Purification and Properties of Acid Carboxypeptidase II from Aspergillus oryzae

Acid carboxypeptidase II from Aspergillus oryzae was purified from the rivanol non-precipitated fraction. The purified enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The optimum activity of the enzyme lay at pH 3.0 for carbobenzoxy-L-glutamyl-l-tyrosine. The enzyme was inhibited by diisopropylphosphorofluoridate and SH reagents such as p-chloromercuribenzoate and monoiodoacetate, but not by such metal chelating agents as ethylenediaminetetraacetate, α, α′-dipyridyl and o-phenanthroline. The molecular weight of the enzyme was estimated to be about 105,000.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

A Novel Preparation of a trans-Fused Urea trans-3,4-Ureylenethiophane-1,1-dioxide

A novel synthesis of trans-3,4-ureylenethiophane-1,1-dioxide (X, R=H) from sulfolene via trans-3,4-diaminothiophane-1,1-dioxide (VII, R₁=R₂=H) is described.     

The Metabolism of Coumarin by a Strain of Pseudomonas

The metabolism of coumarin by a strain of Pseudomonas isolated from soil which utilizes coumarin as a sole carbon source was studied. The metabolic pathway was shown to be coumarin→dihydrocoumarin→melilotic acid→2,3-dihydroxyphenylpropionic acid, based on the results of (1) isolation and identification of metabolic products, (2) survey on the utilization of the postulated intermeidates and (3) examination of enzymatic reaction. An alternative pathway involving o-coumaric acid and 2,3-dihydroxycinnamic acid as intermediates at the metabolism of coumarin was also discussed.

Coumarin reducing enzyme (dihydrocoumarin : NAD[NADP] oxydo-reductase) which catalizes the reduction of coumarin to dihydrocoumarin was partially purified from the extracts of the above strain of Pseudomonas and some properties of the enzyme were investigated. The optimum pH of the reaction was 5.25. The enzyme is highly specific with respect to coumarin, and Km values for coumarin and NADH were 6.6 × 10^?6 m and 4.1 × 10^?5 m, respectively. The enzyme activity was extremely sensitive to sulfhydryl reagents particularly to p-chloromercuribenzoate. 2-Mercaptoethanol or dithiothreitol protected the enzyme from inactivation by low temperature storage. The molecular weight of enzyme was estimated to be about 140,000 by gel permiation Chromatographic method. The enzyme showed a substrate inhibition at higher concentrations of coumarin. This inhibition was noncompetitive with respect to NADH. The enzyme was also inhibited by many coumarin analogues. 3-Hydroxycoumarin showed noncompetitive inhibition with both coumarin and NADH. The mechanism of inhibition for the enzyme is discussed. It is concluded that enzyme protein contains zinc atom and that NADH is attached to zinc in the enzyme reaction.     

Purification and Properties of Acid Carboxypeptidase IV from Aspergillus oryzae

Acid carboxypeptidase IV from Aspergillus oryzae was purified from the rivanol precipitable fraction by column chromatography on DEAE-cellulose, DEAE-Sephadex A–50, hydroxylapatite and P-cellulose and gel filtration through Sephadex G–100. The optimum pH is at pH 3.0 for carbobenzoxy-l-glutamyl-l-tyrosine. The enzyme activity was inhibited by sulfhydryl reagents and diisopropylphosphorofluoridate, but was not inhibited by metal chelating agents. The molecular weight of the enzyme was estimated to be about 43,000 by gel filtration method.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

Inhibition of CIN85-Mediated Invasion by a Novel SH3 Domain Binding Motif in the Lysyl Oxidase Propeptide

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the “Cbl-interacting protein of 85-kDa” (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.     

Fungal Garden Making inside Bamboos by a Non-Social Fungus-Growing Beetle

In fungus-growing mutualism, it is indispensable for host animals to establish gardens of the symbiotic fungus as rapidly as possible. How to establish fungal gardens has been well-documented in social fungus-farming insects, whereas poorly documented in non-social fungus-farming insects. Here we report that the non-social, fungus-growing lizard beetle Doubledaya bucculenta (Coleoptera: Erotylidae: Languriinae) transmits the symbiotic yeast Wickerhamomyces anomalus from the ovipositor-associated mycangium into bamboo internode cavities and disperses the yeast in the cavities to make gardens. Microbial isolation and cryo-scanning electron microscopy observation revealed that W. anomalus was constantly located on the posterior ends of eggs, where larvae came out, and on the inner openings of oviposition holes. Direct observation of oviposition behavior inside internodes revealed that the distal parts of ovipositors showed a peristaltic movement when they were in contact with the posterior ends of eggs. Rearing experiments showed that W. anomalus was spread much more rapidly and widely on culture media and internodes in the presence of the larvae than in the absence. These results suggest that the ovipositors play a critical role in vertical transmission of W. anomalus and that the larvae contribute actively to the garden establishment, providing a novel case of fungal garden founding in non-social insect-fungus mutualism.     

Staphylococcus aureus protein A triggers T cell-independent B cell proliferation by sensitizing B cells for TLR2 ligands

B cells possess functional characteristics of innate immune cells, as they can present Ag to T cells and can be stimulated with microbial molecules such as TLR ligands. Because crude preparations of Staphylococcus aureus are frequently used as polyclonal B cell activators and contain potent TLR2 activity, the scope of this study was to analyze the impact of S. aureus-derived TLR2-active substances on human B cell activation. Peripheral B cells stimulated with chemically modified S. aureus cell wall preparations proliferated in response to stimulation with crude cell wall preparations but failed to be activated with pure peptidoglycan, indicating that cell wall molecules other than peptidoglycan are responsible for B cell proliferation. Subsequent analysis revealed that surface protein A (SpA), similar to BCR cross-linking with anti-human Ig, sensitizes B cells for the recognition of cell wall-associated TLR2-active lipopeptides (LP). In marked contrast to TLR7- and TLR9-triggered B cell stimulation, stimulation with TLR2-active LP and SpA or with crude cell wall preparations failed to induce IgM secretion, thereby revealing qualitative differences in TLR2 signaling compared with TLR7/9 signaling. Notably, combined stimulation with SpA plus TLR2 ligands induced vigorous proliferation of a defined B cell subset that expressed intracellular IgM in the presence of IL-2. Conclusion: S. aureus triggers B cell activation via SpA-induced sensitization of B cells for TLR2-active LP. Combined SpA and TLR2-mediated B cell activation promotes B cell proliferation but fails to induce polyclonal IgM secretion as seen after TLR7 and TLR9 ligation.     

Physiological changes leading to anhydrobiosis improve radiation tolerance in Polypedilum vanderplanki larvae

High tolerance against various extreme environments exhibited by some anhydrobionts might be due to being almost completely desiccated, a state where little or no chemical reactions occur. We have shown that anhydrobiotic larvae of Polypedilum vanderplanki have higher tolerance against both high- and low-linear energy transfer (LET) radiation than hydrated larvae. It is of great interest to know how the desiccating larvae gain radiation tolerance. We therefore examined effects of high-LET radiation on four kinds of larvae: (1) normal hydrated (intact) larva, (2) intermediates between the anhydrobiotic and normal hydrated state, (3) almost completely dehydrated (anhydrobiotic) larvae, and (4) immediately rehydrated larvae that are assumed to have a similar molecular profile to anhydrobiotic larvae. The intermediates and immediately rehydrated larvae survived longer after high-LET radiation than intact larvae, indicating that radiation tolerance could be enhanced even in hydrated larvae. Physiological changes toward anhydrobiosis, e.g. accumulation of protectants or increasing damage repair capacity, correlate with improved radiation tolerance in hydrated larvae. In addition, almost complete desiccation further enhanced radiation tolerance, possibly in a different way from the hydrated larvae.