On the interaction site of serine acetyltransferase in the cysteine synthase complex from Escherichia coli

Cysteine synthase from Escherichia coli is a bienzyme complex comprised of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase A. The site of interaction of a SAT molecule was investigated by gel chromatography and surface plasmon technique using various mutant-type SATs, to better understand the mechanism involved in complex formation. The C-terminus of SAT, Ile 273, along with Glu 268 and Asp 271, was found to be essential for complex formation. The effects of O-acetyl-L-serine and sulfide on the affinity for the complex formation were also studied using a surface plasmon technique.     

Cell polarity: centrosomes release signals for polarization

New findings reveal that, in Caenorhabditis elegans embryos, the centrosome provides signals that induce cell polarization, independently of its function as the microtubule-organizing center.     

Development of an amperometric flow analysis sensor for specific detection of D-psicose

The aim of this study was to develop a simple, rapid and highly sensitive sensor for measuring the rare sugar d-psicose. The proposed system adopts amperometric flow analysis and two consecutive enzyme reactions consisting of a reactor packed with d-tagatose 3-epimerase (DTE)-immobilized beads, which converts d-psicose to d-fructose, and a carbon-paste electrode containing d-fructose dehydrogenase (DFDH). In order to fabricate a robust sensor system, various experimental parameters were optimized including the buffer composition, flow rate for the two enzyme reactions and the size of micro-flow cell. The developed sensor responded linearly to d-psicose concentration in the range from 0.08 to 50mM (R(2)=0.988). The signal/noise ratio was 3.0 for the 0.08 mM d-psicose solution, and the relative standard deviations were 1.7 (n=20) and 2.6% (n=20) for the 10 and 20mM d-psicose solutions, respectively. One round of assay was completed within 8 min. Our results suggest that the sensor can be used not only for the detection of d-psicose in food samples but also for monitoring d-psicose within the environment. Moreover, the sensor system can be applied to the detection of many other rare sugars by using the same measurement principle.     

Prevalence and molecular subtyping of Blastocystis from dairy cattle in Kanagawa,Japan

Blastocystis is an intestinal protist, commonly found in the human population and in a wide range of animals globally. Currently, isolates from mammalian and avian hosts are classified into 17 subtypes (STs) based on phylogeny of the small subunit rRNA gene (SSU rDNA), of which ten (ST1-9, 12) are reported in humans. ST10 is a major ST reported from livestock cattle. However, other STs including ST1, 3, 4, 5, and 6, which have the potential to be transmitted to humans, are also reported from cattle in several countries. Although a survey has been conducted previously in western Japan for livestock cattle, there is no information available regarding other parts of Japan. Therefore, this study surveyed the prevalence of Blastocystis and its STs in cattle from Kanagawa prefecture, eastern Japan. Fecal specimens, collected from 133 dairy cattle on four different farms, were subjected to a short-term xenic in vitro culture and Blastocystis were identified by microscopic examination. Seventy-two cattle were positive for Blastocystis (54.1%). Direct sequences for the partial SSU rDNA were obtained for 45 samples. Based on nucleotide sequence homology search and phylogenetic analysis, 44 isolates were identified as ST14 and one as ST10. Our study confirms the presence of these STs in dairy cattle in Japan for the first time. The STs identified here, ST10 and ST14, support previous findings that Bovidae may be the natural host for both STs.     

A practical approach to N-glycan production by hydrazinolysis using hydrazine monohydrate

Hydrazinolysis is a versatile method to liberate N-linked glycans from glycoproteins. However, the method is usually performed with anhydrous hydrazine, a highly toxic and explosive chemical used in rocket fuel. Thus despite the need to produce functionally important glyco-materials, hydrazinolysis is limited to small scale (e.g., 0.2-1 mL) reactions. In the present study, we report an alternative procedure for hydrazinolysis using hydrazine monohydrate in place of anhydrous hydrazine. The developed procedure was applied to both purified glycoproteins (Taka-amylase and transferrin) and hen egg yolk protein fraction with comparable yields to the traditional method using anhydrous hydrazine. The sialyl linkage of alpha2-6disialobiantennary oligosaccharides proved to be fully stable. The developed procedure facilitated the large-scale preparation of N-linked glycans. The new method should make a substantial contribution to both small- and large-scale production of functional glycans, including therapeutically relevant human-type glycans.     

Structural analysis of lysine‐4 methylated histone H3 proteins using synchrotron radiation circular dichroism spectroscopy

We report structural alterations of histone H3 proteins induced by lysine‐4 (K4) monomethylation, dimethylation, and trimethylation identified by using synchrotron radiation circular dichroism spectroscopy. Compared with unmethylated H3, monomethylation and dimethylation induced increases in α‐helix structures and decreases in β‐strand structures. In contrast, trimethylation decreased α‐helix content but increased β‐strand content. The structural differences among K4‐unmethylated/methylated H3 may allow epigenetic enzymes to discriminate the substrates both chemically and sterically.     

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.     

In vivo cross-linking of nucleosomal histones catalyzed by nuclear transglutaminase in starfish sperm and its induction by egg jelly triggering the acrosome reaction.

A histone heterodimer, designated as p28, which contains an Nepsilon(gamma-glutamyl)lysine cross-link between Gln9 of histone H2B and Lys5 or Lys12 of histone H4, is present in starfish (Asterina pectinifera) sperm. Treatment of sperm nuclei with micrococcal nuclease produced soluble chromatin, which was size-fractionated by sucrose-gradient centrifugation to give p28-containing oligonucleosome and p28-free mononucleosome fractions, indicating that the cross-link is internucleosomal. When sperm nuclei were incubated with monodansylcadaverine, a fluorescent amine, in the presence or absence of Ca(2+), histone H2B was modified only in the presence of Ca(2+). Gln9, in the N-terminal region, was modified, but the other Gln residues located in the internal region were not, suggesting that the modification takes place on the surface of the nucleosome core by the in situ action of a Ca(2+)-dependent nuclear transglutaminase. Treatment of sperm with the egg jelly, which activates Ca(2+) influx to induce the acrosome reaction, resulted in a significant elevation of the p28 content in the nucleus. This is the first demonstration of an in vivo activation of transglutaminase leading to the formation of a cross-link in intracellular proteins.     

Identification of N-substituted 8-azatetrahydroquinolone derivatives as selective and orally active M1 and M4 muscarinic acetylcholine receptors agonists

We designed and synthesized N-substituted 8-azatetrahydroquinolone derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors agonists. Optimization of selected derivatives led to the discovery of compound 7 as a highly potent M₁ and M₄ agonist with weak hERG inhibition. Oral administration of compound 7 improved psychosis-like behavior in rats.     

Stability and flexibility of marginally hydrophobic–segment stalling at the endoplasmic reticulum translocon

Many membrane proteins are integrated into the endoplasmic reticulum membrane through the protein-conducting channel, the translocon. Transmembrane segments with insufficient hydrophobicity for membrane integration are frequently found in multispanning membrane proteins, and such marginally hydrophobic (mH) segments should be accommodated, at least transiently, at the membrane. Here we investigated how mH-segments stall at the membrane and their stability. Our findings show that mH-segments can be retained at the membrane without moving into the lipid phase and that such segments flank Sec61α, the core channel of the translocon, in the translational intermediate state. The mH-segments are gradually transferred from the Sec61 channel to the lipid environment in a hydrophobicity-dependent manner, and this lateral movement may be affected by the ribosome. In addition, stalling mH-segments allow for insertion of the following transmembrane segment, forming an N_cytosol/C_lumen orientation, suggesting that mH-segments can move laterally to accommodate the next transmembrane segment. These findings suggest that mH-segments may be accommodated at the ER membrane with lateral fluctuation between the Sec61 channel and the lipid phase.