Novel enzymatic synthesis of spacer-linked P(k) trisaccharide targeting for neutralization of Shiga toxin.

A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-ß-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.

Expression and purification of bioactive hemagglutinin protein of highly pathogenic avian influenza A (H5N1) in silkworm larvae.

The hemagglutinin (HA) of avian influenza viruses plays a very important role in the infection of host cells. In this study, the HA gene of the highly pathogenic avian influenza H5N1 virus was cloned and expressed in silkworm larvae. The expressed recombinant HA (rHA) was purified using fetuin-agarose chromatography and Superdex 200 10/300 GL gel filtration chromatography, and the identity of purified rHA was confirmed by SDS-PAGE and Western blot. Approximately 500 µg of purified rHA was obtained from a total of 30 silkworm larvae, suggesting the high efficiency of the silkworm expression system. The purified rHA bound to a rabbit polyclonal antibody against influenza A virus H5N1 (avian flu) HA, suggesting its antigenicity and potential application in vaccine development. Gel filtration chromatography showed that purified HA was present in the void volume fractions, indicating that rHA may form an oligomer. The rHA bound to poly{Neu5Acα2,3LacNAcß-O[(CH2)5NHCO]2(CH2)5NH-/γ-PGA}, which mimics an avian type receptor, but did not bind to γ-polyglutamic acid or human type receptor mimic, poly{Neu5Acα2,6LacNAcß-O[(CH2)5NHCO]2(CH2)5NH-/γ-PGA}, suggesting that it could be utilized as a blocking agent against infection by highly pathogenic influenza viruses.

The effect of natural extracellular matrix deposited on synthetic polymers on cultured primary hepatocytes.

It is well known that natural extracellular matrix (ECM) molecules are deposited on the surface of biomaterials during culture of cells and affect cellular behaviors. However, it has not been fully understood what kinds of ECM molecules are deposited on the surface of biomaterials although the cellular behaviors were affected by deposited ECM. In this study, to investigate the effect of deposited natural ECM on behaviors of hepatocytes cultured on biomaterials such as poly (N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide) (PVLA) as a hepatocyte-specific matrix and poly (L-lysine) (PLL) as a non-specific one during the culture of hepatocytes in vitro, we investigated expression pattern of ECM genes and adsorption of ECM molecules onto PVLA- and PLL-coated surfaces. It was found that the expression levels of type I collagen and fibronectin genes in the hepatocytes cultured on PVLA-coated surface were different from them in the hepatocytes cultured on PLL-coated one. Also, the results showed that laminin was dominantly deposited on PVLA-coated surface whereas fibronectin was dominantly deposited on PLL-coated one. Hepatocytes maintained liver-specific functions on PVLA- and laminin-coated surfaces. It is thought that deposited laminin during the culture of hepatocytes affects the liver-specific functions of hepatocytes cultured on PVLA-coated surface.

Kinetic studies of AMP-dependent phosphorolysis of amylopectin catalyzed by phosphorylase b on a 27 MHz quartz-crystal microbalance.

Catalytic cleavage reactions of phosphorylase b were monitored directly on an amylopectin-immobilized 27 MHz quartz-crystal microbalance (QCM). When the inactivated phosphorylase b was injected into a phosphate buffer solution of amylopectin-immobilized QCM (method A), the binding of the enzyme to amylopectin was observed as a frequency decrease (mass increase). Then, when AMP (adenosine monophosphate) was added to activate the enzyme, the frequency gradually increased (mass decreased) due to the phosphorolysis of amylopectin in the presence of phosphates as buffers. When the AMP-activated phosphorylase b was employed (method B), the continuous reaction was observed which includes both the mass increase due to the enzyme binding to amylopectin at first and then the following mass decrease due to the phosphorolysis by the AMP-activated enzyme. All kinetic parameters for the enzyme binding to the substrate (binding and dissociation rate constants, k(on) and k(off), and dissociation constant, K(d)), the AMP binding to the enzyme as activator (K(AMP)), the catalytic rate constant (k(cat)) were obtained from curve fittings of time-courses of frequency (mass) changes. The obtained kinetic parameters were compared with those from Michaelis-Menten kinetics.

In vitro selection of N peptide-binding RNA on a quartz-crystal microbalance.

We report here an in vitro selection of a hairpin loop-RNA bound to N Peptide from bacteriophage lambda on a 27 MHz quartz-crystal microbalance (QCM) to study an interaction between an RNA structure and an RNA-binding peptide. The N Peptide was immobilized on a QCM electrode and specific RNAs binding to the peptide were selected from a GNRNA pentaloop-randomized RNA library with in situ monitoring of selection processes using a QCM. After the 5th round selection, the consensus sequences including a GCGCA loop were obtained from the pentaloop library. This RNA sequence was different from a binding site of a native N Protein (boxB RNA).