Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking

Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1-3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an ∼50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.     

Changes in midgut and brain proteins in Morimus funereus larvae depending on nutritive substrate

The response of Morimus funereus larvae to total starvation and refeeding with qualitatively different nutritive substrates (artificial diets supplemented with yeast as a source of B complex vitamins or with a digestibility reducer-tannic acid) was examined in this paper. Refeeding resulted in a compensatory increase of larval growth. Feeding and refeeding with qualitatively different nutritive substrates affected both quality and quantity of midgut and brain proteins. The observed differences suggest the possible switching of enzyme isoforms in M. funereus midgut and changes in synthesis/secretion of neurohormones, depending on food presence and its nutritional value.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Evaluation of genotoxic effects of Apitol (cymiazole hydrochloride) in vitro by measurement of sister chromatid exchange

Apitol^®, with cymiazole hydrochloride as the active ingredient, is used in bee-keeping against the ectoparasitic mite Varroa destructor. The preparation was evaluated for genotoxicity in cultured human peripheral blood lymphocytes. Sister chromatid exchange, the mitotic index and the cell proliferation index were determined for three experimental concentrations of Apitol^® (0.001, 0.01 and 0.1 mg/ml). All concentrations significantly (p < 0.001) increased the mitotic index (MI = 7.35 ± 0.18%, 8.31 ± 0.20% and 12.33 ± 0.25%, respectively), the proliferative index (PI = 1.83 ± 0.01, 1.84 ± 0.01 and 1.88 ± 0.02, respectively) and the frequency of sister chromatid exchange (SCE = 8.19 ± 1.81, 8.78 ± 1.80 and 13.46 ± 1.88, respectively), suggesting that cymiazole hydrochloride has genotoxic potential.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Functional tyrosine residue in the active center of human dipeptidyl peptidase III

Abstract Human dipeptidyl peptidase III (DPP III) is a member of the metallopeptidase family M49 with an implied role in the pain-modulatory system and endogenous defense against oxidative stress. Here, we report the heterologous expression of human DPP III and the site-directed mutagenesis results which demonstrate a functional role for Tyr318 at the active site of this enzyme. The substitution of Tyr318 to Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate, or of a competitive hydroxamate inhibitor designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases.     

Risk factor analysis and diagnoses of coronary heart disease in patients with hypercholesterolemia from Croatian Zagorje County

Our aim is to determine if there exists a difference in risk factors and diagnosis between patients being treated on internal medicine ward for coronary heart disease who have higher levels of cholesterol in their blood and other patients, without proved higher levels of cholesterol, hospitalized for coronary heart disease. We followed patients hospitalized in General Hospital Zabok for coronary heart disease for the period between 2004-2006y. On admission patients were diagnosed with coronary heart disease based on laboratory markers specific for the disease (CK, troponin, LDH,CRP), ECG and history taking. We analyzed two groups of patients for diagnosis and risk factors on discharge from the hospital: one group with proven hypercholesterolemia, the other with coronary heart disease without hypercholesterolemia. For the duration of the study there were no significant alternations concerning risk factors for coronary heart disease, and hypertension was the most prevalent of these factors in both groups. Values of HDL, as an indirect indicator of coronary heart disease, were lower in both groups for the duration of the study. In group of patients with hypercholesterolemia myocardial infarction with a ST segment elevation, as a discharge diagnosis, was a more prevalent complication of the disease, while for the group of patients without hypercholesterolemia stable angina pectoris was more prevalent and this is explained as atheroma plaque stabilization when there are normal values of blood cholesterol.     

Cloning,Heterologous Expression,Purification and Characterization of M12 Mutant of Aspergillus niger Glucose Oxidase in Yeast Pichia pastoris KM71H

Aspergillus niger glucose oxidase (GOx) genes for wild-type (GenBank accession no. X16061, swiss-Prot; P13006) and M12 mutant (N2Y, K13E, T30 V, I94 V, K152R) were cloned into pPICZαA vector for expression in Pichia pastoris KM71H strain. The highest expression level of 17.5 U/mL of fermentation media was obtained in 0.5 % (v/v) methanol after 9 days of fermentation. The recombinant GOx was purified by cross-flow ultrafiltration using membranes of 30 kDa molecular cutoff and DEAE ion-exchange chromatography at pH 6.0. Purified wt GOx had k _cat of 189.4 s^?1 and K _m of 28.26 mM while M12 GOx had k _cat of 352.0 s^?1 and K _m of 13.33 mM for glucose at pH 5.5. Specificity constants k _cat/K _m of wt (6.70 mM^?1 s^?1) and M12 GOx (26.7 mM^?1 s^?1) expressed in P. pastoris KM71H were around three times higher than for the same enzymes previously expressed in Saccharomyces cerevisiae InvSc1 strain. The pH optimum and sugar specificity of M12 mutant of GOx remained similar to the wild-type form of the enzyme, while thermostability was slightly decreased. M12 GOx expressed in P. pastoris showed three times higher activity compared to the wt GOx toward redox mediators like N,N-dimethyl-nitroso-aniline used for glucose strips manufacturing. M12 mutant of GOx produced in P. pastoris KM71H could be useful for manufacturing of glucose biosensors and biofuel cells.