Elastic and inelastic diffraction changes upon variation of the relative humidity environment of PurE crystals.

The different changes observed in the diffraction patterns of three different crystal forms (hexagonal, trigonal and monoclinic) of PurE (EC 4.1.1.21), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, upon a wide range of changes in the relative humidity environment of the crystals are documented. In addition, the changes in the unit-cell parameters, volume and bulk solvent in the three different crystal forms were systematically followed. In an attempt to explain the elastic (P6(5)22) and inelastic (P3(1)21) changes in the diffraction pattern, refined structures of the three different crystal forms determined at 100 K are presented, with particular emphasis on the tertiary and quaternary structural differences, crystal packing, intermolecular and intramolecular interactions and solvent structure. The refined structures show that the precipitant salts, solvent structure (both ordered and bulk) and conformation of the C-termini all play a role in creating a unique cement at both the intramolecular and intermolecular contacts of the different crystal forms. It is suggested that it is the combination of polyethylene glycol and the structure of the ordered water molecules (first and second layers) as well as the structure of the bulk solvent that are the critical factors in the plasticity of the hexagonal crystal packing as opposed to the inelastic responses of the lower symmetry forms.

Humidity control can compensate for the damage induced in protein crystals by alien solutions.

The use of relative humidity control of protein crystals to overcome some of the shortcomings of soaking ligands (i.e. inhibitors, substrate analogs, weak ligands) into pre-grown apoprotein crystals has been explored. Crystals of PurE (EC 4.1.1.21), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, were used as a test case. The findings can be summarized as follows: (i) using humidity control, it is possible to improve/optimize the diffraction quality of crystals soaked in solutions of organic solvent (DMSO, ethanol) containing ligands/inhibitors; (ii) optimization of the relative humidity can compensate for the deterioration of the diffraction pattern that is observed upon desalting crystals grown in high salt; (iii) combining desalting protocols with the addition of PEG it is possible to achieve very high concentrations of weak ligands (in the 5-10 mM range) in soaking solutions and (iv) fine control of the relative humidity of crystals soaked in these solutions can compensate for the deterioration of crystal diffraction and restore `high-resolution' diffraction for structure-based and fragment-based drug design. It is suggested that these experimental protocols may be useful in other protein systems and may be applicable in academic or private research to increase the probability of obtaining structures of protein-ligand complexes at high resolution.

The structure of an engineered domain-swapped ribonuclease dimer and its implications for the evolution of proteins toward oligomerization.

BACKGROUND: Domain swapping has been proposed as a mechanism that explains the evolution from monomeric to oligomeric proteins. Bovine and human pancreatic ribonucleases are monomers with no biological properties other than their RNA cleavage ability. In contrast, the closely related bovine seminal ribonuclease is a natural domain-swapped dimer that has special biological properties, such as cytotoxicity to tumour cells. Several recombinant ribonuclease variants are domain-swapped dimers, but a structure of this kind has not yet been reported for the human enzyme. RESULTS: The crystal structure at 2 A resolution of an engineered ribonuclease variant called PM8 reveals a new kind of domain-swapped dimer, based on the change of N-terminal domains between the two subunits. The swapping is fastened at both hinge peptides by the newly introduced Gln101, involved in two intermolecular hydrogen bonds and in a stacking interaction between residues of different chains. Two antiparallel salt bridges and water-mediated hydrogen bonds complete a new interface between subunits, while the hinge loop becomes organized in a 3(10) helix structure. CONCLUSIONS: Proteins capable of domain swapping may quickly evolve toward an oligomeric form. As shown in the present structure, a single residue substitution reinforces the quaternary structure by forming an open interface. An evolutionary advantage derived from the new oligomeric state will fix the mutation and favour others, leading to a more extended complementary dimerization surface, until domain swapping is no longer necessary for dimer formation. The newly engineered swapped dimer reported here follows this hypothetical pathway for the rapid evolution of proteins.

Three-dimensional structure of a human pancreatic ribonuclease variant, a step forward in the design of cytotoxic ribonucleases.

We have determined the crystal structure of a human pancreatic ribonuclease or RNase 1 variant at 1.65 A resolution. Five residues in the N-terminal region were substituted by the corresponding amino acids of the bovine seminal RNase. In addition, a Pro to Ser mutation was present at position 50. The substitution of part of the N terminus has been critical both in improving the expression of this enzyme as a recombinant protein and in achieving its crystallisation. The determination of the crystal structure revealed the characteristic RNase fold including a V-shaped beta-sheet and three alpha-helices. It differs from its bovine RNase orthologue mainly in the loop regions. The active-site cleft shows a similar architecture to that of its bovine counterpart, with the essential residues occupying equivalent positions. In the present structure, however, His119 is displaced as it is in the structure of RNase A at high pH. An interaction model of human ribonuclease with the ribonuclease inhibitor, together with inhibition assays, indicate that, in contrast to RNase A, the modification of the loop beta4beta5 is not enough to avoid inhibition. This study represents the first crystallographic approach to the human enzyme, and should constitute an invaluable tool for the design of ribonuclease variants with acquired cytotoxic properties.

Crystallographic analysis reveals the 12-fold symmetry of the bacteriophage phi29 connector particle.

The internal symmetry of the connector or portal particle from the double-stranded DNA bacteriophage phi29 has been examined by X-ray crystallography. This large multimeric structure (420 kDa) is built up by a number of identical subunits of the p10 protein. It connects the head of the virus with the tail and plays a central role in the prohead assembly and DNA packaging. For the first time a bacteriophage connector has been crystallized and X-ray data have been collected up to a resolution of 3.2 A. A self-rotation function has been calculated, unambigously revealing the 12-fold symmetry of the particle and its orientation in the crystal lattice. The orientation has been confirmed by calculating a cross-rotation function using a low resolution model based on electron microscopy reconstructions.

Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution.

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.

Purification, crystallization and preliminary X-ray diffraction studies of the bacteriophage phi29 connector particle.

The connector or portal particle from double-stranded DNA bacteriophage phi29 has been crystallized. This structure, which connects the head of the virus with the tail and plays a central role in prohead assembly and DNA packaging and translocation, is formed by 12 subunits of the p10 protein and has a molecular weight of 430 kDa. The connector structure was proteolysed with endoproteinase Glu-C from Staphylococcus aureus V8, which removes 13 and 18 amino acids from the amino- and carboxy-terminal regions of the p10 protein, respectively. Two crystal forms were grown from drops containing an alcohol solution and paraffin oil. Crystals of form I are monoclinic, space group C2 with cell dimensions a = 416.86 A, b = 227.62 A, c = 236.68 A and beta = 96.3 degrees and contain four connector particles per asymmetric unit. Crystals of form II are tetragonal, space group P4(2)2(1)2 with cell dimensions a = b = 170.2 A, c = 156.9 A and contain half a particle per asymmetric unit. X-ray diffraction data from both native crystal forms have been collected to 6.0 and 3.2 A respectively, using synchrotron radiation. Crystals of form II are likely to have the same packing arrangement as the two-dimensional crystals analyzed previously by electron microscopy.

Alzheimer disease: protective factors.

Approximately 6-8% of all persons aged >65 y have Alzheimer disease and the prevalence of the disease is increasing. Any intervention strategy aimed at decreasing risks or delaying the onset of the disease will therefore have a substantial effect on health care costs. Nutrition seems to be one of the factors that may play a protective role in Alzheimer disease. Many studies suggest that oxidative stress and the accumulation of free radicals are involved in the pathophysiology of the disease. Several studies have shown the existence of a correlation between cognitive skills and the serum concentrations of folate, vitamin B-12, vitamin B-6, and, more recently, homocysteine. However, nutritional factors have to be studied not alone but with the other factors related to Alzheimer disease: genetics, estrogen, antiinflammatory drug use, and socioeconomic variables. The objective of this article was to review recent studies in this field.

Trends in acute myocardial infarction prognosis and treatment in southwestern France between 1985 and 1990 (the MONICA Project-Toulouse).

Since 1984, the Multinational Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA project) has studied progress in acute coronary artery disease (CAD) in defined geographic areas. This study, based in a French MONICA center in Southwestern France, assessed acute myocardial infarction (AMI) frequency, prognosis, and treatment between 1985 and 1990. The age-standardized AMI attack rate showed a slight decrease in men (218.3 of 100,000 in 1985 and 200.1 of 100,000 in 1990, p < 0.05). In men, the AMI age-standardized 28-day case fatality rate decreased from 38% in 1985 to 27% in 1990 (p < 0.001), with a dramatic reduction in the age-standardized in-hospital case fatality rate (21% in 1985 and 11% in 1990, p < 0.001). Furthermore, the in-hospital 28-day case fatality decreased exclusively in male patients with a history of CAD (33% in 1986 and 19% in 1990, p < 0.01). AMI treatment improved between 1986 and 1990 with increased use of antiplatelets (40.7% in 1986, 73.6% in 1989, and 84.8% in 1990, p < 0.001), beta-blockers (29.5% in 1986, 45.7% in 1989, and 57.7% in 1990, p < 0.001), and thrombolytic therapy (23.6% in 1986, 45.3% in 1989, and 39.5% in 1990, p < 0.001). Invasive cardiology technique utilization increased dramatically between 1986 and 1990 with the generalized use of coronary angiography (68.2% in 1986, 89.3% in 1989, and 87.3% in 1990, p < 0.001) and percutaneous transluminal coronary angioplasty (17.7% in 1986, 49.6% in 1989, and 55.3% in 1990, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Disability, psychosocial factors and mortality among the elderly in a rural French population.

The purpose of this work is to identify risk markers of mortality in a cohort of 645 people aged 60 and over. The study was carried out in rural areas in south west France. Data were collected by questionnaire in 1982. Mortality was determined 4 years later; 111 deaths were registered. The analysis of age-adjusted odds ratios (OR) showed strong relationships between mortality and disability (OR = 7.75), compared health (OR = 3.94), self-rated health (OR = 2.47), home comfort (OR = 0.52), physical activity (OR = 0.32), sociability (OR = 0.43) and two subjective well-being items: the feeling of uselessness (OR = 3.51), and the lack of projects for the future (OR = 2.35). By contrast, no significant association was observed with reported morbidity and social support. Two multivariate analyses were performed: the first on longevity using Cox's regression model, the second on mortality using a linear discriminant analysis. The results of these analyses were translated into a simple set of 8 independent risk markers for the identification of a "high risk group" of mortality within 4 years. The sensitivity of this mortality risk indicator was 73% and its specificity 77%.

Ultrastructural aspects of nerves, bones, and vessels in hereditary sensory neuropathy.

This study concerns two children, from the same otherwise normal family, presenting progressive spontaneous amputations of the limbs, suggesting peripheral hereditary sensory neuropathy. Samples from the distal nerves and vessels and the distal tibial epiphyses were examined ultrastructurally. The nerves showed signs of degeneration, and a typical bone necrosis, without osteoclasts, was observed in the 2 cm margin of the samples. Simultaneous modifications of the vessels suggest a neurally initiated vascular reflex resulting in bone destruction. Theories that might explain the observations are discussed.

Predictive value of life events, psychosocial factors and self-rated health on disability in an elderly rural French population.

Increasing evidence suggests that exposure to stressful life events and a variety of psychosocial factors are related to different health outcomes in the elderly. Our purpose is to study the predictive value of each of these items on the ability deterioration of a panel of 645 rural adults, aged 60 and over, living at home and followed for 4 years (1982-1986). This survey was carried out in five rural areas of Haute-Garonne (South-West France). Data were collected from the elderly themselves by questionnaire in 1982 and 1986. An indicator of ability evolution (1982-1986) was constructed for all those surviving and reviewed in 1986. Our study concerned 470 elderly people. Ability deterioration was 55.3% (260 elderly people). The analysis of age-adjusted relative risks (RR) of ability deterioration showed a significant impact of economic level (RR = 2.3), self-rated health (RR = 2.2) and reported morbidity (RR = 2.2). Among the psychosocial factors, we noted the predictive role of a lack of project for the future (RR = 1.7) and mostly of a feeling of uselessness (RR = 9.8), but also of non-participation in association activities for people aged less than 75. All these relationships remain significant after adjustment according to reported morbidity. In contrast, no significant effect was found for social support and life events which occurred during the follow-up period. Logistic discriminant analysis and segmentation analysis were performed. They confirmed the independence of the predictive roles played by age, economic level, reported morbidity and the feeling of uselessness. These results, discussed with bibliographic data, should give a better knowledge of the processes which underlie pathological ageing.

Legg-Calvé-Perthes disease. Histochemical and ultrastructural observations of the epiphyseal cartilage and physis.

UNLABELLED: Biopsy specimens of the lateral aspect of the femoral head and neck were obtained from five children with Legg-Calvé-Perthes disease and were studied using histochemistry and electron microscopy. Beneath the normal articular cartilage there was a thick zone of hyaline (epiphyseal) cartilage containing sharply demarcated areas of hypercellular and fibrillated cartilage with prominent blood vessels. The fibrillated cartilage was strongly positive to alcian blue, weakly positive to periodic acid-Schiff, and positive to aniline blue. The interterritorial matrix in the hypercellular areas was weakly positive to both alcian blue and periodic acid-Schiff. Ultrastructural examination of these areas revealed many irregularly oriented large collagen fibrils and variable amounts of proteoglycan granules. These results suggest that in the fibrillar areas there are: (1) a high proteoglycan content, (2) a decrease in structural glycoproteins, and (3) a different size of collagen fibrils from that of normal epiphyseal cartilage. The hypercellular areas had a decrease in proteoglycans, glycoproteins, and collagen. The lateral physeal margin was often irregular, with a marked reduction of collagen and proteoglycan granules, and contained numerous large lipid inclusions. CLINICAL RELEVANCE: The abnormal areas in the epiphyseal cartilage of patients with Legg-Calvé-Perthes disease have different histochemical and structural properties from normal cartilage and from fibrocartilage. This suggests that the disease could be a localized expression of a generalized, transient disorder of epiphyseal cartilage that is responsible for delayed skeletal maturation. The cartilage lesions are similar to those seen in the vertebral plates in patients with juvenile kyphosis. Whether the epiphyseal cartilage abnormalities are primary or are secondary to ischemia remains uncertain; however, it appears that the collapse and necrosis of the femoral head could result from the breakdown and disorganization of the matrix of the epiphyseal cartilage, followed by abnormal ossification.

Neonatal surgery in clubfoot.

Neonatal surgery for clubfoot is conducted according to the following rules: One must work as close to the lesions as possible. It is important to avoid going beyond abnormal tissues. Dissection should be precise and economical. Opening of Lisfranc's joint or sectioning of the medial ligament must be avoided. One must operate with maximal accuracy. All areas of fibrosis must be excised, but care must be taken not to enter the interarticular space. If a bone deformity is present, one must accept an incomplete correction. One must act as soon as possible. This is the inverse of classic management in which orthopedic surgery was postponed. We prefer early surgery followed by intensive postoperative rehabilitation.

[Cost-effectiveness in diagnosis and treatment of carcinomas of unknown primary origin]. / Analyse Coût/efficacité de la prise en charge diagnostique et thérapeutique des carcinomes d'origine indéterminée.

The aim was to compare, in terms of cost-effectiveness, two diagnostic strategies for finding out the primary site of tumors revealed by metastasis, adopting the hospital's perspective. The observed strategy reflected the usual practices of doctors at the Regional Cancer Center in Toulouse (France), and was based on a sample of 202 patients of this Center. The standardized strategy, which reflected limited diagnostic investigation, was simulated using the same sample of patients to whom we applied the recommendations of local experts. In the low assumption regarding the effectiveness of the standardized strategy, the observed strategy compared to the standardized one raised the life expectancy from 407 to 418 days at an incremental cost of $US 1,236 per patient (1996 values). In this case, one day of additional life induced a cost of $US 112 per patient. In the high assumption, the incremental effectiveness was null and the incremental cost was $US 1,236 per patient. In conclusion, the effectiveness of the observed strategy as compared to the standardized strategy was highly questionable, given that the patients' quality of life was not taken into account.

Chronic recurrent multifocal osteomyelitis (C.R.M.O.). A case report and review of literature.

The case of an 8-year-old boy presenting two episodes of osteomyelitis of the left and right tibial diaphysis is reported. The recurrent course, the negative cultures and the symmetrical aspect of the lesions suggest the diagnosis of recurrent multifocal osteomyelitis. The location of the lesions at the middle of the diaphysis and the histological finding of predominant bone necrosis are unusual.

[Mental and behavior disorders in the aged: attempt at classification and relationship with caregivers' burden]. / Troubles mentaux et comportementaux des personnes âgées: essai de classification et lien avec la charge des aidants.

BACKGROUND: To measure the relation between different disorders of the elderly and the carer's burden. METHOD: A cross-sectional study was carried out by a questionnaire administered to carers of dependent elderly living at home. Mental and behavioral disorders were measured by the Zarit "Memory and behavior problems check list" (MBPC); carer's burden was measured with the Zarit "Burden Inventory" (BI). A principal components analysis was performed to make groups of disorders with the MBPC items and the relation between these groups and the carers' burden was determined by a multiple regression analysis. RESULTS: The analysis was performed with 190 files. Six groups of disorders were individualized: memory and orientation problems, aggressive or dangerous behaviors, regressive behaviors, disruptive behaviors, wandering, mood disturbance and agitation. These 6 factors explain 43.2% of the variance of the carer's burden. Each factor is linked to the BI score and 3 behavioral typologies are specially related to the carers BI: regressive behaviors (R = 0.49), disruptive behaviors (R = 0.51), and mood disturbance with agitation (R = 0.53). CONCLUSION: This study shows that it is possible to differentiate specific behavioral typologies in elderly, which explain a limited part of the carer's burden.

[Disabilities during aging: descriptive approach and risk factors. 4-year longitudinal study of an aged rural population]. / Les incapacités au cours du vieillissement: approche descriptive et facteurs de risque. Enquête longitudinale sur 4 ans dans une population rurale âgée.

A longitudinal survey has been conducted from 1982 to 1986 in a rural population of 645 persons aged 60 and over. The aim of this study is to analyse the evolution of disabilities in this population and the main risk factors. The incidence of disabilities within 4 years is more important in the oldest birth-cohort groups and this result suggests a cumulative effect of the ageing process and chronic diseases. Indeed, the assessment of predictive factors shows that age and reported morbidity are related to the incidence of disabilities, but two other factors are strongly related to the loss of functional abilities after age adjustment: socioeconomic status and the feeling of uselessness. We conclude that disability in the elderly is not an inevitable consequence of the physiological ageing process and that a preventive approach should be based on a multidimensional concept: physical, social and psychological.

[Measuring the burden for carers of dependent elderly with the Zarit inventory]. / Mesure de la charge des aidants de personnes âgées dépendantes par la grille de Zarit.

The aim of this study was to measure the subjective burden felt by persons who care for dependent elderly people living in their home. A questionnaire was administered to 291 persons. The burden is measured by the Zarit "Burden Inventory" in the French version; it is a 22 question scale exploring the consequences of caring activities in the physical, psychological and social area. The burden score is related to the physical dependency and mostly to the behavioural troubles of the elderly. It is not different according to the carer's family relationship but it is related to his family situation, his self-rated health and the desire to institutionalize the elderly person. This synthetic tool seems to measure the quality of life of carers, but longitudinal studies are necessary to measure its predictive value concerning behaviours in this population.

[Predictive factors of institutionalization of dependent elderly persons]. / Facteurs prédictifs de la rupture de la prise en charge à domicile des personnes âgées dépendantes.

BACKGROUND: This study analyses the factors related to the institutionalization of dependent patients living in the community and receiving informal care from relatives. METHODS: The study is based on a 3-year follow-up of a sample of 205 pairs (carereceiver-caregiver). Eligible care receivers were aged 65 years and over, living at home, and physically or mentally dependent. Caregivers were interviewed at inclusion and three years later in order to know whether the elderly was institutionalized or still living at home. RESULTS: Cox proportional hazards analysis indicated that significant predictors of subsequent institutionalization were intention to institutionalize (p = 0.001), caregiver type (p = 0.015), caregiver's subjective perception of patient behavior problems (p = 0.009), and, at a lesser extent, to live in a urban environment (p = 0.051). The functional status and the carereceiver's age were not predictive of institutionalization. CONCLUSION: This study emphasizes the importance of subjective factors and characteristics of the primary caregiver in predicting institutionalization of elderly dependent persons.