A toll-like receptor 3 single nucleotide polymorphism in Japanese patients with sarcoidosis.

Toll-like receptor 3 (TLR3) may be associated with T helper 1 immune response. This study aimed to investigate the role of a functional TLR3 single nucleotide polymorphism (SNP) in sarcoidosis. We genotyped 220 Japanese patients with sarcoidosis and 140 controls for TLR3 SNP rs3775291 to analyze its association with susceptibility to sarcoidosis and assessed its relationship to clinical features in 172 patients over 2 years. The TLR3 rs3775291 genotype was not significantly associated with disease susceptibility. However, patients with cardiac sarcoidosis (CS) significantly more frequently had the TT genotype (p < 0.01) or the T allele (p < 0.05) than those patients without CS. We conclude that TLR3 SNP rs3775291 may affect cardiac involvement in Japanese patients with sarcoidosis.

Demonstration of a 3-dB directional coupler with enhanced robustness to gap variations for silicon wire waveguides.

We demonstrate a robust 3-dB directional coupler which has a narrow silicon wire core and a wide gap. Sensitivity to the gap variation is decreased to one tenth that of a conventional directional coupler. Better spectral stability due to the enhanced robustness to waveguide geometrical fluctuations was experimentally verified.

A CD40 single-nucleotide polymorphism affects the lymphocyte profiles in the bronchoalveolar lavage of Japanese patients with sarcoidosis.

CD40 plays a critical role in adaptive immunity, and alveolar macrophages in patients with sarcoidosis express higher levels of CD40. This study investigated the association of rs1883832, a functional single-nucleotide polymorphism in the CD40 gene with susceptibility to sarcoidosis and phenotypes of sarcoidosis. Genotyping of rs1883832 in 175 Japanese patients with sarcoidosis and 150 age- and sex-matched controls revealed no significant difference between the genotypes of the patient and control groups (CC/CT/TT, 32.8/52.0/14.7% in the patients; 37.3/48.0/14.7% in the controls, P = 0.66; allele C, 59.1% in the patients, 61.3% in the controls, P = 0.57). T-cell and CD4+ cell counts in the bronchoalveolar lavage fluid were significantly higher in the TT genotype group than in the CC and CT genotype group.

Significance of plasma NT-proBNP levels as a biomarker in the assessment of cardiac involvement and pulmonary hypertension in patients with sarcoidosis.

BACKGROUND: Cardiac involvement and pulmonary hypertension (PH) are life-threatening complications in sarcoidosis. OBJECTIVE: This study aimed to investigate the utility of plasma NT-proBNP in the assessment of these conditions in sarcoidosis patients. STUDY DESIGN AND METHODS: A prospective, observational study was performed on 150 consecutive Japanese sarcoidosis patients. Doppler echocardiography was performed in all subjects, and those who were successfully evaluated for PH status were included in the analysis. Cardiac sarcoidosis was diagnosed based on Japanese guidelines, and PH was defined as estimated systolic pulmonary artery pressure (sPAP) > or = 35 mmHg. The diagnostic accuracy of NT-proBNP according to the presence of cardiac sarcoidosis and PH was assessed based on receiver-operator characteristic (ROC) curves. RESULTS: 130 subjects were successfully evaluated for PH status. Of these, 29 met the diagnostic criteria of cardiac sarcoidosis, and 21 were diagnosed with PH. Plasma NT-proBNP levels were significantly higher in patients with cardiac sarcoidosis (p < 0.0001). Stepwise regression analysis showed that presence of cardiac sarcoidosis, decreased ejection fraction and increased sPAP were all independently associated with higher plasma NT-proBNP levels. Plasma NT-proBNP showed good accuracy in identifying patients with cardiac sarcoidosis (area under the ROC curve; AURC = 0.913). However, even when patients with cardiac sarcoidosis were excluded, plasma NT-proBNP levels could not be used reliably to identify patients with PH (AURC = 0.681). CONCLUSION: In patients with sarcoidosis, plasma NT-proBNP levels are a useful biomarker to identify cardiac involvement, but not to identify PH.

Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein.

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.

Gene therapy of liver tumors with human liver-specific nanoparticles.

The development of safe and efficient liver-specific gene delivery approaches offers new perspectives for the treatment of liver disease, in particular, liver cancer. We evaluated the therapeutic potential of hepatotropic nanoparticles for gene therapy of liver tumor. These nanoparticles do not contain a viral genome and display the hepatitis B virus L antigen, which is essential to confer hepatic specificity. It has not been shown whether a therapeutic effect could be obtained using L nanoparticles in a human liver tumor xenograft model. Rats bearing human hepatic (NuE) and non-hepatic tumors were injected with L nanoparticles containing a green fluorescent protein (GFP) expression plasmid. GFP expression was observed only in NuE-derived tumors but not in the non-hepatic tumor. The potential for treatment of liver tumors was analyzed using L nanoparticles containing the herpes simplex virus thymidine kinase gene, in conjunction with ganciclovir pro-drug administration. The growth of NuE-derived tumors in L particle-injected rats was significantly suppressed, but not of the non-hepatic tumor control. In summary, this is the first demonstration that nanoparticles could be used for delivery of therapeutic genes with anti-tumor activity into human liver tumors. This intravenous delivery system may be one of the major advantages as compared to many other viral vector systems.

Enantiomeric specificity at the deacylation process of tryptic catalysis.

Trypsin-specific substrate analogs, 'inverse substrates', carrying chiral acyl group were synthesized. Kinetic analysis of the trypsin-catalyzed hydrolysis of these substrates revealed that the deacylation process is also appropriate for discrimination between enantiomers. The enantiomeric preference during the deacylation process was analyzed by comparing the hydrolytic rates corresponding to eight enantiomeric pairs of the substrates. The spatial requirement of the enzyme active site for catalytic efficiency is discussed, based on the steric characteristics of the optically active acyl residues.

Specific spin-labeling at trypsin active site. Application of 'inverse substrate' to the structural analysis of the active site.

Specific and reversible spin-labeling of trypsin (EC 3.4.21.4) active site was carried out by 'inverse substrate', 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolinyloxyl-p-amidinophenyl esters. The paramagnetic resonance spectra of the labeled trypsin in the form of acyl enzyme intermediate were investigated. The 2T value, separation of the outer hyperfine extrema, was observed to be sensitive to pH of the medium. These results are discussed in terms of pH-dependent conformational change at the vicinity of active site.

'Inverse' substrates for butyrylcholinesterase.

1. 'Inverse'-type substrates for butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8), i.e., p- and o-nitrophenyl esters of (3-carboxypropyl)-trimethylammonium iodide, (4-carboxybutyl)trimethylammonium iodide and (5-carboxypentyl)trimethylammonium iodide were prepared, and their kinetic parameters for butyrylcholinesterase-catalyzed hydrolysis were determined. 2. The hydrolysis of these 'inverse'-type substrates were found to proceed through specific binding with the enzyme and efficient production of acyl enzyme intermediates, a pathway essentially identical with that followed by choline esters, normal type substrates.

Comparison of Streptomyces griseus and bovine trypsin by active site analysis using fluorescent acyl groups.

The preparation of fluorescence labeled acyl enzymes (Streptomyces griseus trypsin) was successfully carried out using specific trypsin substrates, 'inverse substrates'. The topographical analysis of the structures of the area around the active site was carried out by measuring the fluorescence spectra of the acyl enzyme preparations and these results were compared with those of bovine trypsin. It was found that the polarity of the active site vicinity at pH 5 was similar to that of bovine trypsin, whereas considerable differences were noticed at lower pH as a result of pH-induced transformation. Conformational changes of the active site induced by the interaction with the specific ligand were analyzed from the fluorescence spectra. In these responses the two enzymes were quite distinguishable. The two enzymes active sites were also different in the energy transfer experiments. The spatial arrangements of the catalytic residues relative to the intrinsic tryptophan residues were suggested to be substantially different for the two enzymes.

Identification of novel membrane-bound phospholipase D from Streptoverticillium cinnamoneum, possessing only hydrolytic activity.

A membrane-bound phospholipase D (PLD) has been identified and isolated in a soluble form from an actinomycete, Streptoverticillium cinnamoneum. The enzyme has a monomeric structure with a molecular size of about 37 kDa, being the smallest among the enzymes so far reported. The enzyme catalyzes the hydrolysis of phosphatidylethanolamine and phosphatidylserine as preferred substrates, but not the transphosphatidylation reaction of their phospholipid groups to ethanol. Together with the absence of immunochemical cross-reactivity, these enzymatic properties demonstrate that the membrane-bound enzyme is distinct from the extracellular enzyme recently characterized and cloned from the same bacterial strain [C. Ogino et al., J. Biochem. 125 (1999) 263-269] and is therefore regarded as a novel prokaryotic PLD.

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus.

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates.

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.

Engineered plant phosphorylase showing extraordinarily high affinity for various alpha-glucan molecules.

alpha-Glucan phosphorylases are characterized by considerable difference in substrate specificities, even though the primary structures are well conserved among the enzymes from microorganisms, plants, and animals. The higher plant phosphorylase isozyme designated as type L exhibits low affinity for a large, highly branched glucan (glycogen), presumably due to steric hindrance caused by a unique 78-residue insertion located beside the mouth of the active-site cleft, whereas another isozyme without the insertion (designated as type H) shows very high affinity for both linear and branched glucans. Using the recombinant type L isozyme from potato tuber as a starting framework and aiming at altering its substrate specificity, we have genetically engineered the 78-residue insertion and its flanking regions. Firstly, removal of the insertion and connection of the newly formed C- and N-terminals yielded a totally inactive enzyme, although the protein was produced in Escherichia coli cells in a soluble form. Secondly, a chimeric phosphorylase, in which the 78-residue insertion and its flanking regions are replaced by the corresponding region of the type H isozyme, has been shown to exhibit high affinity for branched glucans (Mori, H., Tanizawa, K., & Fukui, T., 1993, J. Biol. Chem. 268, 5574-5581), but when two and four unconserved residues in the N-terminal flanking region of the chimeric phosphorylase were mutated back to those of the type L isozyme, the resulting mutants showed significantly lowered affinity for substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Probing the pyrophosphate-binding site in potato tuber UDP-glucose pyrophosphorylase with pyridoxal diphosphate.

Potato tuber UDP-glucose pyrophosphorylase (EC 2.7.7.9) catalyzes the reversible uridylyl transfer from UDP-glucose to MgPPi forming glucose 1-phosphate and MgUTP, according to an ordered bi-bi mechanism in which UDP-glucose and MgPPi bind in this order. To probe the active site of this enzyme, we have applied pyridoxal 5'-diphosphate, a reactive PPi analogue. The enzyme was rapidly inactivated when incubated with the reagent in the presence of Mg2+ followed by sodium borohydride reduction. The degree of the inactivation was decreased by MgUTP, MgPPi, and glucose 1-phosphate, but enhanced by UDP-glucose. The enhancement was prevented by co-addition of Pi, the competitive inhibitor with respect to PPi. The complete inactivation corresponded to the incorporation of 0.9-1.1 mol of reagent/mol of enzyme monomer. In the presence of UDP-glucose, labels were almost exclusively incorporated into Lys-329. Thus, this residue may be located near the bound MgPPi and its modification is promoted, probably through conformational changes, by the binding of UDP-glucose to the enzyme. The results of the modification by the same reagent of the mutant enzymes in which Lys-329 and Lys-263 are individually replaced by Gln suggest the roles of these lysyl residues in the binding of MgPPi and in the UDP-glucose-induced conformational changes, respectively.

Anhydrosubtilisin-catalyzed peptide synthesis.

Anhydrosubtilisin was found to be a good catalyst for the peptide synthesis though its hydrolytic activity was fully inhibited. Amino acid (peptide) p-chlorophenyl ester (acyl donor) was coupled with amino acid (peptide) amide in the presence of the modified enzyme. The method did not afford by-products resulting from the hydrolysis of the acyl donor. This is the most advantageous characteristic of the method, since the hydrolysis of the acyl donor is unavoidable for the methods using catalytically active proteases. Anhydrosubtilisin was further shown to be useful for the fragment condensation of peptides.

Differentiation of tryptic enzymes based on enantiomeric specificity at the deacylation step.

The chiral specificity of tryptic enzymes at their deacylation step has been determined for the first time by virtue of 'inverse substrates' carrying optically active acyl groups. Differentiation of tryptic enzymes was also successful with these substrates. The stability of acyl-thrombin is substantially higher than those of trypsin and plasmin when the (S)-dihydrocoumarilyl group is applied. This is in contrast to the result with its (R)-antipode in which all three enzymes are not differentiated. The use of chiral p-amidinophenyl esters is proposed as a versatile methodology for the design of specific inhibitors capable of discriminating among tryptic enzymes.

Site-directed mutagenesis of AMP-binding residues in adenylate kinase. Alteration of substrate specificity.

Adenylate kinase is highly specific for AMP as phosphoryl acceptor. We have found that the replacement of Thr39 by Ala in the chicken muscle enzyme, alone or together with the replacement of Leu66 by Ile, caused remarkable increases in CMP and UMP activities with a concomitant decrease in AMP activity; therefore, the resulting mutant enzymes show CMP and UMP activities/AMP activity ratios much higher than the wild-type enzyme. The mutant enzyme in which Ala is substituted for Thr39 has a Vmax value for CMP comparable to that of CMP-UMP kinase.