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This study examined patient-reported sleep quality in a single-center cross-sectional sample of adolescents with solid organ transplants and evaluated associations between sleep quality, psychosocial functioning (ie, depression/anxiety symptoms), and HRQOL. Health disparities associated with minority race/ethnicity and socioeconomic variables were also examined. Sixty-nine adolescents (M = 16.51 years; SD = 1.63) who received a solid organ transplant (kidney: n = 25; liver: n = 24; heart: n = 20) completed self-report measures of sleep quality, psychosocial functioning, and HRQOL. Adolescent transplant recipients endorsed significantly lower levels of sleep quality (ie, falling asleep) compared with previously published norms of healthy peers (t = -3.60; P ≤ .001). Higher sleep quality was significantly associated with fewer anxiety and depressive symptoms (r = -.31 to -.40), and higher physical and psychosocial HRQOL (r = .33-.43). Adolescents from minority backgrounds had significantly worse sleep quality compared with non-Hispanic Whites. Adolescent transplant recipients, particularly those from minority backgrounds, may be at increased risk for experiencing poor sleep quality. Suboptimal sleep is a risk factor for higher levels of anxiety and depressive symptoms, as well as lower levels of physical and psychosocial HRQOL. Sleep is an important modifiable factor that, if improved, may contribute to lower anxiety/depressive symptoms and better HRQOL in adolescent transplant recipients., (© 2019 Wiley Periodicals, Inc.)

Importance: Less than one-third of patients with major depressive disorder (MDD) achieve remission with their first antidepressant., Objective: To determine the relative effectiveness and safety of 3 common alternate treatments for MDD., Design, Setting, and Participants: From December 2012 to May 2015, 1522 patients at 35 US Veterans Health Administration medical centers who were diagnosed with nonpsychotic MDD, unresponsive to at least 1 antidepressant course meeting minimal standards for treatment dose and duration, participated in the study. Patients were randomly assigned (1:1:1) to 1 of 3 treatments and evaluated for up to 36 weeks., Interventions: Switch to a different antidepressant, bupropion (switch group, n = 511); augment current treatment with bupropion (augment-bupropion group, n = 506); or augment with an atypical antipsychotic, aripiprazole (augment-aripiprazole group, n = 505) for 12 weeks (acute treatment phase) and up to 36 weeks for longer-term follow-up (continuation phase)., Main Outcomes and Measures: The primary outcome was remission during the acute treatment phase (16-item Quick Inventory of Depressive Symptomatology-Clinician Rated [QIDS-C16] score ≤5 at 2 consecutive visits). Secondary outcomes included response (≥50% reduction in QIDS-C16 score or improvement on the Clinical Global Impression Improvement scale), relapse, and adverse effects., Results: Among 1522 randomized patients (mean age, 54.4 years; men, 1296 [85.2%]), 1137 (74.7%) completed the acute treatment phase. Remission rates at 12 weeks were 22.3% (n = 114) for the switch group, 26.9% (n = 136)for the augment-bupropion group, and 28.9% (n = 146) for the augment-aripiprazole group. The augment-aripiprazole group exceeded the switch group in remission (relative risk [RR], 1.30 [95% CI, 1.05-1.60]; P = .02), but other remission comparisons were not significant. Response was greater for the augment-aripiprazole group (74.3%) than for either the switch group (62.4%; RR, 1.19 [95% CI, 1.09-1.29]) or the augment-bupropion group (65.6%; RR, 1.13 [95% CI, 1.04-1.23]). No significant treatment differences were observed for relapse. Anxiety was more frequent in the 2 bupropion groups (24.3% in the switch group [n = 124] vs 16.6% in the augment-aripiprazole group [n = 84]; and 22.5% in augment-bupropion group [n = 114]). Adverse effects more frequent in the augment-aripiprazole group included somnolence, akathisia, and weight gain., Conclusions and Relevance: Among a predominantly male population with major depressive disorder unresponsive to antidepressant treatment, augmentation with aripiprazole resulted in a statistically significant but only modestly increased likelihood of remission during 12 weeks of treatment compared with switching to bupropion monotherapy. Given the small effect size and adverse effects associated with aripiprazole, further analysis including cost-effectiveness is needed to understand the net utility of this approach., Trial Registration: clinicaltrials.gov Identifier: NCT01421342.

Purpose: This study aimed to examine differences in transition readiness, self-involvement and parental involvement in completing medical tasks, and general self-efficacy between a sample of older adolescents and young adults (AYAs) with medical conditions and a sample of healthy peers. Relations among these variables were also examined., Methods: The sample included 494 AYAs (mean age = 19.30 years, standard deviation = 1.33) who reported on their levels of transition readiness, self-involvement and parental involvement in completing medical tasks, and general self-efficacy., Results: AYAs with medical conditions reported significantly higher levels of transition readiness and self-involvement in completing medical tasks and lower levels of parent involvement in completing medical tasks than healthy peers. Parent involvement in completing medical tasks indirectly related to transition readiness through AYA self-involvement in completing medical tasks for both AYAs with medical conditions and healthy peers., Conclusions: AYAs with medical conditions appear to have greater transition readiness skills and demonstrate more independence in completing medical tasks than healthy peers. For AYAs with medical conditions and healthy peers, transition readiness appears to be enhanced as parents decrease their involvement in completing AYAs' medical tasks and AYAs increase self-involvement in completing these tasks. AYAs with medical conditions, as well as healthy peers, may benefit from programming delivered in primary care, specialty clinic, or educational settings that focuses on increasing AYAs' involvement in and responsibility for managing their health care., (Copyright © 2017 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

OBJECTIVE : To evaluate levels of executive functioning in a sample of adolescent and young adult (AYA) transplant recipients, and to examine executive functioning in association with barriers to adherence and medication nonadherence.  METHOD : In all, 41 caregivers and 39 AYAs were administered self- and proxy-report measures.  RESULTS : AYA transplant recipients have significant impairments in executive functioning abilities. Greater dysfunction in specific domains of executive functioning was significantly associated with more barriers to adherence and greater medication nonadherence.  CONCLUSION : AYA transplant recipients are at increased risk for executive dysfunction. The assessment of executive functioning abilities may guide intervention efforts designed to decrease barriers to adherence and promote developmentally appropriate levels of treatment responsibility., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Pediatric Psychology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Children and adolescents with Tourette syndrome are at risk for social, emotional, and behavioral difficulties that may negatively influence feelings of self-competence and attitudes toward having this diagnosis. Attending a camp for children with Tourette syndrome may facilitate improvements in overall socio-emotional and behavioral functioning and self-perception. The current study obtained data from 37 campers (76 % male, 24 % female) and 47 caregivers of campers to investigate changes in children's emotional and behavioral functioning, self-perception (i.e., social and general), attitudes towards having Tourette syndrome, and how different they felt from peers after attending a 1-week summer camp for youth with Tourette syndrome using a pre-post design. Results indicated that campers endorsed a significantly improved sense of social self-competence and more favorable attitudes toward having Tourette syndrome post-camp. Campers also tended to endorse feeling less different than peers at camp versus peers in general. Caregivers endorsed significantly less severe symptoms associated with Tourette syndrome for campers after attending camp. No pre-to-post-camp changes were observed for campers' levels of anxiety or obsessive-compulsive symptoms. These preliminary findings suggest that attending camp or having other opportunities to interact with similar peers may promote better perceptions of social self-competence, more favorable attitudes toward having Tourette syndrome, and a stronger sense of affiliation with peers for children with Tourette syndrome.

Objective: To evaluate self-competence, health-related quality of life (HRQOL), and emotional/behavioral functioning in children with Tourette syndrome (TS) compared to normative data and to examine self-competence as a potential protective factor against poorer HRQOL and emotional/behavioral outcomes in this population., Method: Thirty-nine children between the ages of 8 and 17 years and 72 caregivers participated in this study. Participants completed measures of children's HRQOL, emotional/behavioral functioning, and self-competence., Results: Participants reported significantly lower levels of emotional/behavioral functioning and HRQOL compared with norms of healthy children. No significant differences were found in domains of perceived self-competence. Social and general self-competence domains were significantly and positively correlated with most emotional and behavioral outcomes examined. Only social self-competence was significantly correlated with domains of HRQOL., Conclusion: Self-competence, particularly in the social realm, may play a protective role against lower HRQOL and worse emotional and behavioral outcomes in children with TS. Children with this condition may benefit from self-competence-promoting interventions targeting children's perceptions of their own abilities.

This study aimed to evaluate the degree of agreement between parent proxy- and child self-report on measures of child psychosocial functioning and health-related quality of life in children with Tourette syndrome. Participants included 28 children with Tourette syndrome and their parents. All participants provided ratings of children's level of quality of life and psychosocial functioning. Results revealed strong, positive relationships between child self- and parent proxy-reports on all quality of life and psychosocial functioning domains. Parents perceived significantly higher levels of depression compared to their children, whereas children reported significantly lower Physical quality of life compared to their parents. Results suggest that assessment of quality of life and psychosocial functioning should include multiple reporters whenever feasible. Caution should be used when exclusively relying on parent proxy-reports of quality of life and psychosocial functioning, as these reports may not accurately reflect children's difficulties or perceptions of their functioning., (© The Author(s) 2014.)

Age-related macular degeneration (AMD) is a devastating retinal disease that occurs in later life as the retinal pigment epithelium (RPE) cells die, with subsequent photoreceptor degeneration. In the past, RPE transplant surgeries gave evidence that AMD was potentially treatable, but it involved limited amounts of ocular tissue, and the complication rate was high. Then, stem cell transplants offered an unlimited supply of retinal precursors for endogenous repair and exogenous cell replacement. Debate continues as to which type of stem cell is most appropriate for treating AMD. The prospects include adult-derived progenitor stem cells (including progenitor cells from ocular tissues), hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Now the therapy is expanding into phase I human trials. This review examines the collective research contributions toward a clinical model of AMD management with stem cells.

Purpose: Dry eye disease is a common condition that affects millions of people world wide. The common findings of dry eye disease are blurred vision and tear film instability. The purpose of this study was to determine if long-term use of artificial tears altered visual disturbances and tear film instability of dry eye patients., Methods: Contrast sensitivity and optical aberrations were measured in 22 dry eye and 10 normal patients before and after daily use of artificial tears. The contrast sensitivity and optical aberrations were measured in response to the administration of a single drop of artificial tear placed in the eye., Results: The short-term effect (i.e., a few minutes) of a single drop of artificial tear placed in the eye was a decrease in contrast sensitivity and an increase in optical aberrations. Long-term daily use of the artificial tears (i.e., up to 2 weeks) resulted in less of a short-term effect in dry eye patients. No long-term effect was observed for normal subjects. Both contrast sensitivity loss and optical aberrations decreased by 35% per week of artificial tear use for the dry eye patients suggesting that the changes in contrast sensitivity were the result of optical aberrations., Conclusions: The results suggest that the changes in contrast sensitivity with artificial tear administration were the result of optical aberrations. It appears that long-term use of artificial tears may normalize the tear layer of dry eye disease patients.

Vision research involving stem cells is a rapidly evolving field. Animal experiments have shown that in response to environmental cues, stem cells can repopulate damaged retinas, regrow neuronal axons, repair higher cortical pathways, and restore pupil reflexes, light responses and basic pattern recognition. Viable corneas have been grown from stem cells and transplanted into humans. Similarly, human trials to repair damaged retinas in retinitis pigmentosa and age-related macular degeneration patients have produced preliminary successes. This review attempts to place the collective contributions toward stem cell/vision research into a broader clinical model of how stem cells might ultimately be used to restore the entire visual pathway.

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyl1 from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xyn11 from Bacillus agaradhaerens and XylJ from Bacillus sp. 41M-1 share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xyn11 and Xyl1 were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xyn11. Some of these charges were introduced in the structure of Xyl1 by site-directed mutagenesis with substitutions Y16D, S18E, G50R, N92D, A135Q, E139K, and Y186E. Furthermore, the eight additional loop residues of Xyn11 were introduced in the homologous loop of Xyl1. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyl1 mutants. The Y186E substitution inactivated Xyl1, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl1 from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant.

The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli. This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5. The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the surface of cellulose available to a bound enzyme molecule. The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi. Both kcat and kcat/K(m) values at 4 degrees C for the psychrophilic cellulase are similar to the values for Cel5 at 30-35 degrees C, suggesting temperature adaptation of the kinetic parameters. The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data. The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site. No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures.

Xyl1 from Streptomyces sp. S38 belongs to the low molecular mass family 11 of endo-beta-1,4-xylanases. Its three-dimensional structure has been solved at 2.0 A and its optimum temperature and pH for enzymatic activity are 60 degrees C and 6.0, respectively. Aspergillus kawachii xylanase XynC belongs to the same family but is an acidophilic enzyme with an optimum pH of 2.0. Structural comparison of Xyl1 and XynC showed differences in residues surrounding the two glutamic acid side chains involved in the catalysis that could be responsible for the acidophilic adaptation of XynC. Mutations W20Y, N48D, A134E, and Y193W were introduced by site-directed mutagenesis and combined in multiple mutants. Trp 20 and Tyr 193 are involved in substrate binding. The Y193W mutation inactivated Xyl1 whereas W20Y decreased the optimum pH of Xyl1 to 5.0 and slightly increased its specific activity. The N48D mutation also decreased the optimum pH of Xyl1 by one unit. The A134E substitution did not induce any change, but when combined with N48D, a synergistic effect was observed with a 1.4 unit decrease in the optimum pH. Modeling showed that the orientations of residue 193 and of the fully conserved Arg 131 are different in acidophilic and "alkaline" xylanases whereas the introduced Tyr 20 probably modifies the pKa of the acid-base catalyst via residue Asn 48. Docking of a substrate analog in the catalytic site highlighted striking differences between Xyl1 and XynC in substrate binding. Hydrophobicity calculations showed a correlation between acidophilic adaptation and a decreased hydrophobicity around the two glutamic acid side chains involved in catalysis.

The role of the mobile loop comprising residues 60-66 in metallo-beta-lactamases has been studied by site-directed mutagenesis, determination of kinetic parameters for six substrates and two inhibitors, pre-steady-state characterization of the interaction with chromogenic nitrocefin, and molecular modeling. The W64A mutation was performed in IMP-1 and BcII (after replacement of the BcII 60-66 peptide by that of IMP-1) and always resulted in increased K(i) and K(m) and decreased k(cat)/K(m) values, an effect reinforced by complete deletion of the loop. k(cat) values were, by contrast, much more diversely affected, indicating that the loop does not systematically favor the best relative positioning of substrate and enzyme catalytic groups. The hydrophobic nature of the ligand is also crucial to strong interactions with the loop, since imipenem was almost insensitive to loop modifications.

The VIM metallo-beta-lactamases are emerging resistance determinants, encoded by mobile genetic elements, that have recently been detected in multidrug-resistant nosocomial isolates of Pseudomonas aeruginosa and other Gram-negative pathogens. In this work a T7-based expression system for overproduction of the VIM-2 enzyme by Escherichia coli was developed, which yielded approximately 80 mg of protein per litre of culture. The enzyme was mostly released into the medium, from which it was recovered at >99% purity by an initial ammonium sulphate precipitation followed by two chromatography steps, with almost 80% efficiency. Determination of kinetic parameters of VIM-2 under the same experimental conditions previously used for VIM-1 (the first VIM-type enzyme detected in clinical isolates, which is 93% identical to VIM-2) revealed significant differences in K(m) values and/or turnover rates with several substrates, including penicillins, cephalosporins and carbapenems. Compared with VIM-1, VIM-2 is more susceptible to inactivation by chelators, indicating that the zinc ions of the latter are probably more loosely bound. These data indicated that at least some of the amino acid differences between the two proteins have functional significance. Molecular modelling of the two enzymes identified some amino acid substitutions, including those at positions 223, 224 and 228 (in the BBL numbering), that could be relevant to the changes in catalytic behaviour.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases whose production by pathogenic bacteria makes their broad activity spectrum especially frightening. X-ray structures of several zinc beta-lactamases have revealed the coordination of the two metal ions, but their mode of action remains unclear. Geometry optimisation of stable complexes along the reaction pathway of benzylpenicillin hydrolysis highlighted a proton shuttle occurring from D120 of the Bacillus cereus beta-lactamase to the beta-lactam nitrogen via Zn2 which is central to the network. First, the Zn1 ion has a structural role maintaining Zn-bound waters, WAT1 and WAT2, either directly or through the Zn1 tetrahedrally coordinated histidine ligands. The Zn2 ion has a more catalytic role, stabilising the tetrahedral intermediate, accepting the beta-lactam nitrogen atom as a ligand. The role of Zn2 and the flexibility in the coordination geometry of both Zn ions is of crucial importance for catalysis.

Background: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme., Results: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme., Conclusions: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Background: Concern about the safety of laser pointers has increased as these devices have become readily available to the public, which includes children who may use them improperly. According to federal (Food and Drug Administration) standards, laser pointers are class 3a devices, and power output cannot exceed 5.0 mW., Methods: We tested a sample consisting of 29 red diode laser pointers obtained from various sources that were equipped with fresh batteries. Power output was measured with an optical power meter with the sensor 5.0 cm from the laser. The laser was turned on for 60 s and a reading was taken every 10 s. This procedure was repeated five times for every laser., Results: The power output of unaltered lasers ranged from 0.7 mW to a high of 3.9 mW. Alterations to some of the lasers increased the output slightly above 5.0 mW., Conclusions: When the laser pointers in our sample were used as packaged, they did not exceed the federal standards of 5.0 mW.

The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-beta-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The beta-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed., (Copyright 2000 Wiley-Liss, Inc.)

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 +/- 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0 degrees-20 degrees C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

This study was conducted to determine the level of automobile window tint that causes a significant reduction of vision for automobile drivers. Contrast sensitivity was measured on 20 participants, of whom 10 were age 20 to 29 years and 10 were age 60 to 69 years, through a stock automobile window (control) and two windows darkened with plastic film. For the younger drivers, a car window with 37% transmittance did not significantly reduce contrast sensitivity, but a darker tint of 18% transmittance reduced contrast sensitivity at higher spatial frequencies. For the older drivers, a tint of 37% transmittance significantly reduced mid-to- high spatial frequency contrast sensitivity. The typical state standard (no tint with less than 35% transmittance) would thus seem to be appropriate for younger drivers; however, further examination of the standard may be necessary in regard to older drivers. Actual or potential applications of this research include guidelines and regulations regarding tinting of automobile windows.

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.

Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.

The study of the interactions between the Tyr280Phe mutant of the Streptomyces R61 DD-peptidase, various substrates and beta-lactam antibiotics shows that Tyr280 is involved not only in the formation of the acylenzyme with the peptide substrate and beta-lactam antibiotics, but also and specifically in the catalysis of the transpeptidation reaction. Surprisingly, this residue does not belong to the conserved structural and functional elements which characterise the penicillin-recognising enzymes.

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed.

The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the 'positive inside rule' approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane alpha-helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.

The primary structure of an elastase from the Antarctic fish Notothenia neglecta (NE) was elucidated by molecular cloning and cDNA sequence analysis. The cDNA of interest was isolated from a cDNA library obtained from Notothenia's pyloric caeca. The amino acid sequence identity with mammalian elastases ranges between 53 and 64%, but interestingly reaches 79% with one isoform (CEB) of two recently isolated cod elastases. The most interesting changes distinguishing the model of NE, predicted from the three dimentional structure of the native porcine elastase (PE), concern the catalytic crevice located in the inter-domains region. These features might be involved in the adaptation to cold of the Antarctic elastase.

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.

A heat-labile beta-lactamase has been purified from culture supernatants of Psychrobacter immobilis A5 grown at 4 degrees C and the corresponding chromosomal ampC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C beta-lactamases. The kinetic parameters of P. immobilis beta-lactamase for the hydrolysis of some beta-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.

Bacterial resistance to beta-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of beta-lactamases, enzymes that efficiently hydrolyze the amide bond of the beta-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine beta-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new beta-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of beta-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the "classical" TEM-1 beta-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179. The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.

Few studies have been done on the absorption and release of chemical vapors by high water content hydrogel lenses. In this study we investigated the absorption of ammonia vapors by this type of contact lens (CL). Ten high water content hydrogel lenses were exposed to vapor generated in a cuvette by 1 drop of aqueous ammonia. The contact lens was transferred to a 0.9% saline solution containing a colorometric indicator for ammonia and the amount of ammonia released into the saline was measured. We found that significant quantities of ammonia were absorbed into the lenses and the relation between average concentration of ammonia vapor and amount absorbed per contact lens was nearly linear from 50 parts per million (ppm) to 250 ppm ammonia vapor. For concentrations greater than 250 ppm there appears to be a saturation effect. Our findings indicate that high water contact lenses will absorb ammonia and release it into a solution similar to tears.

DD-peptidases and beta-lactamases share several common properties, including the formation of an acylenzyme intermediate in their catalytic pathways. In their interactions with beta-lactam antibiotics, the stability of this intermediate is much higher with the peptidases than with the beta-lactamases. The structural factors responsible for this difference have not been identified. The evolution of beta-lactamases is taking place before our eyes, since mutants are constantly selected which can hydrolyze the molecules newly introduced as "beta-lactamase resistant" in the chemotherapeutic arsenal.

The non-catalytic C-terminal regions of the N-acetylmuramidase (lysozyme) of Clostridium acetobutylicum and N-acetylmuramoyl(D-lactyl)-L-alanine amidases CwlA of Bacillus subtilis, ORFL3 and CwlL of Bacillus licheniformis were previously reported to have similarities with the amino acid sequence of the non-catalytic N-terminal module of the Streptomyces albus G Zn DD-peptidase. This peptidase is a bipartite protein of known three-dimensional structure. Its non-catalytic N-terminal module possesses, exposed at the surface, an elongated crevice which is defined by a loop-helix-loop-helix motif that consists of two repeats, each 16 amino acid residues long, connected by a heptapeptide and whose design is compatible with its possible functioning as a substrate recognition and binding site. Amino acid alignments suggest that cavities nearly identical in shape to that present in the non-catalytic module of the S. albus peptidase, are borne by the C-terminal regions of the CwlA amidase (in one copy), the lysozyme and the ORFL3 and CwlL amidases (in two copies). Since a common feature of the five enzymes is their substrate, the bacterial cell wall peptidoglycan, we interpret the striking similarity of their non-catalytic N- or C-terminal modules to suggest that these modules are involved in the binding of these exocellular enzymes to their insoluble wall substrate.

Several bicyclic imidazolidinones structurally-related to penicillins have been prepared from penam precursors. The compounds were compared to benzylpenicillin as active reference, using the methods of theoretical chemistry. Structures 7 and 8, in which the acyl group of the side-chain was anchored, via a "one-atom spacer", at position N-7 of the imidazolidinone ring, appeared as good penicillin mimics. However, they were found devoid of significant antibacterial activity.

Trauma is the cause of 5 to 10% of all cases of osteoarthritis of the hip. One third of patients with a dislocation or fracture-dislocation of the hip or an acetabular fracture develop osteoarthritis of the hip within one to 20 years. Symptoms may be minor or absent during the interval. In addition to severe injuries, simple contusions resulting for instance from a blow to the greater trochanter or knee or from forced abduction of the hip (e.g., splits) can also result in osteoarthritis of the hip. We developed a set of five imputability criteria, of which three are essential: 1) documentation of the exact nature of the trauma; 2) absence of osteoarthritis in the uninjured hip; 3) consistency between the estimated duration of osteoarthritis and the date of the injury. Causality assessment problems which arise when a trauma worsens or accelerates preexisting osteoarthritis are discussed on the basis of clinical and roentgenographic findings. Mechanical and biochemical factors involved in the pathophysiology of contusion-related osteoarthritis of the hip are briefly reviewed.

The interactions between imipenem and four monobactams and three class A beta-lactamases have been studied in detail. Despite their reputation as being beta-lactamase-stable, some of these compounds were significantly hydrolysed by the enzymes. The results obtained with the Streptomyces albus G beta-lactamase have been analysed in the light of molecular modelling studies. The discussion is extended to include other so-called beta-lactamase-stable antibiotics to demonstrate that this appellation can often be misleading.

The interactions between three class A beta-lactamases and compounds bearing a methoxy side chain on the alpha-face of the beta-lactam ring (cefoxitin, moxalactam and temocillin) have been studied. When compared with the situation prevailing with good substrates, both acylation and deacylation steps appeared to be severely impaired. Molecular modelling studies of the structures of the Henri-Michaelis complexes and of the acyl-enzymes indicate a major displacement of the crystallographically observed water molecule which connects the glutamate-166 and serine-70 side chains and underline the role of this water molecule in both reaction steps.

The main Indices of Quality of Life used in rheumatology and their aims and applications are surveyed. Their use in osteoarthritis implies a few issues that are addressed in a critical manner. Indices of Quality of Life are useful to measure deficiency resulting from osteoarthritis by comparison with other rheumatic diseases, as rheumatoid arthritis, connective tissue diseases, fibromyalgia... They are sensitive enough for apraising the domains improved by a radical treatment such as joint replacement. However, their scores are only slightly changing in most of the drug trials, in which the more simple functional indices seem demonstrative enough. Last but no least, they are notably time consuming for both the patient and the physician.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.

In previous studies, several amino acids of the active site of class A beta-lactamases have been modified by site-directed mutagenesis. On the basis of the catalytic mechanism proposed for the Streptomyces albus G beta-lactamase [Lamotte-Brasseur, Dive, Dideberg, Charlier, Frère & Ghuysen (1991) Biochem. J. 279, 213-221], the influence that these mutations exert on the hydrogen-bonding network of the active site has been analysed by molecular mechanics. The results satisfactorily explain the effects of the mutations on the kinetic parameters of the enzyme's activity towards a set of substrates. The present study also shows that, upon binding a properly structured beta-lactam compound, the impaired cavity of a mutant enzyme can readopt a functional hydrogen-bonding-network configuration.