Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (maltoporin) channel of Escherichia coli. II. Effect on maltose and maltooligosaccharide binding kinetics.

The 3-D structure of the maltooligosaccharide-specific LamB channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by tyrosine 118. Y118 was replaced by site-directed mutagenesis by 10 other amino acids (alanine (A), isoleucine (I), asparagine (N), serine (S), cysteine (C), aspartic acid (D), arginine (R), histidine (H), phenylalanine (F), and tryptophan (W)) including neutral ones, negatively and positively charged amino acids to study the effect of their size, their hydrophobicity index, and their charge on maltose and maltooligosaccharide binding to LamB. The mutants were reconstituted into lipid bilayer membranes and the stability constants for binding of maltose, maltotriose, maltopentaose, and maltoheptaose to the channel were measured using titration experiments. The mutation of Y118 to any other non-aromatic amino acid led to a substantial decrease of the stability constant of binding by factors between about two and six. The highest effect was observed for the mutant Y118A. Replacement of Y118 by the two other aromatic amino acids, phenylalanine (F) and tryptophan (W), resulted in a substantial increase of the stability constant maximally by a factor of almost 400 for the Y118W mutant. The carbohydrate-induced block of the channel function was used for the study of current noise through the different mutant LamB channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of sugar binding. The results suggest that both rate constants were affected by the mutations. For most mutants, with the exception of Y118F and Y118W, k(1) decreased and k(-1) increased, whereas the opposite was found for the aromatic amino acid mutants. The results suggest that tyrosine 118 has a crucial effect on carbohydrate transport through LamB

Site-directed mutagenesis of tyrosine 118 within the central constriction site of the LamB (Maltoporin) channel of Escherichia coli. I. Effect on ion transport.

The three-dimensional structure of the malto-oligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by a tyrosine residue (Y118). Y118 was replaced by site-directed mutagenesis by ten other amino acids (alanine, isoleucine, asparagine, serine, cysteine, aspartic acid, arginine, histidine, phenylalanine, and tryptophane) including neutral ones, negatively and positively charged amino acids to study the effect of their size, hydrophobicity, and charge on ion transport through LamB. The mutant proteins were purified to homogeneity. They were reconstituted into lipid bilayer membranes and single-channel conductance and ion selectivity were measured to get insight into the mechanism of ion transport through LamB. The mutation of Y118 to any other nonaromatic amino acid led to a substantial increase of the single-channel conductance by more than a factor of six at maximum. The highest effect was observed for Y118D. Additionally, a nonlinear relationship between the salt concentration in the aqueous phase and the channel conductance was observed for this mutant, indicating strong discrete charge effects on ion conductance. For all other mutants, with the exception of Y118R, linear relationships were found between single-channel conductance and bulk aqueous concentration. The individual hydrophobicity indices of the amino acids introduced inside the central constriction of the LamB channel had a somewhat smaller effect on the single-channel conductance as compared with the effect of their size and charge

CymA of Klebsiella oxytoca Outer Membrane: Binding of Cyclodextrins and Study of the Current Noise of the Open Channel

CymA, the outer membrane component of the cyclodextrin (CD) uptake and metabolism system of Klebsiella oxytoca, was reconstituted into lipid bilayer membranes. The channel properties of this unusual porin were studied in detail. The binding of CDs to the channel resulted in its complete block for ion transport. This result allowed the detailed investigation of carbohydrate binding, and the stability constants for the binding of cyclic and linear carbohydrates to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the carbohydrates. Highest stability constant was observed for α-cyclodextrin (α-CD; K = 32,000 1/M) followed by β-cyclodextrin (β-CD; K = 1970 1/M) and γ-cyclodextrin (γ-CD; K = 310 1/M). Linear maltooligosaccharides bound also to CymA but with much smaller stability constants as compared to cyclic ones. The noise of the current through CymA in multi- and single-channel experiments was investigated using fast Fourier transformation. The current through the open channels had a rather high spectral density, which was a Lorentzian function of the frequency up to 2000 Hz. Upon addition of cyclic dextrins to the aqueous phase the spectral density decreased in a dose-dependent manner, which made it impossible to evaluate the binding kinetics. Experiments with single CymA-channels demonstrated the channel is highly asymmetric concerning channel flickers and current noise

Obesity and smoking predict the results of two-stage exchange in septic revision hip arthroplasty: A cohort study.

BACKGROUND Prosthetic joint infection (PJI) is deemed to be the most serious complication following total hip arthroplasty. Obesity and smoking are known risk factors for PJI. However, the influence of these variables on infection free survival, of septic revision hip arthroplasty, is yet to be explored. The aim of this study was to determine the effect of obesity and smoking on the outcome of two-stage prosthetic exchange surgery. PATIENTS AND METHODS A consecutive series of 97 hips in 94 patients (69 male, 25 female, mean age 66±12 years), undergoing two-stage revision surgery for hip PJI, were investigated retrospectively, after a mean follow-up of 60 (24-170) months. Survival was estimated using Kaplan-Meyer curves. A multivariate cox-regression model was applied to test for the influence of smoking or obesity (BMI≥30) after adjusting 16 potential patient-dependant variables. HYPOTHESIS The study hypothesis was that smoking and high BMI are predictors for the failure of septic revision hip arthroplasty. Failure of septic revision hip arthroplasty was defined as failure to eradicate the infection or eradication of the infection but failure to preserve the arthroplasty. RESULTS Kaplan-Meier showed a cumulative survival proportion of 80.4%(standard error S.E 4%), of the definitive implant, at 5 years. Obese patients (BMI≥30) and smokers had a significantly lower 5-year survival of 60.9% (S.E 1%) and 50.6% (S.E 1.4%), respectively (p=0.001). DISCUSSION Obesity and smoking are both factors determining infection free survival in two-stage revision hip arthroplasty. Clinicians should be aware of potential complications and anticipate a higher likelihood of conversion to a Girdlestone resection or even amputation in this group of patients. LEVEL OF EVIDENCE III, retrospective cohort study

The moduli space of three qutrit states

19 pages, revtex4, three figures; revised versionInternational audienceWe study the invariant theory of trilinear forms over a three-dimensional complex vector space, and apply it to investigate the behaviour of pure entangled three-partite qutrit states and their normal forms under local filtering operations (SLOCC). We describe the orbit space of the SLOCC group SL(3,\C)^{\times 3} both in its affine and projective versions in terms of a very symmetric normal form parameterized by three complex numbers. The parameters of the possible normal forms of a given state are roots of an algebraic equation, which is proved to be solvable by radicals. The structure of the sets of equivalent normal forms is related to the geometry of certain regular complex polytopes