Microwave initiated synthesis of polymethylmethacrylate grafted guar (GG-g-PMMA), characterizations and applications

Guar gum was grafted with polymethylmethacrylate (PMMA) by microwave initiated method. The synthesis was optimized for highest percentage grafting, by varying the microwave irradiation time and monomer (methyl methacrylate) concentration. The various grades of grafted product (GG-g-PMMA) were characterized through standard physicochemical characterization techniques (intrinsic viscosity measurement, elemental analysis, FTIR spectroscopy & SEM morphology study). Further, flocculation efficacy of this grafted polysaccharide was studied by standard 'Jar test' procedure in 0.5% kaolin suspension, to assess its application as flocculating agent for wastewater treatment and mineral ore beneficiation. Correlation between percentage grafting and intrinsic viscosity was also studied for the possibility of using it as a tool to control the intrinsic viscosity in the final product. This leads to the possibility of application of the grafted product as superior viscosifier than guar gum.     

Structural insight into the role of the human melanocortin 3 receptor cysteine residues on receptor function

Melanocortin-3 receptor (MC3R), expressed in the hypothalamus and limbic systems of the brain, as well as by peripheral sites, plays an important role in the regulation of energy homeostasis and other physiological functions. Past work shows that MC3R-deficiency resulted in fat mass increase, feeding efficiency increase, hyperleptinemia and mild hyperinsulinemia in mice and human. MC3R belongs to G-protein coupled receptor (GPCR) family and many studies indicate that some cysteine residues in GPCR play key roles in maintaining receptor tertiary structure and function. In this study, we examined the role of cysteine residues in MC3R on receptor function. Human MC3R (hMC3R) has eighteen cysteine residues where they are located in the extracellular loops (ELs), the transmembrane domains (TMs) and the intracellular loops (ILs). We replaced these cysteines with serine and expressed these receptors in HEK-293 cells which lack endogenous MC3R. Our results indicate that five cysteines in eighteen of the hMC3R are important for hMC3R function. Mutations, C305S, C311S, and C313S in EL3, resulted in significant decrease in receptor expression and receptor function while two other mutations C115S and C162S in TM3 significantly decreased NDP-MSH binding affinity and potency. These results suggest that extracellular cysteine residue 305, 311 and 313 are crucial for receptor expression and the transmembrane cysteine residue, C115 and 162 are important for ligand binding and signaling. These findings provide important insights into the importance of cysteine residues of hMC3R on receptor tertiary structure and function.     

Structural stability and unfolding transition of β-glucosidases: a comparative investigation on isozymes from a thermo-tolerant yeast

The folding of proteins in the milieu of the cellular environment involves various interactions among the residues of the polypeptide chain and the microenvironment where it resides. These interactions are responsible for stabilizing the protein molecule, and disruption of the same provides information about the stability of the molecule. β-Glucosidase isozymes, despite having high homology in their primary and tertiary designs, show deviations in their properties such as unfolding, refolding, and stability. In a comparative study on two large cell-wall-bound isozymes, β-glucosidase I (BGLI) and β-glucosidase II (BGLII) from a thermo-tolerant yeast, Pichia etchellsii, we have investigated guanidine hydrochloride (GdnHCl)-induced, alkali-induced, and thermal-unfolding transitions using CD and fluorescence spectroscopy and high sensitivity differential scanning calorimetry. Using spectral parameters (MRE 222 nm) to monitor the conformational transitions of the GdnHCl-induced unfolding phenomenon, it was observed that the midpoints of unfolding, apparent C _m, occurred at 1.2 M ± 0.05 and 0.8 M ± 0.03 GdnHCl, respectively, for BGLI and BGLII. The alkali-induced unfolding process indicated that BGLI showed a mid-transition point at pH 11 ± 0.17, while for BGLII it was at pH 10 ± 0.40, further indicating BGLI to be more stable to alkali denaturation than BGLII. In the case of thermal unfolding, the midpoint of transition was observed at 63 ± 0.12°C for BGLI and at 58 ± 0.55°C for BGLII. Analysis by high sensitivity differential scanning calorimeter supported the unfolding data in which BGLI showed higher melting temperature, T _m, (56.07°C ± 0.34) than BGLII (54.02°C ± 0.36). Our results clearly indicate that BGLI is structurally more rigid and stable than BGLII.     

Functional and molecular characterization of maxi K+ -channels (BK(Ca)) in buffalo myometrium

Large conductance potassium channels (BK(Ca) channels) play a central role in maintaining myometrial tone, thus activation of these channels proved to have therapeutic potential in preterm labor. Present study aims to unravel the presence of BK(Ca) (maxi-K) channels in buffalo myometrium. Tension experiments, mRNA and protein expression studies were done to characterize BK(Ca) channels in buffalo myometrium. Isolated myometrial preparations exhibited rhythmic spontaneity with regular pattern of amplitude and frequency. Selective blockers of BK(Ca) channels iberiotoxin (IbTx; 100nM) and tetraethylammonium (TEA; 1mM) produced excitatory effects as evidenced by increase in amplitude and frequency of myogenic activity. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimi-dazol-2-one (NS-1619; 10(-7)-10(-4)M), a BK(Ca) channel opener, produced concentration-dependent relaxation of myometrium with pD(2) of 5.02±0.19 and R(max) of 31.35±3.5% (n=5). TEA significantly antagonized NS-1619-induced relaxation (pD(2) of 4.72±0.12 and R(max) of 22.72±1.78%; n=5). IbTx also significantly shifted the dose response curve of NS-1619 towards right (pD(2) of 3.98±0.16; n=4) without significant change in the per cent maximal response. Further, RT-PCR study detected mRNA encoding BK(Ca) α-subunit and Western blot analysis detected its protein expression in myometrium. Based on the results of the present investigation, it is suggested that BK(Ca) channels are present in the buffalo myometrium and are open in the resting state. Thus, their activation by potassium channel opener/β(2)-adrenoceptor agonist (tocolytic drug) may lead to uterine relaxation in preterm labor.     

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.     

Kinetic behavior of liquefaction of Japanese beech in subcritical phenol

Non-catalytic liquefaction of Japanese beech (Fagus crenata) wood in subcritical phenol was investigated using a batch-type reaction vessel. After samples were treated at 160 °C/0.9 MPa–350 °C/4.2 MPa for 3–30 min, they were fractionated into a phenol-soluble portion and phenol-insoluble residues. These residues were then analyzed for their chemical composition. Based on the obtained data, the kinetics for liquefaction was modeled using first-order reaction rate law. Subsequently, the liquefaction rate constants of the major cell wall components including cellulose, hemicellulose, and lignin were determined. The different kinetic mechanisms were found to exist for lignin and cellulose at two different temperature ranges, lower 160–290 °C and higher 310–350 °C, whereas for hemicellulose, it was only liquefied in the lower temperature range. Thus, the liquefaction behaviors of these major cell wall components highlighted hemicellulose to be the most susceptible to liquefaction, followed by lignin and cellulose.