaps and independent replications, FA can be used to identify the latent factors underlying the correlation structure among the observed two-color microarray data. An orthogonal design will reflect the principal components associated with each experimental factor. We applied CFA to a microarray study performed to investigate cisplatin resistance in four ovarian cancer cell lines, which only differ in their degree of cisplatin resistance. RESULTS: Two latent factors, coinciding with principal components, representing the differences in cisplatin resistance between the four ovarian cancer cell lines were easily identified. From these two factors 315 genes associated with cisplatin resistance were selected, 199 genes from the first factor (False Discovery Rate (FDR): 19%) and 152 (FDR: 24%) from the second factor, while both gene sets shared 36. The differential expression of 16 genes was validated with reverse transcription-polymerase chain reaction. CONCLUSION: Our results show that FA is an efficient method to analyze two-color microarray data provided that there is a pre-defined hypothesis reflected in an orthogonal design.

Antineoplastic Agents, Alkylating↗

[The analysis of physicians' work: announcing the end of attempts at in vitro fertilization].

The purpose of this empirical study is to analyze modalities of announcing the end of attempts at in vitro ferti-lization to women who, for various reasons, were not able to have a child after several trials. What are the problems physicians face when, in the course of their work, they make these announcements? How do they give (or not give) support to these women who have placed so much hope in this technique? These are some of the questions that led the authors to conduct this empirical study within the framework of a clinical and qualitative approach to work psychology. Within this framework, work is conceptualised as a complex activity that involves the subject, both bodily and through his various modes of socialisation. The field of clinical and quali-tative approach to work psychology situations focuses on different ways of expressing distress related to contradictory work demands, as the activity is being performed; it also focuses on those creative processes used by the subject to cope with those internal and external conflicts that hinder task performance. A review of the literature and preliminary observations led us to postulate that the problems physicians are faced with when they announce the end of attempts at in vitro fertilisation (IVF) are linked to several conflicts between work values (that are specific to the medical world) and the recognition of work failure: termination of attempts at IVF. The popu-lation that participated in this research project belongs to a network of private practitioners who work with the in-house team of a Parisian clinic. But the group is not uniform and some physicians perform IVFs more frequently than others. Our qualitative study involved 10 semi-directive interviews of approximately 1 1/2 hours each, which were recorded and transcribed. Initial instructions focused on a concrete description of situations of abandonment of attempts at IVF, in terms of their preparation, development, and the way they are experienced . Interviews therefore centred on specific and limited practitioner activity. Each transcription was submitted to a Qualitative Analysis of Discourse, followed by a comparative analysis of the 10 transcriptions. We propose an original method of Qualitative Analysis of Discourse, to be applied to semi-structured clinical interviews. This method seeks to analyse the structure of the resulting egocentric monologue in research si-tuations of semi-directive interviewing. The method of Quali-tative Analysis of Discourse involves three steps, but only the first two were applied in this work: a) identification of sequences of discourse; b) analysis of relationships between statements; c) stylistic analysis of figures of speech. Our first set of analyses showed that seve-ral markers increase in physicians' discourse when they describe difficult and/or conflict-laden consultation situations: logical connectors, impersonal pronouns, reported discourse, anti-cipations regarding the interviewer's judgement. The logical balance of the discourse therefore appears threatened when pro-blems inherent in the work demands involved in ending IVF attempts are mentioned. As a whole, these markers underscore the importance of the implicit dimension of discourse (inferences, presuppositions, hints, allusions, etc.), thus reflecting complex speech that attempts to negotiate between subjective positions and shared cultural values. A comparative analysis of the markers identified in the 10 interviews revealed four areas, each involving nervous tension poles, that are suggestive of cognitive-emotional dissonance in the task to be performed. Some factors increase professional distress while others temper it. They act upon the work situation itself on the one hand, and on the working relationship between physician and patient on the other. 1. Areas of tension relating to the task to be performed. The first area contrasts individual with collective decision-making. The independent status which characterises private medical practice increases self-esteem in cases of success but weakens it when IVF attempts fail. In addition, it goes against collective involvement in the work situation, yet such involvement may act as a strong moderating factor for the experience of distress. The second area contrasts work that is well done with recognition by peers. Indeed, in the hierarchy of medical values, recognition by peers that work has been well performed is anchored in successful healing (in the broad sense of the term), whereas in situations of abandonment of IVF attempts, ending the attempt is considered by everyone to be a failure, even if it has been well conducted . The third area opposes objective medical practice to a necessarily subjective medical involvement. The scientific and ideal values which characterise medicine reflect its objective and scientific orientation, but IVF situations are a reminder that medicine is not an exact science and that it can make mistakes. There are numerous special individual cases which reduce certainty that a decision to terminate IVF is well-founded. The fourth area distinguishes between work that is considered to be well done and work considered to be well conducted . Personal estimation of work that is well done is based on the impression that the maximum feasible has been done . But in IVF situations, constant uncertainty leads to professional over-involvement (examinations, verifications, changes of protocols). Work that is poorly done is work that does not cure or that brings no relief. As a result, work that consists in ending IVF attempts, even if it is well conducted , remains a subjective failure for everyone since it does not bring a cure (pregnancy). 2. Areas of tension in the physician-patient relationship. The first area contrasts women's irrational desire with possible support from their husbands, when the time has come to announce the end of the attempts. But this voice/presence of husbands is consi-dered desirable and important only when attempts have failed, so that husbands are not encouraged to participate in the protocols except to help restrain their wives' over reactions . The second area opposes respect for the patient role with demands made by women. Lack of respect for the patient role, by making demands or by refusing to follow advice, particularly when IVF attempts are abandoned, crystallises all the resentment experienced by physicians in difficult work situations. Two cognitive-emotional worlds, more or less tuned to one another over the course of the IVF, start to clash and lose all mutual understanding: the medical world and the patient's subjective world. The third area results from the second one. It contrasts a listening physician with a powerful one. Physicians are very concerned that their relationship with their patients be one of partnership. But this (idealised) equilibrium is abruptly disrupted by the end of the attempts, inasmuch as it is the physician who has the power to stop these attempts and who decides to do so. The unveiling of this reality of a power relationship becomes a source of suffering and contradicts expressed surface values. The fourth area contrasts an attitude of ongoing patient support based on a belief in success with an attitude of patient support based on the prediction of a possible failure. Indeed, for a patient to be supported in a way physicians would consider right and adequate , the abandonment of IVF attempts should be anticipated in advance so that the physician can prepare both himself and the patients for the high risk of failure. But physicians insist on the fact that medical work can only succeed if they believe in it . As a result, the more energy the physician puts into launching the initial phase of IVF, the greater the feeling of self-accomplishment during the first phase of IVF; but conversely, the weaker the efficacy of the process of seeing the patient through the end of the attempts, the stronger the fee-ling of subjective distress at work will be. Overall, it is a para-doxical work situation for physicians to have to anticipate the interruption of IVF attempts and to have to prepare for seeing the patient through this abandonment. This situation creates conflicts of representations and values within their very practice and generates distress at work. It is worth noting that some moderating factors could alleviate their sense of suffering and contribute to improving their work experience: a) the deve-lopment of a protocol for seeing patients through the end of IVF attempts, which would make abandonment part of a job well done for physicians; b) regular participation by the spouse in these protocols; c) making all decisions to end IVF attempts a collective process, in order to avoid placing exclusive responsibility on the treating physician. The limitations of this study are inherent both in the qualitative nature of the data that involve a small number of physicians, and in the specificity of this population that works within a poorly structured network. On the other hand, our method of Qualitative Analysis of Discourse can be applied to all types of discourse obtained in research situations, provided the discourse is produced through semi-directive or non-directive interviews.

Affect↗

Gamma knife radiosurgery as a primary treatment for prolactinomas.

OBJECT: The purpose of this study was to estimate the efficacy of gamma knife radiosurgery (GKS) in controlling tumor growth and endocrinopathy associated with prolactinomas. METHODS: Between 1993 and 1997, 164 of 469 patients with pituitary adenomas treated by GKS harbored prolactinomas. The dose to the tumor margin ranged from 9 to 35 Gy (mean 31.2 Gy), and the visual pathways were exposed to a dose of less than 10 Gy. The mean tumor diameter was 13.4 mm. The mean follow-up time for 128 cases was 33.2 months (range 6-72 months). Tumor control was observed in all but two patients who underwent surgery 18 and 36 months, respectively, after GKS. Clinical cure was achieved in 67 cases. Clinical improvement was noted with a decrease in the hyperprolactinemia after GKS. Nonetheless, in 31 (29%) of 108 patients who were followed for more than 2 years no improvement in serum prolactin levels was demonstrated, although this could be normalized by bromocriptine administration after treatment. Nine infertile women became pregnant 2 to 13 months after GKS and all gave birth to normal children. There was no visual deterioration related to GKS. Five women experienced premature menopause. In these patients there was subtotal disappearance of the tumor and an empty sella developed. CONCLUSIONS: Gamma knife radiosurgery as a primary treatment for prolactinomas can be safe and effective both for controlling tumor growth and for normalization of prolactin hypersecretion. A higher margin dose (> or = 30 Gy) seemed to be associated with a better clinical outcome. Gamma knife radiosurgery may make prolactinomas more sensitive to the bromocriptine.

Humans↗