Novel preparation method for surfactant-coated enzymes using W/O emulsion

Summary A novel preparation method for surfactant-coated enzymes has been developed using a W/O emulsion. The enzymatic activity of chymotrypsin in isooctane significantly increased with the coating of surfactants. The surfactant-coated chymotrypsin showed a high enzymatic activity for amidation, although powdered chymotrypsin did not show the activity. Further, the coated enzyme showed a remarkably high storage stability.     

Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47

Abstract Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene. The amount of alpha-toxin produced by the B. brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B. subtilis transformant carrying the toxin gene. Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B. brevis 47 transformant were identical to those of wild-type alpha-toxin.     

Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

The polymerization of proteins can create newly active and large bio‐macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine‐tag (Y‐tag) through a flexible linker at the N‐ and/or C‐termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y‐tagged HRPs with molecular O₂ to form a tyrosyl‐free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP‐catalyzed self‐crosslinking reaction in the presence of H₂O₂. The addition of H₂O₂ in the self‐polymerization of Y‐tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site‐selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y‐tagged HRPs and HRP‐protein G (Y‐HRP‐pG) units catalyzed by TL shows a higher signal in enzyme‐linked immunosorbent assay (ELISA) than the genetically pG‐fused HRP, Y‐HRP‐pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.     

X-ray crystallographic and biochemical characterization of the inhibitory action of an imidazole-dioxolane compound on heme oxygenase

Heme oxygenase (HO) catalyzes the regiospecific cleavage of the porphyrin ring of heme using reducing equivalents and O2 to produce biliverdin, iron, and CO. Because CO has a cytoprotective effect through the p38-MAPK pathway, HO is a potential therapeutic target in cancer. In fact, inhibition of the HO isoform HO-1 reduces Kaposi sarcoma tumor growth. Imidazole-dioxolane compounds have recently attracted attention because they have been reported to specifically inhibit HO-1, but not HO-2, unlike Cr-containing protoporphyrin IX, a classical inhibitor of HO, that inhibits not only both HO isoforms but also other hemoproteins. The inhibitory mechanism of imidazole-dioxolane compounds, however, has not yet been characterized. Here, we determine the crystal structure of the ternary complex of rat HO-1, heme, and an imidazole-dioxolane compound, 2-[2-(4-chlorophenyl)ethyl]-2-[(1H-imidazol-1-yl)methyl]-1,3-dioxolane. This compound bound on the distal side of the heme iron, where the imidazole and 4-chlorophenyl groups were bound to the heme iron and the hydrophobic cavity in HO, respectively. Binding of the bulky inhibitor in the narrow distal pocket shifted the distal helix to open the distal site and moved both the heme and the proximal helix. Furthermore, the biochemical characterization revealed that the catalytic reactions of both HO-1 and HO-2 were completely stopped after the formation of verdoheme in the presence of the imidazole-dioxolane compound. This result should be mainly due to the lower reactivity of the inhibitor-bound verdoheme with O2 compared to the reactivity of the inhibitor-bound heme with O2.     

Stimulation of oncogenic metabotropic glutamate receptor 1 in melanoma cells activates ERK1/2 via PKCepsilon

Metabotropic glutamate receptor 1 (Grm1, formerly mGluR1) is a G protein coupled receptor (GPCR) normally expressed and functional in the central nervous system. Studies of our transgenic mouse melanoma model (TG-3) revealed that ectopic expression of Grm1 in melanocytes is sufficient to induce melanoma development in vivo [P.M. Pollock, K. Cohen-Solal, R. Sood, J. Namkoong, J.J. Martino, A. Koganti, H. Zhu, C. Robbins, I. Makalowska, S.S. Shin, Y. Marin, K.G. Roberts, L.M. Yudt, A. Chen, J. Cheng, A. Incao, H.W. Pinkett, C.L. Graham, K. Dunn, S.M. Crespo-Carbone, K.R. Mackason, K.B. Ryan, D. Sinsimer, J. Goydos, K.R. Reuhl, M. Eckhaus, P.S. Meltzer, W.J. Pavan, J.M. Trent, S. Chen, Nat. Genet. 34 (2003) 108-112.]. We have established and characterized several cell lines in vitro from independent mouse melanoma tumors [Y.E. Marín, J. Namkoong, S.S. Shin, J. Raines, K. Degenhardt, E. White, S. Chen, Neuropharmacol. 49 (2005) 70-79.]. These cell lines are useful tools in the studies of signaling events that may be mediated by Grm1 in transformed melanocytes. Here we show that stimulation of Grm1 by l-quisqualate, a group I metabotropic glutamate receptor agonist, results in inositol triphosphate (IP3) accumulation, and the activation of ERK1/2 in these cell lines. IP3 accumulation and ERK1/2 activation were inhibited by pretreatment of the tumor cells with a Grm1-specific antagonist (LY367385) or by dominant negative mutants of Grm1, demonstrating the specificity of these events. We also show that ERK1/2 activation by Grm1 was PKC-dependent, but cAMP and PKA-independent. PKCepsilon was shown to play a pivotal role in Grm1-mediated ERK1/2 phosphorylation. Insights into the signaling cascades mediated by Grm1 in melanoma cells may aid in the identification of key molecular targets for the future design of combined therapies for melanoma.     

The evolution of cranial form and function in theropod dinosaurs: insights from geometric morphometrics

Theropod dinosaurs, an iconic clade of fossil species including Tyrannosaurus and Velociraptor, developed a great diversity of body size, skull form and feeding habits over their 160+ million year evolutionary history. Here, we utilize geometric morphometrics to study broad patterns in theropod skull shape variation and compare the distribution of taxa in cranial morphospace (form) to both phylogeny and quantitative metrics of biting behaviour (function). We find that theropod skulls primarily differ in relative anteroposterior length and snout depth and to a lesser extent in orbit size and depth of the cheek region, and oviraptorosaurs deviate most strongly from the "typical" and ancestral theropod morphologies. Noncarnivorous taxa generally fall out in distinct regions of morphospace and exhibit greater overall disparity than carnivorous taxa, whereas large-bodied carnivores independently converge on the same region of morphospace. The distribution of taxa in morphospace is strongly correlated with phylogeny but only weakly correlated with functional biting behaviour. These results imply that phylogeny, not biting function, was the major determinant of theropod skull shape.     

Biochemical properties of alligator (Alligator mississippiensis) bone collagen

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) isolated and purified from alligator (Alligator mississippiensis) bone were studied for molecular size, amino acid profile, secondary structure, and denaturation temperature by SDS-PAGE, HPLC, circular dichroism, and viscometry. Two collagen subunits, alpha1 and alpha2 were identified by SDS-PAGE. The molecular masses for alpha1 and alpha2 chains of ASC were 124 kDa and 111 kDa, respectively. The molecular masses were 123 kDa for alpha1 and 110 kDa for alpha2 chains of the PSC preparation. The molecular masses for ([alpha1](2) alpha2) of ASC and PSC were 359 kDa and 356 kDa, respectively. The major composition of alligator bone ASC and PSC was found to be typical type I collagen. The amino acid profiles of alligator ASC and PSC were similar to amino acid profile of subtropical fish black drum (Pogonias cromis, Sciaenidae) bone. Comparison of amino acid profiles with shark cartilage PSC, showed differences in alanine, hydroxylysine, lysine, and histidine contents. The denaturation temperatures (T(d)) of alligator ASC and PSC collagen measured by viscometry were 38.1 and 38.2 degrees C, respectively. Thermal denaturation temperatures, measured by melting point using circular dichroism, were 37.6 and 37.9 degrees C, respectively. Taken together, these results suggest that alligator bone collagen may find a wide range of applications in biological research, functional foods and nutraceuticals, and biomedical and pharmaceutical research.     

Role of side-edge site of sphingomyelinase from Bacillus cereus

Bacillus cereus sphingomyelinase (Bc-SMase) belongs to the Mg(2+)-dependent neutral sphingomyelinase (nSMase) which hydrolyzes sphingomyelin (SM) to produce phosphocholine and ceramide, and acts as an extracellular hemolysin. Bc-SMase has two metal ion-binding sites in a long horizontal cleft across the molecule, with one Mg(2+) in the central region of the cleft and one divalent metal ion at the side-edge of the cleft. The role of the Mg(2+) at the side-edge of the long horizontal cleft in Bc-SMase remains unresolved. The replacement of Asn-57, Glu-99, and Asp-100 located in close proximity to Mg(2+) at the side-edge with alanine resulted in a striking reduction in binding to and hydrolysis of sphingomyelin in membranes of sheep erythrocytes or SM-liposomes but that of Phe-55 did not. However, the replacement of these residues had little effect on the enzymatic activity. N57A, E99A, and D100A contained 2 mol of Mg(2+) per mol of protein, and the wild type and F55A contained 3 mol. A crystal analysis showed that N57A with Mg(2+) had no metal ion at the side-edge. These results indicate that the Mg(2+) at the side-edge of Bc-SMase plays an important role in the binding to membranes.