Unusual Incorporation of Neutral and Low Water-Soluble Guest Molecules into Layered Double Hydroxides: The Case of Cucurbit [6 and 7]uril Inclusion Hosts

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Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

On the loss of regularity for a class of weakly hyperbolic operators

In this paper we determine bounds for the optimal loss of regularity in the Sobolev scale for a class of weakly hyperbolic operators. (C) 2009 Elsevier Inc. All rights reserved.

The influence of oscillations on global existence for a class of semi-linear wave equations

The goal of this paper is to study the global existence of small data solutions to the Cauchy problem for the nonlinear wave equation u(tt) - a(t)(2) Delta u = u(t)(2) - a(t)(2)vertical bar del u vertical bar(2). In particular we are interested in statements for the 1D case. We will explain how the interplay between the increasing and oscillating behavior of the coefficient will influence global existence of small data solutions. Copyright c 2011 John Wiley & Sons, Ltd.

On a class of abstract neutral functional differential equations

By using the theory of semigroups of growth a, we discuss the existence of mild solutions for a class of abstract neutral functional differential equations. A concrete application to partial neutral functional differential equations is considered. (C) 2011 Elsevier Ltd. All rights reserved.

Existence of solutions for a class of abstract differential equations with nonlocal conditions

We discuss the existence of mild, classical and strict solutions for a class of abstract differential equations with nonlocal conditions. Our technical approach allows the study of partial differential equations with nonlocal conditions involving partial derivatives or nonlinear expressions of the solution. Some concrete applications to partial differential equations are considered. (C) 2010 Elsevier Ltd. All rights reserved.

Democracy versus Republic: Inclusion and Desire in Social Struggles

This paper argues that the idea of inclusion is linked to the democratic tradition rather than to the republican one. By analyzing the origins and meaning of these two concepts, the author holds that democracy is rather linked to desire and republic to will (and to the expression of desire), and concludes that, since North Atlantic political tradition has not given a key role to desire, democracy, in order to overcome the difficulties it has been encountering in all parts of the world, should take more account of desire and of the social struggles it brings to the fore.

A comparison of observed versus estimated baseline creatinine for determination of RIFLE class in patients with acute kidney injury

Methods. Data from the Beginning and Ending Supportive Therapy for the Kidney (BEST Kidney) study, a prospective observational study from 54 ICUs in 23 countries of critically ill patients with severe AKI, were analysed. The RIFLE class was determined by using observed (o) pre-morbid and estimated (e) baseline SCr values. Agreement was evaluated by correlation coefficients and Bland-Altman plots. Sensitivity analysis by chronic kidney disease (CKD) status was performed. Results. Seventy-six percent of patients (n = 1327) had a pre-morbid baseline SCr, and 1314 had complete data for evaluation. Forty-six percent had CKD. The median (IQR) values were 97 mu mol/L (79-150) for oSCr and 88 mu mol/L (71-97) for eSCr. The oSCr and eSCr determined at ICU admission and at study enrolment showed only a modest correlation (r = 0.49, r = 0.39). At ICU admission and study enrolment, eSCr misclassified 18.8% and 11.7% of patients as having AKI compared with oSCr. Exclusion of CKD patients improved the correlation between oSCr and eSCr at ICU admission and study enrolment (r = 0.90, r = 0.84) resulting in 6.6% and 4.0% being misclassified, respectively. Conclusions. While limited, estimating baseline SCr by the MDRD equation when pre-morbid SCr is unavailable would appear to perform reasonably well for determining the RIFLE categories only if and when pre-morbid GFR was near normal. However, in patients with suspected CKD, the use of MDRD to estimate baseline SCr overestimates the incidence of AKI and should not likely be used. Improved methods to estimate baseline SCr are needed.

Centrifugal processing of cell debris and inclusion bodies from recombinant Escherichia coli

The settling characteristics of cell debris and inclusion bodies prior to, and following, fractionation in a disc-stack centrifuge were measured using Cumulative Sedimentation Analysis (CSA) and Centrifugal Disc photosedimentation (CDS). The impact of centrifuge feedrate and repeated homogenisation on both cell debris and inclusion body collection efficiency was investigated. Increasing the normalised centrifuge feedrate (Q/Sigma) from 1.32 x 10(-9) m s(-1) to 3.97 x 10(-9) m s(-1) leads to a 36% increase in inclusion body paste purity. Purity may also be improved by repeated homogenisation. Increasing the number of homogeniser passes results in smaller cell debris size whilst leaves inclusion body size unaltered. At a normalised centrifuge feedrate of 2.65 x 10(-9) m s(-1), increasing the number of homogeniser passes from two (2) to ten (10) improved overall inclusion body paste purity by 58%. Grade-efficiency curves for both the cell debris and inclusion bodies have also been generated in this study. The data are described using an equation developed by Mannweiler (1989) with parameters of k = 0.15-0.26 and n = 2.5-2.6 for inclusion bodies, and k = 0.12-0.14 and n = 2.0-2.2 for cell debris. This is the first accurate experimentally-determined grade efficiency curve for cell debris. Previous studies have simply estimated debris grade efficiency curves using an approximate debris size distribution and grade efficiency curves determined with 'ideal particles' (e.g. spherical PVA particles). The findings of this study may be used to simulate and optimise the centrifugal fractionation of inclusion bodies from cell debris.

A computerized tomography scan method for calculating the hernia sac and abdominal cavity volume in complex large incisional hernia with loss of domain

Preoperative progressive pneumoperitoneum (PPP) is a safe and effective procedure in the treatment of large incisional hernia (size > 10 cm in width or length) with loss of domain (LIHLD). There is no consensus in the literature on the amount of gas that must be insufflated in a PPP program or even how long it should be maintained. We describe a technique for calculating the hernia sac volume (HSV) and abdominal cavity volume (ACV) based on abdominal computerized tomography (ACT) scanning that eliminates the need for subjective criteria for inclusion in a PPP program and shows the amount of gas that must be insufflated into the abdominal cavity in the PPP program. Our technique is indicated for all patients with large or recurrent incisional hernias evaluated by a senior surgeon with suspected LIHLD. We reviewed our experience from 2001 to 2008 of 23 consecutive hernia surgical procedures of LIHLD undergoing preoperative evaluation with CT scanning and PPP. An ACT was required in all patients with suspected LIHLD in order to determine HSV and ACV. The PPP was performed only if the volume ratio HSV/ACV (VR = HSV/ACV) was a parts per thousand yen25% (VR a parts per thousand yen 25%). We have performed this procedure on 23 patients, with a mean age of 55.6 years (range 31-83). There were 16 women and 7 men with an average age of 55.6 years (range 31-83), and a mean BMI of 38.5 kg/m(2) (range 23-55.2). Almost all patients (21 of 23 patients-91.30%) were overweight; 43.5% (10 patients) were severely obese (obese class III). The mean calculated volumes for ACV and HSV were 9,410 ml (range 6,060-19,230 ml) and 4,500 ml (range 1,850-6,600 ml), respectively. The PPP is performed by permanent catheter placed in a minor surgical procedure. The total amount of CO(2) insufflated ranged from 2,000 to 7,000 ml (mean 4,000 ml). Patients required a mean of 10 PPP sessions (range 4-18) to achieve the desired volume of gas (that is the same volume that was calculated for the hernia sac). Since PPP sessions were performed once a day, 4-18 days were needed for preoperative preparation with PPP. The mean VR was 36% (ranged from 26 to 73%). We conclude that ACT provides objective data for volume calculation of both hernia sac and abdominal cavity and also for estimation of the volume of gas that should be insufflated into the abdominal cavity in PPP.

A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences'. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response(2,3). The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV(4), led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells(5). However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue(6). Here we show that a recombinant MCMV,in which. the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV, We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC dass I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

High-titer antichromatin antibody is associated with proliferative class IV of lupus nephritis

The antichromatin antibody (aCT) has been described as a useful marker for lupus nephropathy. The relevance of its nephritogenic potential may be appropriately evaluated in the context of renal histopathology. Therefore, the present study investigated the relationship of aCT with a particular histopathologic class of lupus nephritis (LN). Seventy-eight consecutive patients with systemic lupus erythematosus (ACR criteria) and active nephritis who underwent renal biopsy from 1999 to 2004 and with available frozen serum sample obtained at the time of biopsy were selected. aCT was measured by ELISA, and anti-dsDNA was measured by indirect immunofluorescence (IIF) and by ELISA. All renal biopsies were revised in a blinded manner by the same expert renal pathologist. Charts were extensively reviewed for demographic and renal features obtained at the time of biopsy. The prevalence of aCT (>= 20 U) was 59% with a mean titer of 74.3 +/- 38.7U. Both aCT-positive and aCT-negative groups of patients had similar age, gender distribution, duration of lupus, and duration of renal disease. Anti-dsDNA was detected by IIF in 29.5% and by ELISA in 42.3% of the patients. Concomitant presence of both antibodies was observed in 63% (29/46) [anti-dsDNA by ELISA] and 45.6% (21/46) [anti-dsDNA by IIF] of the patients. Lower serum levels of C3 (73% vs. 40%, P=0.0058) and C4 (82% vs. 46.7%, P=0.0021) were more commonly observed in aCT >= 20 U patients compared to the aCT-negative group. It is important to note that the use of a higher cut-off value (>= 40 U) for aCT test revealed a predominance of class IV LN (58% vs. 33%, P=0.039) in aCT >= 40 U compared to aCT<40 U group. The mean levels of proteinuria, serum albumin, and creatinine were markedly altered but were comparable in both groups (P >= 0.05). One fourth (26.3%) of the 19 patients with class IV LN and aCT >= 40 U had no detectable anti-dsDNA (ELISA). These data suggest that high-titer aCT seems to be a valuable biomarker for proliferative class IV of LN.

Anti-Group A Streptococcal Vaccine Epitope STRUCTURE, STABILITY, AND ITS ABILITY TO INTERACT WITH HLA CLASS II MOLECULES

Streptococcus pyogenes infections remain a health problem in several countries due to poststreptococcal sequelae. We developed a vaccine epitope (StreptInCor) composed of 55 amino acids residues of the C-terminal portion of the M protein that encompasses both T and B cell protective epitopes. The nuclear magnetic resonance (NMR) structure of the StreptInCor peptide showed that the structure was composed of two microdomains linked by an 18-residue alpha-helix. A chemical stability study of the StreptInCor folding/unfolding process using far-UV circular dichroism showed that the structure was chemically stable with respect to pH and the concentration of urea. The T cell epitope is located in the first microdomain and encompasses 11 out of the 18 alpha-helix residues, whereas the B cell epitope is in the second microdomain and showed no alpha-helical structure. The prediction of StreptInCor epitope binding to different HLA class II molecules was evaluated based on an analysis of the 55 residues and the theoretical possibilities for the processed peptides to fit into the P1, P4, P6, and P9 pockets in the groove of several HLA class II molecules. We observed 7 potential sites along the amino acid sequence of StreptInCor that were capable of recognizing HLA class II molecules (DRB1*, DRB3*, DRB4*, and DRB5*). StreptInCoroverlapping peptides induced cellular and humoral immune responses of individuals bearing different HLA class II molecules and could be considered as a universal vaccine epitope.

Correlation between CD34(+) and exercise capacity, functional class, quality of life and norepinephrine in heart failure patients

Background: A previous study associated CD34(+) levels with NYHA functional class in heart failure patients. The aim of this study was to correlate CD34(+) levels to exercise capacity, functional class, quality of life and norepinephrine in heart failure patients. Methods: Twenty three sedentary patients (52 +/- 7 years, 78% male) answered the Minnesota Living with Heart Failure Questionnaire and rested for 20 minutes before an investigator collect a blood sample. After this, patients performed a cardiopulmonary exercise test to determine the heart rate at anaerobic and ventilatory threshold and oxygen consumption at peak effort, at anaerobic and ventilatory threshold. One other blood sample was collected during the peak effort to investigate the norepinephrine and CD34(+) levels. Results: Rest percentage of CD34(+) did not show correlation with: left ventricle ejection fraction (r = 0.03, p = 0.888), peakVO(2) (r = 0.32, p = 0.13), VO(2) at anaerobic threshold (VO(2)AT) (r = 0.03, p = 0.86), VO(2) at ventilatory threshold (VO(2)VT) (r = 0.36, p = 0.08), NYHA functional class (r = -0.2, p = 0.35), quality of life (Minnesota) (r = -0.17, p = 0.42). CD34(+) did not show correlation, either, with: peak VO(2) (r = 0.38, p = 0.06), VO(2)AT (r = 0.09, p = 0.65), VO(2)VT (r = 0.43, p = 0.4), NYHA functional class (r = -0.13, p = 0.54), quality of life (r = 0.00, p = 0.99). Conclusions: CD34(+) levels did not correlate with exercise capacity, functional class, quality of life and norepinephrine. Percentage of CD34(+) levels did not increase during the cardiopulmonary exercise test in heart failure patients. (Cardiol J 2009; 16, 5: 426-431)