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.     

Rate of recalls in congenital hypothyroidism based upon a regional survey in Isfahan, Iran, using serum T4 and TSH analyses: comparison of two different recall methods

AIMS: To evaluate and compare the recall rate in congenital hypothyroidism screening project in Isfahan, first using an approach involving measures of both TSH and T4 and then using TSH alone. METHODS: From June 2002 to January 2005, serum TSH and T4 level of referred neonates were measured at 3rd to 7th day of birth through venous sampling. If neonates' serum TSH was >20 mIU/l or T4 was <6.5 microg/dl by the first protocol, or TSH was >20 mIU/l by the second protocol, they were recalled. TSH and T4 were measured using an immunoradiometric assay and radioimmunoassay, respectively. Neonates with TSH > 10 and T4 < 6.5 on their second measurement were considered as congenitally hypothyroid. RESULTS: Serum T4 and TSH of 29,425 neonates by first and 57,235 neonates by second recall approach were measured. Recall rate was higher in the first protocol (2.2% vs. 0.6%, p < 0.05). Most of the recalled neonates in the first protocol were recalled for low T4 level (p < 0.05). The prevalence of CH was 1 in 350 livebirths. CONCLUSION: Although the recall rate was in the acceptable range by either approach, the TSH alone protocol seems to be a more sensitive and practical approach with the least recall burden and considering the high prevalence of CH in our region merit adaptation of widespread screening for CH using TSH measurements from heel stab blood spotted on filter paper.     

Changes in extra cellular matrix remodelling and re-expression of fibronectin and tenascin-C splicing variants in human myocardial tissue of the right atrial auricle: implications for a targeted therapy of cardiovascular diseases using human SIP format antibodies

Cardiovascular diseases are accompanied by changes in the extracellular matrix (ECM) including the re-expression of fibronectin and tenascin-C splicing variants. Using human recombinant small immunoprotein (SIP) format antibodies, a molecular targeting of these proteins is of therapeutic interest. Tissue samples of the right atrial auricle from patients with coronary artery disease and valvular heart disease were analysed by PCR based ECM gene expression profiling. Moreover, the re-expression of fibronectin and tenascin-C splicing variants was investigated by immunofluoerescence labelling. We demonstrated changes in ECM gene expression depending on histological damage or underlying cardiac disease. An increased expression of fibronectin and tenascin-C mRNA in association to histological damage and in valvular heart disease compared to coronary artery disease could be shown. There was a distinct re-expression of ED-A containing fibronectin and A1 domain containing tenascin-C detectable with human recombinant SIP format antibodies in diseased myocardium. ED-A containing fibronectin showed a clear vessel positivity. For A1 domain containing tenascin-C, there was a particular positivity in areas of interstitial and perivascular fibrosis. Right atrial myocardial tissue is a valuable model to investigate cardiac ECM remodelling. Human recombinant SIP format antibodies usable for an antibody-mediated targeted delivery of drugs might offer completely new therapeutic options in cardiac diseases.     

Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase

A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit, consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS⁺). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS⁺, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway. [BMB Reports 2012; 45(12): 748-753]     

Stabilization of Bacillus licheniformis alpha-amylase by specific antibody which recognizes the N-terminal fragment of the enzyme

Bacillus licheniformis alpha-amylase (BLA) is an industrially important extracellular enzyme with a number of applications. In the present work, an investigation was carried out on the tryptolytic digestion of BLA which produced two fragments, TF18K and TF38K, and no further fragments could be seen after 6h incubation of BLA with trypsin. The fragments were isolated by preparative gel electrophoresis and reverse phase HPLC. The N-terminal sequencing of fragments showed that trypsin attacks on Arg(127)-Val(128) peptide bond in BLA. Intrinsic and acrylamide quenching fluorescence experiments and Far-UV circular dichroism studies showed that substantial changes in the secondary and tertiary structures of the TF18K and TF38K have occurred. Subsequently, polyclonal antibody was raised against TF18K. After purification of the antibody by protein A Sepharose, thermal stability of BLA in the presence of this antibody was determined. Results showed that the presence of antiTF18K leads to significant stabilization of BLA. For example, after 30 min incubation at 90 degrees C, residual activity of the enzyme in the presence of antibody (40 microg/ml) was determined as 40% while the enzyme showed no activity in the absence of antibody after incubating in the same condition. In addition, it has been proved that calcium enhances the thermal stability of BLA and a synergistic stabilization of BLA has been seen with antiTF18K and calcium, simultaneously.     

Enhancement of thermostability and resistance against autolysis in a zinc metalloprotease

Neutral proteases are inactivated at higher temperatures because of autolysis. It appears that autolysis involves some specific solvent‐exposed regions that become prone to local unfolding as temperature increases. Accordingly, we designed surface‐located mutations at the N‐terminal loops (A56P and T73F) of the neutral protease from Salinivibrio proteolyticus and compared the thermostability and autolysis as well as structural properties of wild‐type (WT) and mutant proteins. Circular dichroism in far‐UV region and intrinsic fluorescence data indicated that compactness of protein increases upon mutation. It was revealed that the catalytic efficiency (k_cat/K_m) of the enzyme is improved in mutants and optimum temperature of mutants increases relative to WT enzyme. It was also shown that the mutant enzymes are more resistant against autolysis and their thermostability and kinetic parameters are also changed compared to WT protein. This study shows that the stability of enzyme against autolysis and temperature may be increased even by changing only a single amino acid, which in turn is important from application point of view.     

Contrasting soil microbial responses to fertilization and tillage systems in canola rhizosphere

Information regarding the simultaneous evaluation of tillage and fertilization on the soil biological traits in canola production is not available. Therefore, field experiments were conducted in 2007–2010 in a split plot based on randomized complete block design with three replications. Main plots consisted of conventional tillage (CT); minimum tillage (MT) and no tillage (NT). Six strategies of fertilization including (N₁): farmyard manure (cattle manure); (N₂): compost; (N₃): chemical fertilizers; (N₄): farmyard manure + compost; (N₅): farmyard manure + compost + chemical fertilizers and (N₆): control, were arranged in sub plots. Results showed that the addition of organic manure increased the soil microbial biomass. No tillage system increased microbial biomass compared to other tillage systems. The activities of all enzymes were generally higher in the N₄ treatment. The activity of phosphatase and urease tended to be higher in the no tillage treatment compared to the CT and MT treatments.     

Chondrotinase ABC I thermal stability is enhanced by site-directed mutagenesis: a molecular dynamic simulations approach

Chondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of growth in the adult central nervous system. Use of the enzyme chondroitinase ABC I (ChABC I) as a strategy to reduce CSPG inhibition in experimental models of spinal cord injury has led to observations of its remarkable capacity for repair. More importantly, ChABC therapy has been demonstrated to promote significant recovery of function to spinal injured animals. Despite this incomparable function of ChABC I, its clinical application has been limited because of its thermal instability as reported in the literature. In a recent study by Nazari-Robati et al., thermal stability of ChABC I was improved by protein engineering using site-directed mutagenesis method. Here, in this study, molecular dynamics simulations were used to take a closer look into the phenomenon leading to the experimentally observed thermal stability improvement followed by the corresponding site-directed mutagenesis. We concluded that the mutations induce local flexibility along with a re-conformation into the native structure which consequently increase the protein thermal stability.