The response of electrons to structural changes.

The properties of a molecule are determined by the distribution of its electrons. This distribution can be described by the charge density, which is readily obtained from the wave functions derived by ab initio molecular orbital calculations. The charge density may be analyzed in a number of different fashions to give information about the effects of substituents, structural changes, and electronic excitation on the properties of molecules; one common procedure makes use of projection density or charge difference plots. Charge density also may be partitioned among atoms, and by numerical integration over appropriate volume elements one may obtain atomic charges, dipoles, kinetic energies, and other properties of the atoms in a molecule. Many chemical phenomena have been analyzed in terms of charge densities.

Proton NMR study of the influence of heme vinyl groups on the formation of the isomeric forms of sulfmyoglobin.

The formation of sulfmyoglobin has been investigated for myoglobin reconstituted with hemins having vinyls replaced by hydrogens to determine the participation of the vinyl groups in the reaction processes. Green complexes are produced in all cases, proving that vinyls are not obligatory for the formation of sulfproteins. In the presence of the 4-vinyl group, the 1H NMR spectra of the met-cyano derivatives indicate the formation of three green species; however, the most stable of these products is not formed in the absence of this group, confirming reaction of the 4-vinyl in this species. Two new red extractable sulfmyoglobin derivatives are formed in the absence of the 4-vinyl group.

[Development over 3 years of asthma recently diagnosed in a cohort of children (ASMA study). Factors related to improvement in clinical status]. / Evolution sur 3 ans de l'asthme récemment diagnostiqué dans une cohorte d'enfants (étude ASMA). Facteurs liés à l'amélioration de l'état clinique.

OBJECTIVE: The objective of the ASMA study was to describe the evolution of light to moderate asthma, newly or recently (12 Pounds months) diagnosed in private pneumology centers, and to search for the predictive factors. METHODS: In 1995, 251 private pneumologists, throughout Metropolitan France, recruited 396 asthmatic children, 6 to 12 years old (64% boys). The 334 patients eligible for the study were examined every 4 months during 3 years (a mean of 6 controls were conducted out of the expected 9). The data were collected on standardized questionnaires completed by the physicians and notebooks filled-in by the patients the week before each control. This questionnaire comprised two asthma 'control' criteria: "control" of the clinical state, defined as asthma attacks < 1 per week AND nocturnal awakening < 1 per week AND absence of asthma symptoms between attacks on every control visit; "control" of the need for b2 mimetics on request, defined as the non-use throughout the week preceding the control visit. RESULTS: The global clinical state of the cohort rapidly improved once care was initiated: the proportion of children exhibiting at least one attack of asthma per week rapidly dropped to 43% on inclusion and to 13% on the first control visit (4 months), 10% on the second control visit, and then fluctuated at around 8% up until the last control visit. A similar evolution was noted regarding nocturnal asthma attacks. The proportion of patients with prescriptions for inhaled corticosteroids and long-lasting b2-mimetics increased over the three years of follow-up. Analysis of the factors related to the individual 'control' of the clinical state showed a negative effect in family histories of asthma (father) and the presence of smokers in the home, but above all a positive effect of compliance to treatment and particularly its understanding (OR = 2.5; p = 0.03) and respect of the doses (OR = 2.7; p < 0.01). The positive effect of compliance was confirmed by analysis of the factors related to the use of b2 mimetics on request. CONCLUSION: Smoking should be avoided in the home. Compliance to treatment could be improved by making sure that the patients and their parents fully understand the disease and its treatment, and by persuading them to strictly follow the treatments prescribed.

[Factors predicting the severity and the degree of symptom control of asthma in a recently diagnosed cohort of adult patients (A.S.M.A study)]. / Facteurs liés à la sévérité et au contrôle de l'asthme récemment diagnostiqué dans une cohorte de patients adultes.

INTRODUCTION: The aim of this cohort study was to describe the evolution of recently diagnosed (<12 months) asthma, and to identify outcome predictive factors. This paper describes the evolution of an adult cohort and the factors related to asthma severity and control. METHODS: In 1995, 251 chest specialists from throughout France, recruited 347 asthmatic adults (subjects with severe asthma were excluded). 220 eligible patients were examined every four months over a three year period. Data (socio-demographic characteristics, asthma history, results of atopy testing and lung function tests, treatment, drug compliance, respiratory infections, changes in lifestyle and environment, and major life events) were collected by means of detailed standardised questionnaires completed by physicians. Asthma severity, recorded one year after study inclusion, and asthma control, assessed at each follow-up visit in the second and third year, were defined according to the international guidelines. RESULTS: The clinical status of these adult patients generally improved rapidly. Asthma severity correlated closely with allergy, with a history of childhood asthma and with sensitisation to indoor allergens. After adjusting for severity, poor asthma control was associated with poor compliance, with respiratory infections, and, to a lesser extent, with animals inside the home. CONCLUSIONS: This cohort study highlights the association of asthma severity with allergy, and of poor asthma control with poor compliance and respiratory infections.

[Asthma in private pneumology practice]. / Asthme en pneumologie libérale.

We assessed asthma severity in patients attending private practice chest specialists, studied the factors related to classification by physicians, and described medications prescribed. 545 chest specialists scattered throughout France, included the patients examined from 3 to 28 May 1993 (N = 14,865). Besides a classification of asthma severity in four classes (mild, moderate, moderately severe, and severe), questionnaires included 20 questions on the history and characteristics of asthma, lung function level and medications prescribed. The proportion of mild asthmatics was 55% among the 3,620 children (aged 6 to 15 years), 42% among the 6,479 young adults (aged 16 to 45 years), and 18% among the 4,766 older adults (aged 46 to 75 years). Followed-up patients were considered more severe than new patients among adults, but not among children. The factors related to asthma severity were impaired FEV1, history of hospitalization, critical care and emergency visits for asthma, limitation of physical activities, and, to a latter extent, symptoms between exacerbations, frequent asthma attacks and daily use of beta 2-agonists. Anti-inflammatory drugs were prescribed to practically all patients from grade 2 (moderate): steroids increased whereas sodium cromoglycate and nedocromil decreased with increasing severity. This study provides a valuable estimate of the classification and medications prescribed to asthmatic patients examined by 50% of private practice chest specialists in France.

[Prognostic factors in operated non-small cell cancer of the lung. Study from a randomized therapeutic trial]. / Facteurs pronostiques du cancer du poumon opéré non à petites cellules: étude à partir d'un essai thérapeutique randomisé.

This article presents the results of a prognostic study of primary resected lung cancer (non-small cell). The data result from a randomised clinical trial of immunotherapy with a non-specific adjuvant; the follow-up was between four to seven years. Thirty-five clinical, biological and anatomo-pathological parameters were gathered at the time of inclusion in the trial. The response criteria used were survival without recurrence and total survival. A multivariate analysis using the Cox's model was carried out for each criterion. At the reference date of the 1st April 1985, 125 relapses and 132 deaths were counted amongst 219 patients; there was only one patient lost to follow-up and only 39 missing data were observed. The negative therapeutic results of the immunotherapy used were confirmed by this new intermediate analysis. The rate of survival without recurrence at 5 years was 43% and the overall survival at five years was 42%. The use of Cox's model to show the prognostic information at the 5% level for survival without recurrence could be summarised by five factors: main staging (the prognostic factor), leucocytosis, the cutaneous reaction to proteus, Karnofsky index and presence of physical signs. For stages I and II the outcome was identical and no factor was predictive at the 5% level. For stage III the cutaneous reaction to proteus and leucocytosis were prognostic. For overall survival, the prognostic information at the 5% level could be summarised by five factors: staging (main prognostic factor), leucocytosis, Karnofsky index, presence of physical signs and lymphocytosis. For stages I and II whose outcome was identical only Karnofsky index and lymphocytosis were predictive at the 5% level.(ABSTRACT TRUNCATED AT 250 WORDS)

[Negative results of a randomized therapeutic trial of nonspecific immunotherapy in primary, surgically-treated non-small cell bronchial cancer]. / Résultats négatifs d'un essai thérapeutique randomisé d'immunothérapie non spécifique dans le cancer bronchique primitif non microcellulaire opéré.

Between March 1978 and May 1981, 219 patients suffering from non-small cell primary bronchial carcinoma underwent surgical excision which was intended to be curative. Three weeks later the patients were randomised into two groups: 1. A control group, with no other treatment following excision (110 patients). 2. A non-specific immunotherapy group (109 patients). The immunostimulant used was an aqueous suspension of heat killed mycobacterium smegmatis administered subcutaneously once a month. The trial was analysed on December 1, 1982. There were 117 recurrences and 112 deceased. There was no significant difference as regard survival without relapse or overall survival; all causes of death were included.

[Color doppler echography in the exploration of vasculogenic impotence]. / Echographie-doppler couleur dans l'exploration de l'impuissance d'origine vasculaire.

102 patients with suspected vasculogenic impotence were evaluated with color doppler sonography. Measurement of normal systolic and diastolic velocities were obtained from the cavernosal arteries of patients responding by a full erection after intra-cavernosal injection of 20 mg of Papaverine. A correlation with cavernosometry was obtained in 61 patients and with selective internal pudendal arteriography in 11. The 10 patients with abnormal arteriograms had a systolic velocity < 25 cm/sec. 13 out of the 15 patients with an end diastolic velocity > 5 cm/sec had a venous leak defined by a maintenance flow rate of erection during cavernosometry > 25 ml/mn. End diastolic velocity is an excellent index of the function of the veno-occlusive system, provided the systolic velocity remains at a normal value. In case of arterial insufficiency, a diastolic flow < 5 cm/sec is of no value and cavernometry is mandatory to detect a mixed arterio-venous impotence. The addition of color doppler sonography permitted a more rapid detection of vessels and an easily reproducible measurement of velocities which makes color doppler sonography an excellent screening test for examining patients with potential vasculogenic impotence.

Early expression of a collagenase-like hatching enzyme gene in the sea urchin embryo.

The hatching enzyme is a developmentally regulated protease secreted at the blastula stage by the sea urchin embryo to digest its protective envelope. A nearly full-length cDNA clone (HE6) encoding the entire sequence of the hatching enzyme was isolated from a prehatching blastula lambda gt11 cDNA library. The 1761 bp open reading frame codes for a preprohatching enzyme with an 18 amino acid signal sequence, a 148 amino acid activation peptide and a 421 amino acid mature enzyme which has homologies with the mammalian collagenases. Transcripts of the hatching enzyme gene are not detected in the unfertilized egg, they accumulate during the cleavage stages and disappear at hatching. This transient expression results from a transcriptional control. Thus the hatching enzyme mRNA is not a maternal gene product but a transcript synthesized at a very early stage from the zygotic genome.

Purification and characterization of the sea urchin embryo hatching enzyme.

The sea urchin hatching enzyme provides an interesting model for the control of gene expression during early development. In order to study its properties and developmental regulation, the hatching enzyme of the species Paracentrotus lividus has been purified. The fertilization envelopes of the embryos were digested before hatching by a crude culture supernatant previously made. The enzyme was then solubilized by 1 M NaCl and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and purified by hydrophobic chromatography on Procion-agarose. A 470-fold increase in specific activity was obtained. The kinetic parameters of the proteolytic activity using dimethylcasein as substrate are: Km = 120 micrograms x ml-1, Vm = 200 mumol x min-1 x mg-1, and kcat = 180 s-1 at 500 mM NaCl, 10 mM CaCl2, pH 8.0, at 35 degrees C. The purified enzyme is highly active on fertilization envelopes: at 20 degrees C and 500 mM NaCl, 10 mM CaCl2, pH 8.0, 100 ng of enzyme completely denudes embryos in about 20 min under standard conditions. The molecular mass of the enzyme was estimated as 57 kDa by gel filtration, 51 kDa by gel electrophoresis, and 52 kDa by amino acid analysis. The hatching enzyme was shown to be a glycoprotein which autolyzes to a 30-kDa inactive form. Antibodies raised against the 51- or 30-kDa forms reacted with both these forms. Immunoblotting experiments showed that the hatching supernatants contain important amounts of the autolyzed species.

Spatial and temporal expression pattern during sea urchin embryogenesis of a gene coding for a protease homologous to the human protein BMP-1 and to the product of the Drosophila dorsal-ventral patterning gene tolloid.

A cDNA clone coding for a sea urchin embryonic protein was isolated from a prehatching blastula lambda gt11 library. The predicted translation product is a secreted 64 x 10(3) Mr enzyme designated as BP10. The protein contains several domains: a signal peptide, a putative propeptide, a catalytic domain with an active center typical of a Zn(2+)-metalloprotease, an EGF-like domain and two internal repeats similar to repeated domains found in the C1s and C1r serine proteases of the complement cascade. The BP10 protease is constructed with the same domains as the human bone morphogenetic protein BMP-1, a protease described as a factor involved in bone formation, and as the recently characterized product of the tolloid gene which is required for correct dorsal-ventral patterning of the Drosophila embryo. The transcription of the BP10 gene is transiently activated around the 16- to 32-cell stage and the accumulation of BP10 transcripts is limited to a short period at the blastula stage. By in situ hybridization with digoxygenin-labelled RNA probes, the BP10 transcripts were only detected in a limited area of the blastula, showing that the transcription of the BP10 gene is also spatially controlled. Antibodies directed against a fusion protein were used to detect the BP10 protein in embryonic extracts. The protein is first detected in early blastula stages, its level peaks in late cleavage, declines abruptly before ingression of primary mesenchyme cells and remains constant in late development. The distribution of the BP10 protein during its synthesis and secretion was analysed by immunostaining blastula-stage embryos. The intracellular localization of the BP10 staining varies with time. The protein is first detected in a perinuclear region, then in an apical and submembranous position just before its secretion into the perivitelline space. The protein is synthesized in a sharply delimited continuous territory spanning about 70% of the blastula. Comparison of the size and orientation of the labelled territory in the late blastula with the fate map of the blastula stage embryo shows that the domain in which the BP10 gene is expressed corresponds to the presumptive ectoderm. Developing embryos treated with purified antibodies against the BP10 protein and with synthetic peptides derived from the EGF-like domain displayed perturbations in morphogenesis and were radialized to various degrees. These results are consistent with a role for BP10 in the differentiation of ectodermal lineages and subsequent patterning of the embryo. On the basis of these results, we speculate that the role of BP10 in the sea urchin embryo might be similar to that of tolloid in Drosophila. We discuss the idea that the processes of spatial regulation of gene expression along the animal-vegetal in sea urchin and dorsal-ventral axes in Drosophila might have some similarities and might use common elements.

[The anterior subcostal transperitoneal approach to the kidney. Report on a series of enlarged nephrectomies (author's transl)]. / La voie d'abord antérieure trans-péritonéale sous-costale du rein. A propos d'une série de Néphrectomies élargies.

An anterior subcostal transperitoneal approach was employed in a series of 50 operations, including 43 enlarged nephrectomies for cancer. The advantages of this technique are that it gives direct access to the aortic diaphragmatic orifice and the large vessels and upper pole of the kidney, it is simple to enlarge, and it is solid. It has only minor inconveniences. As only the abdominal cavity is opened, the patient's respiratory function is preserved, especially in the elderly and those with respiratory insufficiency. With a lower mortality and morbidity, a shorter period of hospitalisation, and lower costs, it enables the same enlarged excision to be performed as during thoraco-phrenolaparotomy. It can also be employed for other types of operation on the kidneys and renal vessels, the adrenals, or the lateral aortic glands.

[Tumor grafts in the upper urinary tract secondary to tumors of the bladder epithelium]. / Greffe de tumeur dans la voie excrétrice supérieure à partir des tumeurs de l'épithelium vésical.

A retrospective study of cases seen between 1975 and 1985 showed 24 tumors of upper urinary tract secondary to primary bladder localizations. These secondary localizations developed during evolution of multi-resected recurring bladder tumors, within a mean period of 15 months after discovery of a secondary vesicorenal reflux. This reflux is acquired constantly during the course of these tumors and it is undoubtedly responsible for the onset of these upper tract tumoral grafts. These findings suggest the need for urographic surveillance combined with regular endoscopic examinations of patients with bladder tumors, for prevention of reflux during endoscopic resections and for treatment of any reflux detected.

Spatial expression of the hatching enzyme gene in the sea urchin embryo.

The sea urchin embryo at the blastula stage hatches from its protective fertilization envelope which is degraded by a secreted protease, the hatching enzyme. We have previously purified the hatching enzyme from Paracentrotus lividus (Lepage and Gache (1989). J. Biol. Chem. 264, 4787-4793), cloned its cDNA, and analyzed the temporal expression of its gene (Lepage and Gache (1990). EMBO J. 9, 3003-3012). We study here the temporal and spatial expression of the hatching enzyme gene in whole embryos by immunolabeling with an affinity-purified polyclonal antibody and by in situ hybridization using nonradioactive RNA probes. The timing of expression is consistent with our data on the activation of the gene, the mRNA accumulation in the blastula, and the role of the enzyme. Immunolabeling was observed only in blastula stage embryos; neither before the 128-cell stage nor after hatching. The distribution of the enzyme varies with time from a diffuse labeling around the nucleus to a punctate localization between the nucleus and the apical face of the blastomeres, and finally at the time of hatching, to a submembranous apical location. Not all the cells of an embryo are labeled. The presence of the hatching enzyme is restricted to a sharply delimited continuous territory spanning about two-thirds of the blastula. The orientation of this territory has been determined with respect to the animal-vegetal axis of the embryo using as a landmark the subequatorial pigmented band of the P. lividus species. The synthesis of the hatching enzyme only takes place in the animal-most two-thirds of the blastula. By in situ hybridization, the mRNA coding for the hatching enzyme is only detected in early blastulas, in a limited area having the same size and shape as the territory in which the protein is found. Thus the hatching enzyme gene is likely to be spatially controlled at the transcriptional level: its expression is restricted to a region of the blastula that corresponds roughly to the presumptive ectoderm territory. To date, the hatching enzyme gene products constitute the earliest molecular markers of the sea urchin embryo spatial organization along the primordial egg axis.

Structure of the sea urchin hatching enzyme gene.

The sea urchin embryo develops from an encased to a free-living larva by secreting at an early stage the hatching enzyme, a metalloprotease which hydrolyses a protective envelope derived from the egg extracellular matrix. Genomic clones containing the entire hatching enzyme gene were isolated from a lambda phage sea urchin library and the complete sequence of the transcription unit was determined. The hatching enzyme gene spans 6.3 kb and comprises 9 exons. The exon/intron organization of the hatching enzyme gene is similar but not identical to those of the vertebrate collagenases and stromelysins. The position and/or phase of several introns are different even in the N-terminal moiety where similarity between echinoderm and vertebrate enzymes was first detected. The active-center domain is encoded by a 1-1 class exon whose sequence, length and borders are highly conserved and might be considered as coding for a protein module. Adjacent to the active-center exon, the hatching enzyme gene has an additional 1-1 exon which codes for a threonine-rich region. This provides further evidence that the matrix-degrading metalloproteinases evolved by shuffling exons of the 1-1 class. Phylogeny analysis indicates a close relationship between the sea urchin and vertebrate enzymes.

Guidelines versus clinical practice in the treatment of chronic obstructive pulmonary disease.

The main purpose of this study was to assess whether pharmacological treatments prescribed by respiratory physicians to patients with chronic obstructive pulmonary disease (COPD) were consistent with the guidelines. The treatments prescribed by respiratory physicians to 631 consecutive patients with COPD, compared to 879 asthmatics were prospectively recorded. All subjects underwent peak expiratory flow rate measurement, spirometry and assessment of recent evolution and dyspnoea (visual analogue and Medical Research Council scales). Patients with COPD received more treatments than asthmatics (mean+/-SD: 2.6+/-0.5 versus 2.2+/-0.4, p<0.0001). Treatments administered to patients with COPD were beta2-agonists in 78% (versus 94% in asthmatics), anticholinergic agents (AC) in 56% (versus 16% in asthma), methylxanthines in 31% (versus 15% in asthma) and inhaled corticosteroids in 76% (versus 85% in asthma). Intensity of treatment was influenced by disease severity for all treatments except AC. In conclusion, pharmacological treatment of chronic obstructive pulmonary disease by respiratory physicians is only partially consistent with current guidelines, with a high proportion of inhaled corticosteroid prescriptions and a relative under-use of anticholinergic agents; this most likely reflects the persistent uncertainties of physicians, and emphasizes that more efforts are required to improve implementation of chronic obstructive pulmonary disease guidelines and assess the efficacy and cost-effectiveness of recommended strategies.

Structure of the gene encoding the sea urchin blastula protease 10 (BP10), a member of the astacin family of Zn2+-metalloproteases.

Blastula protease 10 (BP10), a metalloprotease of the astacin family, is secreted at the blastula stage by the sea urchin embryo. The BP10 gene shows a precise temporal and spatial regulation during embryogenesis. It has been cloned from a sea urchin lambda genomic library and the transcription unit has been entirely sequenced. It spans 6kb and contains seven exons (2.8 kb) and six introns (3.2 kb). Sequence comparison and phylogeny analysis show that BP10 belongs to a sub-family of molecular proteins which all play a role during development. In the two cases where the exon/intron organization of the gene is known (BP10 and tolloid), the modular structure of the protein is not reflected at the gene level, which indicates that this sub-family probably did not evolve by exon shuffling.

Cell-autonomous expression and position-dependent repression by Li+ of two zygotic genes during sea urchin early development.

The expression of two zygotic genes (HE and BP10) during sea urchin embryogenesis was previously found to be early, transient, spatially restricted and controlled at the transcriptional level. Here we studied how the expression of these genes is affected when cell interactions are abolished by dissociating blastomeres and when development is perturbed by treatment with Li+. We found that in isolated blastomeres, transient transcriptional activity (HE) is unchanged and both genes apparently function in the appropriate cell type. Thus HE/BP10 expression is largely cell-autonomous and should rely on maternal factors unevenly distributed in the egg. Treatment with lithium does not affect the temporal control but decreases the transcriptional activity and the size of the domain of expression of the HE/BP10 genes. As the Li+ concentration increases, the border of the domain is progressively shifted towards the animal pole. This alteration of the spatial pattern is the earliest molecular evidence of a change in cell fate detectable only much later by morphological criteria, and reveals a gradient of sensitivity to Li+ along the animal--vegetal axis. These results suggest that the activity of the HE/BP10 genes is strongly dependent on spatially organized maternal information controlling early development.

nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate.

We report the isolation and identification of a new mutation affecting pigment cell fate in the zebrafish neural crest. Homozygous nacre (nac(w2)) mutants lack melanophores throughout development but have increased numbers of iridophores. The non-crest-derived retinal pigment epithelium is normal, suggesting that the mutation does not affect pigment synthesis per se. Expression of early melanoblast markers is absent in nacre mutants and transplant experiments suggested a cell-autonomous function in melanophores. We show that nac(w2) is a mutation in a zebrafish gene encoding a basic helix-loop-helix/leucine zipper transcription factor related to microphthalmia (Mitf), a gene known to be required for development of eye and crest pigment cells in the mouse. Transient expression of the wild-type nacre gene restored melanophore development in nacre(-/-) embryos. Furthermore, misexpression of nacre induced the formation of ectopic melanized cells and caused defects in eye development in wild-type and mutant embryos. These results demonstrate that melanophore development in fish and mammals shares a dependence on the nacre/Mitf transcription factor, but that proper development of the retinal pigment epithelium in the fish is not nacre-dependent, suggesting an evolutionary divergence in the function of this gene.

Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning.

Interaction between distinctly specified cells in adjacent compartments establishes organizing centres that control growth and specify cell fate in the developing limbs of Drosophila. Localized expression of the secreted Hedgehog protein (Hh) by cells in the posterior compartment induces expression of the secreted signalling molecules decapentaplegic (dpp) or wingless (wg) in nearby anterior cells. wg and dpp in turn organize spatial pattern in the wing and leg imaginal discs. The Hh signal is thought to act by antagonizing the ability of the patched (ptc) gene product to repress wg and dpp expression. Here we present evidence that removing activity of the gene encoding cyclic AMP-dependent protein kinase A (pka) is functionally equivalent to removing ptc activity or to providing cells with the Hh signal. These findings suggest that cyclic AMP-dependent protein kinase A is a component of the signal transduction pathway through which Hh and Ptc direct localized expression of dpp (or wg) and establish the compartment boundary organizer.