No association between CCL2 gene polymorphisms and risk of inflammatory demyelinating diseases in a Korean population.

Inflammatory demyelinating disease (IDD), which includes multiple sclerosis (MS) and neuromyelitis optica (NMO), affects the central nervous system. Chemokine ligand 2 (CCL2/MCP-1) is considered an important contributor to the development or progression of IDD. However, genetic association studies of Asian populations are lacking. In this study, we investigated a possible association between CCL2 polymorphisms (rs1024611, rs28730833, and rs2857657) and a Korean population (178 IDD patients and 237 healthy controls) using multiple logistic regression models. However, we did not find any association, which was consistent with other studies in Caucasian populations. In conclusion, our results suggest that CCL2 variants may not contribute to the pathogenesis of IDD.

Effects of growth differentiation factor 9 and bone morphogenetic protein 15 on the in vitro maturation of porcine oocytes.

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are members of the transforming growth factor-ß (TGF-ß) family, and their roles in oocyte maturation and cumulus expansion are well known in the mouse and human, but not in the pig. We investigated GDF9 and BMP15 expressions in porcine oocytes during in vitro maturation. A significant increase in the mRNA levels of GDF9 and BMP15 was observed at germinal vesicle breakdown, with expression levels peaking at metaphase I (MI), but decreasing at metaphase II (MII). GDF9 and BMP15 protein localized to the oocyte cytoplasm. While treatment with GDF9 and BMP15 increased the expression of genes involved in both oocyte maturation (c-mos, cyclinb1 and cdc2) and cumulus expansion (has2, ptgs2, ptx3 and tnfaip6), SB431542 (a TGFß-GDF9 inhibitor) decreased meiotic maturation at MII. Following parthenogenetic activation, the percentage of blastocysts in SB431542 treatment was lower than in the control (41.3% and 74.4%, respectively). Treatment with GDF9 and BMP15 also increased the mRNA levels of maternal genes such as c-mos [a regulatory subunit of mitogen-activated protein kinase (MAPK)], and cyclinb1 and cdc2 [regulatory subunits of maturation/M-phase-promoting factor (MPF)]; however, SB431542 significantly decreased their mRNA levels. These data were supported by poly (A)-test PCR and protein activity analyses. Our results show that GDF9 and BMP15 participate in cumulus expansion and that they stimulate MPF and MAPK activities in porcine oocytes during in vitro maturation.

Development of discrimination SNP markers for Hanwoo (Korean native cattle).

In the Korean meat market, the native cattle, Hanwoo beef, are preferred over imported beef and domestic Holstein beef despite its relatively high price. In order to hold the beef industry accountable and support consumers' right to know, correct beef-origin labeling is required. For this purpose, we developed 90 single-nucleotide polymorphism markers to discriminate between Hanwoo and other breeds including Holstein using 1602 cattle DNAs. The probability of discrimination was found to be 100% in a subsequent validation set consisting of 632 DNAs. Our study suggests that improved beef-origin discrimination can be achieved by using a combined genetic model that takes into account small genetic differences among a large number of markers. These markers could be useful for discriminating between Hanwoo and imported breeds including domestic Holsteins, and would contribute to the prevention of falsified beef origin.

Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome.

Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIalpha and IIIbeta, respectively). Although the central domain II (the 'DDE' domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIalpha also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIalpha or IIIbeta domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIalpha or IIIbeta function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.

Synthesis of tetrakis(multifluoro-4-pyridyl)porphin derivatives as acetylcholinesterase inhibitors.

New tetrakis(multifluoro-4-pyridyl)porphin derivatives (2-4) and water soluble porphyrin (5) were synthesized to investigate their interactions with acetylcholinesterase from electric eel. These compounds have been found to be the potent reversible inhibitors of the enzyme with Ki values of microM range. In addition, porphyrin (5) showed broad spectrum of anticancer activities.

Synthesis of the quinoline-linked triazolopyrimidine analogues and their interactions with the recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. Triazolopyrimidine sulfonamide (TP) is a mixed-type inhibitor of ALS with respect to both pyruvate and thiamine pyrophosphate. In this study, we synthesized new substituted quinoline-linked TP analogues and several TP analogues which contained either unsubstituted aminoquinolines or amino isoquinolines. In addition, we examined the interactions of both the wild-type and the sulfonylurea-resistant recombinant tobacco ALS enzymes in a highly pure and active form with the quinoline-linked TP analogues, respectively. The wild-type tobacco ALS was extremely sensitive to inhibition by the quinoline-linked TP analogues. In contrast, the mutant tobacco ALS was insensitive to both the quinoline-linked triazolopyrimidine and the sulfonylurea herbicides. The results indicate that the ability of the quinoline-linked TP analogues to inhibit ALS is highly sensitive to substitution at the ortho position (C-7) and to the position of the ring nitrogen around the sulfonamide functionality (C-8).

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition.

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.

Altering the DNA-binding specificity of Mu transposase in vitro.

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.

Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic complementation groups.

This study examines the contribution of domain II beta of bacteriophage Mu transposase (A protein), a subdomain of the central catalytic domain II, to the transposition reaction. The properties of several point mutations implicate a role for this domain in facilitating metal-assisted assembly of the synaptic complex, as well as in intramolecular DNA strand transfer. Point mutations as well as deletions in domain II beta can be complemented by those in domain II alpha but not those in domain III alpha. Thus, residues within subdomains II alpha and II beta belong to different catalytic complementation groups.

Soluble overexpression in Escherichia coli, and purification and characterization of wild-type recombinant tobacco acetolactate synthase.

Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine. The wild-type ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T. The resulting recombinant plasmid pGEX-ALS2 was used to transform Escherichia coli strain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST). The fusion product GST-wALS was purified in a single step on a glutathione-Sepharose column. The purified GST-wALS was sensitive to a sulfonylurea herbicide, and was lost its sensitivity to end products, L-valine, L-leucine and L-isoleucine. These results suggest that the purified recombinant tobacco ALS was functionally active, and that the sulfonylureas may not bind to the feedback regulatory site on the plant ALS.

Detection, quantitation, and localization of bovine angiogenin by immunological assays.

Bovine angiogenin (bAng) is a potent blood vessel inducing protein found in bovine serum and milk. Antisera have been raised against bAng. Western blot analysis for bAng indicated that the polyclonal antibody recognized bAng specifically, and no cross-reactivity with bovine RNase A, a protein homologous to bAng, was observed. The sandwich enzyme-linked immunosorbent assay for bAng was sensitive to 10 pg of bAng, and this assay was able to quantitate bAng in bovine serum (100-180 ng/mL) and milk (4-8 micrograms/mL). Strong positive immunohistochemical reactions were detected in alveolar cells, the secretion of alveolar cells and excretory ducts in sections of cow mammary gland, epithelial cells of visceral peritoneum and bile-duct in sections of cow liver, and epithelial cells and mucous glands in sections of cow gallbladder. These results suggest that epithelial cells and secretory cells are major sites of angiogenin synthesis.

Kinetic and structural probing of the precleavage synaptic complex (type 0) formed during phage Mu transposition. Action of metal ions and reagents specific to single-stranded DNA.

In an earlier kinetic study (Wang, Z., and Harshey, R. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate-limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn2+ and Co2+ ions, like Mg2+ and Mn2+ ions, can efficiently cleave the type 0 complex. However, unlike Mg2+ and Mn2+ ions, Zn2+ and Co2+ ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition.

Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand transfer.

We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.

DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex. Relevance of DNA phasing and att site cleavage.

The requirements for negatively supercoiled DNA substrates, the cis-acting transposition enhancer and the Escherichia coli HU protein during the phage Mu transposition reaction are relaxed under DMSO-assay conditions. We have used these modified assay conditions to extend studies on the transposition pathway. We show here that linear DNA fragments containing the right end of Mu (attR) and Mu A protein mutually promote the assembly of "high-order" complexes held together by non-covalent protein-DNA and protein-protein interactions. A large subset of these complexes is competent in mediating strand transfer. DNA fragments containing the left end of Mu (attL) as well as non-Mu DNA can be used as targets during strand transfer. The R1 and R2 subsites within attR are required, but R3 is dispensable, in the protein-DNA oligomerization steps as well as in the strand transfer reaction. Proper phasing and spacing between R1 and R2 are central to the reaction. A single base-pair change in the terminal nucleotide that renders attR non-cleavable prevents the assembly of stable high-order complexes, showing that strand cleavage and stabilization of high-order complexes are tightly coupled events. Conversely, pre-cleavage at the attL site allows it to function in the assembly process, albeit at a much lower efficiency than attR. In the presence of HU, the reactivity of pre-cleaved attL is enhanced significantly.

Effects of naturally occurring flavonoids on mitogen-induced lymphocyte proliferation and mixed lymphocyte culture.

In this investigation, 34 structurally different flavonoids including derivatives of chalcone, flavanone, flavan-3-ol, flavone, flavonol, and their glycosides were evaluated for in vitro suppression of mitogen-induced lymphocyte proliferation and mixed lymphocyte culture from mouse spleen. Flavonoids, mainly derivatives of flavone and flavonol, clearly demonstrated the suppressive effects on lymphocyte proliferation at higher than 10(-6) M depending on the structures of flavonoid molecules, although their suppressive activities were less than that of cyclosporin A or prednisolone. Various glycosidic substitutions to A- and/or C-ring of the flavonoid aglycones were found to eliminate the suppressive activities of their aglycones, regardless of sugar compositions and positions of substitutions. In concanavalin A-induced lymphocyte proliferation, derivatives of flavone and flavonol having 2,3-unsaturation and at least 1 hydroxyl group showed the suppressive activity. In lipopolysaccharide-induced lymphocyte proliferation, only myricetin was active among flavonoids tested at the concentrations up to 10(-5) M. In mixed lymphocyte culture, some derivatives of flavone and flavonol with 2,3-unsaturation were active and especially flavone derivatives showed the higher suppressive activities than those of the flavonol derivatives.

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs.

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with beta-glucosidase, beta-GIcNAcase, and alpha-mannosidase, respectively. Swainsonine specifically inhibited alpha-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited beta-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.

Inhibition of the alpha-L-arabinofuranosidase III of Monilinia fructigena by 1,4-dideoxy-1,4-imino-L-threitol and 1,4-dideoxy-1,4-imino-L-arabinitol.

1. 1,4-Dideoxy-1,4-imino-L-threitol was synthesized and the synthesis of 1,4-dideoxy-1,4-imino-L-arabinitol was improved. 2. Both compounds are competitive inhibitors of Monilinia fructigena alpha-L-arabinofuranosidase III, the additional hydroxymethyl group in the arabinitol contributing about 17.8 kj/mol (4.25 kcal/mol) to the Gibbs free energy of binding. 3. The affinities (1/Ki) of both compounds vary with pH in a classical bell-shaped way, the pKa value being that of the acid-catalytic group on the enzyme [5.9; Selwood & Sinnott (1988) Biochem. J. 254, 899-901] and the pKb values being those of the free inhibitors, 7.6 and 7.8 respectively. 4. On the basis of these and literature data we suggest that efficient inhibition of a glycosidase at its pH optimum by an appropriate iminoalditol will be found when the pKa of the iminoalditol is below that of the acid-catalytic group of the target enzyme.

Inhibition of alpha-L-fucosidase by derivatives of deoxyfuconojirimycin and deoxymannojirimycin.

Deoxyfuconojirimycin (1,5-dideoxy-1,5-imino-L-fucitol) is a potent, specific and competitive inhibitor (Ki 1 x 10(-8) M) of human liver alpha-L-fucosidase (EC 3.2.1.51). Six structural analogues of this compound were synthesized and tested for their ability to inhibit alpha-L-fucosidase and other human liver glycosidases. It is concluded that the minimum structural requirement for inhibition of alpha-L-fucosidase is the correct configuration of the hydroxy groups at the piperidine ring carbon atoms 2, 3 and 4. Different substituents in either configuration at carbon atom 1 (i.e. 1 alpha- and beta-homofuconojirimycins) and at carbon atom 5 may alter the potency but do not destroy the inhibition of alpha-L-fucosidase. The pH-dependency of the inhibition by these amino sugars suggests very strongly that inhibition results from the formation of an ion-pair between the protonated inhibitor and a carboxylate group in the active site of the enzyme. Deoxymannojirimycin (1,5-dideoxy-1,5-imino-D-mannitol) is also a more potent inhibitor of alpha-L-fucosidase than of alpha-D-mannosidase. This can be explained by viewing deoxymannojirimycin as beta-L-homofuconojirimycin lacking the 5-methyl group. Conversely, beta-L-homo analogues of fuconojirimycin can also be regarded as derivatives of deoxymannojirimycin. This has permitted deductions to be made about the structural requirements of inhibitors of alpha- and beta-D-mannosidases.

Structure-activity relationship of swainsonine. Inhibition of human alpha-mannosidases by swainsonine analogues.

The inhibitory properties of a series of synthetic epimers and analogues of swainsonine towards the multiple forms of human alpha-mannosidases were studied in vitro and in cells in culture. Of the five epimers tested, only the 8a-epimer and 8,8a-diepimer of swainsonine were specific and competitive inhibitors (Ki values of 7.5 x 10(-5) and 2 x 10(-6) M respectively) of lysosomal alpha-mannosidases in vitro and induced storage of mannose-rich oligosaccharides in human fibroblasts in culture. The structures of these storage products indicated that processing alpha-mannosidases had also been inhibited. This was consistent with the observed inhibition in vitro of these enzymes by these compounds. In contrast, the 8-epimer, 1,8-diepimer and 2,8a-diepimer of swainsonine had no appreciable effect on any alpha-mannosidases. The corresponding open-chain analogues of swainsonine, namely 1,4-dideoxy-1,4-imino-D-mannitol, of the 8a-epimer, namely 1,4-dideoxy-1,4-imino-D-talitol, and of the 8,8a-diepimer, namely 1,4-dideoxy-1,4-imino-L-allitol, were weaker competitive inhibitors of lysosomal alpha-mannosidase, with Ki values of 1.3 x 10(-5), 1.2 x 10(-4) and 1.2 x 10(-4) M respectively. These analogues also proved less effective at inducing oligosaccharide accumulation and in disturbing glycoprotein processing. These compounds offer the opportunity to determine which alterations in the chirality of the swainsonine molecule affect its inhibitory specificity. A comparison of their biological activities has identified reagents that will be useful for studying steps in the biosynthesis and catabolism of glycoproteins and that may be of potential value in chemotherapy.