Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.     

Ca2+-Dependent Maturation of Subtilisin from a Hyperthermophilic Archaeon, Thermococcus kodakaraensis: the Propeptide Is a Potent Inhibitor of the Mature Domain but Is Not Required for Its Folding

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 is a member of the subtilisin family. T. kodakaraensis subtilisin in a proform (T. kodakaraensis pro-subtilisin), as well as its propeptide (T. kodakaraensis propeptide) and mature domain (T. kodakaraensis mat-subtilisin), were independently overproduced in E. coli, purified, and biochemically characterized. T. kodakaraensis pro-subtilisin was inactive in the absence of Ca²⁺ but was activated upon autoprocessing and degradation of propeptide in the presence of Ca²⁺ at 80°C. This maturation process was completed within 30 min at 80°C but was bound at an intermediate stage, in which the propeptide is autoprocessed from the mature domain (T. kodakaraensis mat-subtilisin*) but forms an inactive complex with T. kodakaraensis mat-subtilisin*, at lower temperatures. At 80°C, approximately 30% of T. kodakaraensis pro-subtilisin was autoprocessed into T. kodakaraensis propeptide and T. kodakaraensis mat-subtilisin*, and the other 70% was completely degraded to small fragments. Likewise, T. kodakaraensis mat-subtilisin was inactive in the absence of Ca²⁺ but was activated upon incubation with Ca²⁺ at 80°C. The kinetic parameters and stability of the resultant activated protein were nearly identical to those of T. kodakaraensis mat-subtilisin*, indicating that T. kodakaraensis mat-subtilisin does not require T. kodakaraensis propeptide for folding. However, only ~5% of T. kodakaraensis mat-subtilisin was converted to an active form, and the other part was completely degraded to small fragments. T. kodakaraensis propeptide was shown to be a potent inhibitor of T. kodakaraensis mat-subtilisin* and noncompetitively inhibited its activity with a K_i of 25 ± 3.0 nM at 20°C. T. kodakaraensis propeptide may be required to prevent the degradation of the T. kodakaraensis mat-subtilisin molecules that are activated later by those that are activated earlier.     

Achromobacter protease I-catalyzed conversion of porcine insulin into human insulin

We have established a procedure for converting porcine insulin into human insulin using a serine protease from $\text{Achromobacter}\text{lyticus}$ M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with $\text{Achromobacter}$ protease. The coupling between DAI and Thr-OBu^t was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBu^t) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBu^t-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin.     

Template-directed polymerization of oligoadenylates using cyanogen bromide

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

Inactivation of Buckwheat α-Glucosidase with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide

The amino acid residue(s) involved in the activity of buckwheat α-glucosidase was modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of glycine ethyl ester. The modification resulted in the decrease in the hydrolytic activity of the enzyme following pseudo-first order kinetics. Competitive inhibitors, such as Tris and turanose, protected the enzyme against the inactivation. Protection was provided also by alkali metal, alkaline-earth metal and ammonium ions, though these cations are non-essential for the activity of the enzyme. Turanose or K⁺ protected one carboxyl group per enzyme from the modification with carbodiimide and glycine ethyl ester. Free sulfhydryl group of the enzyme was also partially modified with carbodiimide, but the inactivation was considered to be mainly attributed to the modification of essential carboxyl group rather than to that of free sulfhydryl group.     

Changes of Hydrolytic Activities of Buckwheat α-Glucosidase by Acetylation with N-Acetylimidazole

A treatment of buckwheat α-glucosidase with N-acetylimidazole brought about the acétylation of 6.4 tyrosyl residues, 0.38 free sulfhydryl groups and about 50% of free amino groups, and the decrease in the hydrolytic activities toward maltooligosaccharides (G₂~G₈, G13) and soluble starch. The affinities for the substrate other than maltose were diminished by the modification and the extent of the reduction of the affinities was apparently dependent on the degree of polymerization of maltooligosaccharides, while the affinity for phenyl α- maltoside was increased. The treatment of the acetylated enzyme with hydroxylamine resulted in the complete restration of the affinities for all substrates tested. It seems that these facts were due to the acétylation of several tyrosyl residues located in or near certain subsites of the enzyme. About 25 % of the hydrolytic activity remained inert in spite of the deacetylation with hydroxylamine, which was assumed to be attributed to the partial modification of free sulfhydryl group localized closely near the catalytic site of the enzyme.     

Flavonol glycosides of Phlomis spectabilis

Four flavonol glucosides, one new, have been isolated from a methanolic extract of Phlomis spectabilis. Their structures were established as the 3-glucosides and 3-(6″-(E)-p-coumaroyl)glucosides of kaempferol and of kaempferol 7,4′-dimethyl ether.     

Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HII

Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi-Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9-7.6 degrees C in T(m). A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable.     

KNApSAcK family databases: integrated metabolite-plant species databases for multifaceted plant research

A database (DB) describing the relationships between species and their metabolites would be useful for metabolomics research, because it targets systematic analysis of enormous numbers of organic compounds with known or unknown structures in metabolomics. We constructed an extensive species-metabolite DB for plants, the KNApSAcK Core DB, which contains 101,500 species-metabolite relationships encompassing 20,741 species and 50,048 metabolites. We also developed a search engine within the KNApSAcK Core DB for use in metabolomics research, making it possible to search for metabolites based on an accurate mass, molecular formula, metabolite name or mass spectra in several ionization modes. We also have developed databases for retrieving metabolites related to plants used for a range of purposes. In our multifaceted plant usage DB, medicinal/edible plants are related to the geographic zones (GZs) where the plants are used, their biological activities, and formulae of Japanese and Indonesian traditional medicines (Kampo and Jamu, respectively). These data are connected to the species-metabolites relationship DB within the KNApSAcK Core DB, keyed via the species names. All databases can be accessed via the website http://kanaya.naist.jp/KNApSAcK_Family/. KNApSAcK WorldMap DB comprises 41,548 GZ-plant pair entries, including 222 GZs and 15,240 medicinal/edible plants. The KAMPO DB consists of 336 formulae encompassing 278 medicinal plants; the JAMU DB consists of 5,310 formulae encompassing 550 medicinal plants. The Biological Activity DB consists of 2,418 biological activities and 33,706 pairwise relationships between medicinal plants and their biological activities. Current statistics of the binary relationships between individual databases were characterized by the degree distribution analysis, leading to a prediction of at least 1,060,000 metabolites within all plants. In the future, the study of metabolomics will need to take this huge number of metabolites into consideration.