Functional motions of Candida antarctica lipase B: a survey through open-close conformations

Candida antarctica lipase B (CALB) belongs to psychrophilic lipases which hydrolyze carboxyl ester bonds at low temperatures. There have been some features reported about cold-activity of the enzyme through experimental methods, whereas there is no detailed information on its mechanism of action at molecular level. Herein, a comparative molecular dynamics simulation and essential dynamics analysis have been carried out at three temperatures (5, 35 and 50 °C) to trace the dominant factors in the psychrophilic properties of CALB under cold condition. The results clearly describe the effect of temperature on CALB with meaningful differences in the flexibility of the lid region (α5 helix), covering residues 141-147. Open- closed conformations have been obtained from different sets of long-term simulations (60 ns) at 5 °C gave two reproducible distinct forms of CALB. The starting open conformation became closed immediately at 35 and 50 °C during 60 ns of simulation, while a sequential open-closed form was observed at 5 °C. These structural alterations were resulted from α5 helical movements, where the closed conformation of active site cleft was formed by displacement of both helix and its side chains. Analysis of normal mode showed concerted motions that are involved in the movement of both α5 and α10 helices. It is suggested that the functional motions needed for lypolytic activity of CALB is constructed from short-range movement of α5, accompanied by long-range movement of the domains connected to the lid region.     

Conformation and activity of lysozyme on binding to two types of gold nanorods: a comparative study

The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration.     

Synthesis and anti-Helicobacter pylori activity of 5-(nitroaryl)-1,3,4-thiadiazoles with certain sulfur containing alkyl side chain

A series of 5-(nitroaryl)-1,3,4-thiadiazoles bearing certain sulfur containing alkyl side chain similar to pendent residue in tinidazole molecule were synthesized and evaluated against Helicobacter pylori using disk diffusion method. The synthesized compounds were also evaluated for their antibacterial, antifungal and cytotoxic effects. Study of the structure-activity relationships of this series of compounds indicated that both the structure of the nitroaryl unit and the pendent group on 2-position of 1,3,4-thiadiazole ring dramatically impact the anti-H. pylori activity. While compound 7a containing 2-[2-(ethylsulfonyl)ethylthio]-side chain from nitrothiophene series was the most potent compound tested against clinical isolates of H. pylori, however, nitroimidazoles 6c and 7c were found to be more promising compounds because of their respectable anti-H. pylori activity besides less cytotoxic effects.     

Interaction of lysozyme with gold nanorods: conformation and activity investigations

Gold nanorods, with their unique morphology and optical properties have offered new prospects for biomedical and biosensing applications. Herein, the interaction between gold nanorods and a model protein has been monitored using spectroscopic techniques. The enzyme retains high fraction of its native structure with a slight increase in the helical content at the expense of β-turns. Kinetic investigations revealed notable increase of enzyme affinity for substrate without significant decrease in the V_max. Emission spectra of tryptophan residues in the presence of chaotropic agent highlighted the maintenance of internal quenching due to the induced compactness. Comparison of the gold nanorod treated lysozyme with free enzyme revealed higher thermodynamic stability under denaturing condition. Results from this study encourage the possibility of utilizing gold nanorods as promising nanocarrier candidates for a new generation of drug delivery applications.     

Keratins and epidermolysis bullosa simplex

Keratin intermediate filaments play an important role in maintaining the integrity of the skin structure. Understanding the importance of this subject is possible with the investigation of keratin defects in epidermolysis bullosa simplex (EBS). Nowadays, in addition to clinical criteria, new molecular diagnostic methods, such as next generation sequencing, can help to distinguish the subgroups of EBS more precisely. Because the most important and most commonly occurring molecular defects in these patients are the defects of keratins 5 and14 (KRT5 and KRT14), comprehending the nature structure of these proteins and their involved processes can be very effective in understanding the pathophysiology of this disease and providing new and effective therapeutic platforms to treat it. Here, we summarized the various aspects of the presence of KRT5 and KRT14 in the epidermis, their relation to the incidence and severity of EBS phenotypes, and the processes with which these proteins can affect them.     

Diversity and Probiotic Potential of Lactic Acid Bacteria Isolated from Horreh,a Traditional Iranian Fermented Food

The aim of this study was to evaluate the probiotic potential of lactic acid bacteria (LAB) strains isolated from Horreh. Some probiotic properties, e.g., resistance to acid, bile tolerance, antibacterial activity, and antibiotic susceptibility, were investigated. A total of 140 Gram-positive and catalase-negative isolates from Horreh were subjected to identification and grouping by cultural methods and the 16S rRNA sequencing. The new isolates were identified to be Lactobacillus (fermentum, plantarum, and brevis) Weissella cibaria, Enterococcus (faecium and faecalis), Leuconostoc (citreum and mesenteroides subsp. mesenteroides) and Pediococcus pentosaceus. Probiotic potential study of LAB isolates showed that Lb. plantarum and Leu. mesenteroides subsp. mesenteroides isolates were able to grow at pH 2.5 and 3.5. Lactobacillus plantarum (isolate A44) showed the highest cell hydrophobicity (84.5%). According to antibacterial activity tests, Listeria innocua and Staphylococcus aureus were the most sensitive indicators against the selected LAB strains, while Escherichia coli and Bacillus cereus were the most resistant. In addition, all the isolated LAB species were resistant to vancomycin. The results of the present study suggested that the Lactobacillus fermentum and plantarum isolated from Horreh, characterized in this study, have potential use for industrial purposes as probiotics.     

Effect of pH on pulmonary vascular tone, reactivity, and arachidonate metabolism

We studied the effects of perfusate pH on pulmonary vascular tone, reactivity, and thromboxane and prostacyclin synthesis in isolated buffer-perfused rabbit lungs. Extracellular acidosis did not affect base-line vascular tone, but alkalosis had a biphasic effect. Increasing the perfusate pH from 7.40 to 7.65 caused vasodilation, whereas raising pH to 7.70-8.10 caused vasoconstriction. Removing calcium (Ca2+) from the perfusate completely prevented the vasoconstriction caused by alkalosis. Perfusate pH strikingly affected pulmonary vascular reactivity. Acidosis inhibited the vasoconstriction caused by thromboxane and potassium chloride (KCl) but did not affect the response to angiotensin II. Alkalosis, in contrast, augmented the vasoconstriction caused by thromboxane and angiotensin II but reduced the vasoconstriction caused by KCl. Changes in pH also altered thromboxane and prostacyclin synthesis after the infusion of exogenous arachidonic acid (AA) or the endogenous release of AA by the lipid peroxide tert-butyl hydroperoxide.     

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol

BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.     

Construction of a recombinant allergen-producing probiotic bacterial strain: Introduction of a new line for a live oral vaccine against Chenopodium album pollen allergy

Background:

During the last two decades, significant advances have been made in the fields of lactococcal genetics and protein expression. Lactococcus lactis (L. lactis) is an effective vector for protein expression and can be used as an antigen delivery system. Hence, L. lactis is an ideal candidate for mucosal immunotherapy. Profilin (Che a 2), the major allergen in Chenopodium album, is one of the most important causes of allergic diseases in desert and semi-desert areas, especially in Iran, Saudi Arabia, and Kuwait that was cloned and expressed in L. lactis for the first time.

Methods:

To construct L. lactis that expressed Che a 2, a DNA sequence was cloned and used to transform bacteria. Expression of Che a 2 was analyzed via monitoring of related RNA and protein. Hydrophobicity, adherence to HT-29 cells, antibiotic resistance, resistance to gastrointestinal contents, pH, and bile salt in recombinant and native L. lactis were evaluated.

Results:

Immunoblot analyses demonstrated that recombinant Che a 2 is expressed as a 32 kDa dimeric protein immunological studies showed it can bind human IgE. Both native and recombinant bacteria were sensitive to low pH and simulated gastric conditions. Bacterial survival was reduced 80-100% after 2 h of exposure to pH 1.5-2. Both native and recombinant bacteria were able to grow in 0.3 and 2% bile salts. After incubation of recombinant L. lactis in simulated gastric and intestinal juices for one and two hours, respectively, cell survival was reduced by 100%. Adhesion capability in both strains was minimal and there were no significant differences in any of our tests between native and recombinant bacteria.

Conclusion:

Successfully recombinant L. lactis with capability of expression Che a 2 was produced and revealed it is sensitive to gastrointestinal contents. Key Words: Recombinant L. lactis, Probiotic bacteria, Chenopodium pollen allergen, Oral vaccines     

Anti-Helicobacter pylori activity of the methanolic extract of Geum iranicum and its main compounds

Geum iranicum Khatamsaz, belonging to the Rosaceae family, is an endemic plant of Iran. The methanol extract of the roots of this plant showed significant activity against one of the clinical isolates of Helicobacter pylori which was resistant to metronidazole. The aim of this study was the isolation and evaluation of the major compounds of G. iranicum effective against H. pylori. The compounds were isolated using various chromatographic methods and identified by spectroscopic data (1H and 13C NMR, HMQC, HMBC, EI-MS). An antimicrobial susceptibility test was performed employing the disk diffusion method against clinical isolates of H. pylori and a micro dilution method against several Gram-positive and Gram-negative bacteria; additionally the inhibition zone diameters (IZD) and minimum inhibitory concentrations (MIC) values were recorded. Nine compounds were isolated: two triterpenoids, uvaol and niga-ichigoside F1, three sterols, beta-sitosterol, beta-sitosteryl acetate, and beta-sitosteryl linoleate, one phenyl propanoid, eugenol, one phenolic glycoside, gein, one flavanol, (+)-catechin, and sucrose. The aqueous fraction, obtained by partitioning the MeOH extract with water and chloroform, was the most effective fraction of the extract against all clinical isolates of H. pylori. Further investigation of the isolated compounds showed that eugenol was effective against H. pylori but gein, diglycosidic eugenol, did not exhibit any activity against H. pylori. The subfraction D4 was the effective fraction which contained tannins. It appeared that tannins were probably the active compounds responsible for the anti-H. pylori activity of G. iranicum. The aqueous fraction showed a moderate inhibitory activity against both Gram-positive and Gram-negative bacteria. The MIC values indicated that Gram-positive bacteria including Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis are more susceptible than Gram-neagative bacteria including Escherichia coli and Pseudomonas aeruginosa.