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.

Early onset of organ failure is the best predictor of mortality in acute pancreatitis.

BACKGROUND: APACHE II is a multifactorial scoring system for predicting severity in acute pancreatitis (AP). Organ failure (OF) has been correlated with mortality in AP. OBJECTIVE: To evaluate the usefulness of APACHE II as an early predictor of severity in AP, its correlation with OF, and the relevance of an early establishment of OF during the course of AP. PATIENTS AND METHODS: From January 1999 to November 2001, 447 consecutive cases of AP were studied. APACHE II scores and Atlanta criteria were used for defining severity and OF. RESULTS: Twenty-five percent of patients had severe acute pancreatitis (SAP). APACHE II at 24 h after admission showed a sensitivity, specificity, and positive and negative predictive value of 52, 77, 46, and 84%, respectively, for predicting severity. Mortality for SAP was 20.5%. Seventy percent of patients who developed OF did so within the first 24 hours of admission, and their mortality was 52%. Mortality was statistically significant (p< 0.01) if OF was established within the first 24 hours after admission. CONCLUSIONS: APACHE II is not reliable for predicting outcome within the first 24 hours after admission and should therefore be used together with other methods. OF mostly develops within the first days after admission, if ever. The time of onset of OF is the most accurate and reliable method for predicting death risk in AP.

Crystallization and preliminary X-ray analysis of the DNA decamers d(CCGGATCCGG) and d(CCGGCGCCGG).

The DNA decamers d(CCGGATCCGG) and d(CCGGCGCCGG) have been crystallized for X-ray analysis in order to investigate the effects of changing the two central base pairs of the DNA fragment d(CCGGGACCGG). Previous studies have already demonstrated that the structure of the former DNA fragment contains a DNA Holliday junction. Crystals were obtained at 293 K by the hanging-drop vapour-diffusion technique using the Nucleic Acid Mini Screen. Over a period of two weeks, hexagonal plates appeared. For the DNA fragment d(CCGGATCCGG), the crystals belong to space group P3(1), with unit-cell parameters a = b = 33.54, c = 46.39 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 2.2 A. In the case of the DNA fragment d(CCGGCGCCGG) the crystals belong to the space group C2, with unit-cell parameters a = 65.35, b = 24.07, c = 37.34 A, beta = 109.97 degrees, and diffract to 2.0 A.

Crystallization and preliminary X-ray analysis of the antimalarial and cytotoxic alkaloid cryptolepine complexed with the DNA fragment d(CCTAGG)2.

Crystals of the indoloquinoline alkaloid cryptolepine complexed with the DNA fragment d(CCTAGG)(2) have been grown by the hanging-drop technique at 293 K using ammonium sulfate as the precipitating agent. Over a period of three weeks, yellow tapering bullet-shaped crystals grew to maximum dimensions of 0.2 x 0.1 x 0.1 mm. The crystals belong to space group P6(4), with unit-cell parameters a = b = 29.960, c = 39.64 A, alpha = beta = 90, gamma = 120 degrees, and diffract to 1.4 A.

[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.

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 human RNase 1 delta N7 at 1.9 A resolution.

Human pancreatic ribonuclease 1 (RNase 1) is considered to be the human counterpart of bovine pancreatic RNase A. Truncation of seven amino-acid residues from the amino-terminal sequence resulted in RNase 1 Delta N7, which has a reduced ribonucleolytic activity and a lower affinity for the human placental RNase inhibitor (PRI). This RNase 1 variant has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data to 1.9 A resolution. The molecule displays the alpha + beta folding topology typical of members of the RNase A superfamily. The main distinct features found in RNase 1 Delta N7 are basically located in three loops affecting the fitting of the enzyme to the active site of subtilisin and the shape of the B2 subsite. These changes, taken with the lack of the catalytically active residue Lys7, may explain the reduced affinity of RNase 1 Delta N7 for PRI and the low ribonucleolytic activity of the protein when compared with the native enzyme.

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.

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.

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.

Alarm communication network to help carers of the elderly for safety purposes: a survey of a project.

Over years, radical changes in engineering technology have taken place due to greatly improved instrumentation, enhanced computing power, new sensors and upgraded transmission mediums making it possible to achieve in the field what once could only be done on laboratory models. In daily life, the use of an active self-testing instrumentation endowed with a self-diagnostic capability built into the structural fabric of a machine permits us to monitor the initiation of breakdown, corrosion and erosion. This has become particularly important in such means of transportation as planes, boats and automobiles where advanced instrumentation, signal processing and pattern recognition systems operate in fairly conventional structures. By capitalizing on the technological progress in the field of instrumentation and data processing, help can be contemplated for elderly people in nursing homes through detection of abnormal situations. The continued assistance to elderly people is achieved by utilizing a technological system respectful of the elderly's privacy and operating without their active participation.

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.

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.

Multisensor system and artificial intelligence in housing for the elderly.

To improve the safety of a growing proportion of elderly and disabled people in the developed countries, a multisensor system based on Artificial Intelligence (AI), Advanced Telecommunications (AT) and Information Technology (IT) has been devised and fabricated. Thus, the habits and behaviours of these populations will be recorded without disturbing their daily activities. AI will diagnose any abnormal behavior or change and the system will warn the professionals. Gerontology issues are presented together with the multisensor system, the AI-based learning and diagnosis methodology and the main functionalities.

[Social situations and health care of immigrant foreign populations in France over the course of the last 5 decades]. / Situations sociales et besoins de santé des populations étrangères immigrées en France au cours des 5 dernières décennies.

The goal of this study is to define possible characteristics in social and medical needs with immigrant foreigners who arrived in France during the last fifty years; and because of the grave situation, it should be necessary to take appropriate and special action. Except identical needs related to similar social and economic situations shared by french and foreign people, the immigration introduces new risk factors: related to ethnologic expression of the illness and cultural breaking, a special assistance is claimed; ethnopsychiatry is one of many possible solutions.

[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.

[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.

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.