Site-directed mutagenesis and spectroscopic studies of the iron-binding site of (S)-2-hydroxypropylphosphonic acid epoxidase.

(S)-2-Hydroxylpropanylphosphonic acid epoxidase (HppE) is a novel type of mononuclear non-heme iron-dependent enzyme that catalyzes the O2 coupled, oxidative epoxide ring closure of HPP to form fosfomycin, which is a clinically useful antibiotic. Sequence alignment of the only two known HppE sequences led to the speculation that the conserved residues His138, Glu142, and His180 are the metal binding ligands of the Streptomyces wedmorensis enzyme. Substitution of these residues with alanine resulted in significant reduction of metal binding affinity, as indicated by EPR analysis of the enzyme-Fe(II)-substrate-nitrosyl complex and the spectral properties of the Cu(II)-reconstituted mutant proteins.

Kinetic and spectroscopic characterization of ACMSD from Pseudomonas fluorescens reveals a pentacoordinate mononuclear metallocofactor.

The enzyme alpha-amino-beta-carboxy-muconic-epsilon-semialdehyde decarboxylase (ACMSD) plays an important role in the biodegradation of 2-nitrobenzoic acid in microorganisms and in tryptophan catabolism in humans. We report that the overexpressed ACMSD enzyme from Pseudomonas fluorescens requires a divalent metal, such as Co(II), Fe(II), Cd(II), or Mn(II), for catalytic activity and that neither a redox reagent nor an organic cofactor is required for the catalytic function. The metal ions can be taken up in either cell or cell-free preparations for generating the active form of ACMSD.

The treatment of relapsing primary nephrotic syndrome in children.

Objective: To explore better therapy and reduce the rate of re-relapse of primary nephritic syndrome in children who had been treated with corticosteroids but relapsed.

Methods: Eighty relapsers were enrolled from Jan. 1994 to Apr.

Mouse intraflagellar transport proteins regulate both the activator and repressor functions of Gli transcription factors.

Intraflagellar transport (IFT) is an active event in which cargo is transported along microtubules by motor proteins such as kinesin and dynein. IFT proteins are required for the formation and maintenance of flagella and cilia. We have previously shown that mouse mutants for two IFT proteins, IFT88 and IFT172, as well as Kif3a, a subunit of mouse kinesin 2, exhibit ventral spinal cord patterning defects that appear to result from reduced hedgehog (Hh) signaling.

The mechanism of inactivation of 3-hydroxyanthranilate-3,4-dioxygenase by 4-chloro-3-hydroxyanthranilate.

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders.

[Molecular mapping of a bacterial blight resistance gene Xa-25 in rice].

Xa-25 was a bacterial blight resistance gene identified in a somaclonal mutant HX-3. A doubled-haploid (DH) population including 129 stable lines was derived from anther culture of a typical japanica 02428 and a typical indica HX-3 cross. The bacterial blight strain Zhe173, a typical bacterial blight strain in Yangtze River valley, was used to test the resistance or susceptible of the DH population lines, and the results showed that the resistance lines and susceptible lines were 62 and 67, respectively.

Enzymatic mechanism of Fe-only hydrogenase: density functional study on H-H making/breaking at the diiron cluster with concerted proton and electron transfers.

The mechanism of the enzymatic hydrogen bond forming/breaking (2H(+) + 2e<==>H(2)) and the plausible charge and spin states of the catalytic diiron subcluster [FeFe](H) of the H cluster in Fe-only hydrogenases are probed computationally by the density functional theory. It is found that the active center [FeFe](H) can be rationally simulated as [[H](CH(3)S)(CO)(CN(-))Fe(p)(CO(b))(mu-SRS)Fe(d)(CO)(CN(-))L], where the monovalence [H] stands for the [4Fe4S](H)(2+) subcluster bridged to the [FeFe](H) moiety, (CH(3)S) represents a Cys-S, and (CO(b)) represents a bridging CO. L could be a CO, H(2)O, H(-), H(2), or a vacant coordination site on Fe(d).

Deciduoid mesothelioma in the pelvic cavity.

A very rare case of deciduoid mesothelioma in the pelvic cavity is presented. A 24-year-old woman (gestational stage: 28 weeks and 6 days) was admitted because of a tumor mass in the abdominal cavity. A well-circumscribed and fibrously encapsulated tumor mass was revealed in the Douglas cavity.

Hedgehog signalling in the mouse requires intraflagellar transport proteins.

Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88.

FGF17b and FGF18 have different midbrain regulatory properties from FGF8b or activated FGF receptors.

Early patterning of the vertebrate midbrain and cerebellum is regulated by a mid/hindbrain organizer that produces three fibroblast growth factors (FGF8, FGF17 and FGF18). The mechanism by which each FGF contributes to patterning the midbrain, and induces a cerebellum in rhombomere 1 (r1) is not clear. We and others have found that FGF8b can transform the midbrain into a cerebellum fate, whereas FGF8a can promote midbrain development.

Biochemical and spectroscopic studies on (S)-2-hydroxypropylphosphonic acid epoxidase: a novel mononuclear non-heme iron enzyme.

The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase.

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions.

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans.

Human lens thioredoxin: molecular cloning and functional characterization.

Purpose: To molecularly clone the human lens thioredoxin (TXN) gene, characterize the recombinant protein (rTrx1) and study the regulation expression of thioredoxin (Trx1) in human lens epithelial cells under oxidative stress.

Methods: The human TXN gene was cloned from a human lens cDNA library. Trx1 activity was measured by insulin reduction assay.

MauG, a novel diheme protein required for tryptophan tryptophylquinone biogenesis.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH.

Interconversion of two oxidized forms of taurine/alpha-ketoglutarate dioxygenase, a non-heme iron hydroxylase: evidence for bicarbonate binding.

Taurinealpha-ketoglutarate (alphaKG) dioxygenase, or TauD, is a mononuclear non-heme iron hydroxylase that couples the oxidative decarboxylation of alphaKG to the decomposition of taurine, forming sulfite and aminoacetaldehyde. Prior studies revealed that taurine-free TauD catalyzes an O(2)- and alphaKG-dependent self-hydroxylation reaction involving Tyr-73, yielding an Fe(III)-catecholate chromophore with a lambda(max) of 550 nm. Here, a chromophore (lambda(max) 720 nm) is described and shown to arise from O(2)-dependent self-hydroxylation of TauD in the absence of alphaKG, but requiring the product succinate.

O2- and alpha-ketoglutarate-dependent tyrosyl radical formation in TauD, an alpha-keto acid-dependent non-heme iron dioxygenase.

Taurine/alpha-ketoglutarate dioxygenase (TauD), a non-heme mononuclear Fe(II) oxygenase, liberates sulfite from taurine in a reaction that requires the oxidative decarboxylation of alpha-ketoglutarate (alphaKG). The lilac-colored alphaKG-Fe(II)TauD complex (lambda(max) = 530 nm; epsilon(530) = 140 M(-)(1) x cm(-)(1)) reacts with O(2) in the absence of added taurine to generate a transient yellow species (lambda(max) = 408 nm, minimum of 1,600 M(-)(1) x cm(-)(1)), with apparent first-order rate constants for formation and decay of approximately 0.25 s(-)(1) and approximately 0.

Clinical study on labor pain relief using the combined spinal-epidural analgesia and inhaling nitrous oxide.

Objective: To study the pain relief effectiveness of the combined spinal-epidural analgesia (CSEA) and the inhalation of nitrous oxide, and the influences on the mothers and infants.

Methods: The 300 cases of pregnant women were randomly divided into 3 groups: CSEA group, nitrous oxide group and control group. The nitrous oxide group was that pregnant women inhaled nitrous oxide premixed with oxygen (50%:50%), the pregnant women of the CSEA group were injected fentanyl and bupivacaine in the subarachnoid and epidural space, analgesic was not used in the control group.

Cloning and Expression of aroG Gene of E. coli and Its Co-expression with pheA and tyrB Genes.

3-Deoxy-D-arabino-heptulonate-7-phosphate synthetase (DAHP) is one of the key enzymes in phenylalanine biosynthesis pathway. In E. coli, DAHP is encoded by aroG Gene.

[Reversing malignant phenotypes of liver cancer cell lines with antisense gene to human telomerase reverse transcriptase].

Objective: To explore the effect of antisense gene to human telomerase reverse transcriptase (hTRT) on reversing malignant phenotypes of liver cancer cell lines.

Methods: Sense and antisense eukaryotic expressing vector of hTRT gene was transfected into human liver cancer line HepG(2) with the DOTAP liposomal transfection method. Changes of cellular malignant phenotypes through proliferation capacity, telomerase activity, cloning formation in soft agar, invasive capacity in Borden's chamber model and tumorigenicity in nude mice were examined.