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To improve the toughness of epoxy polymers, cross-linked rubber (CLR) particles or core–shell rubber (CSR) particles can be blended into the epoxy matrix. Particle cavitation is thought to be the main toughening mechanism of rubber-modified epoxies. Although CLR-modified epoxies have not been studied in depth previously, it is expected that the toughening mechanism differs from that of CSR-modified epoxies because void formation is less likely to occur. We formed the CLR- and CSR-modified epoxy resins using nanosized acrylonitrile butadiene CLR particles, which showed good compatibility with the epoxy matrix and the CSR particles with a rubbery core surrounded by a glassy shell. The toughening mechanism was examined by comparing the fracture behavior, fracture surfaces, and process zones via tensile and fracture toughness testing, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and transmission optical microscopy (TOM). There was a difference in the characteristics of the R-curves between the CLR- and CSR-modified epoxies. In addition, the SEM and TEM observations indicated that there was a difference in the fracture mechanism.

Purpose. To investigate the repeatability and reproducibility of intraocular pressure (IOP) and central corneal thickness (CCT) measurements using a noncontact tono/pachymeter (NT-530P) and to assess the correlation of CCT with IOP.Methods. Forty-six eyes of healthy volunteers were measured by two examiners. Three consecutive measurements per eye were performed. Repeatability was assessed using the coefficient of variation, and reproducibility was assessed using Bland-Altman plots. Linear correlations were used to determine agreement between CCT and noncorrected IOP and CCT and corrected IOP, which was calculated using a formula built into the NT-530P.Results. The coefficient of variation for IOP was 6.4% and for CCT was 0.4%. The 95% limits of agreement between examiners were−0.17±1.42 mmHg (range:−2.95to 2.61 mmHg) for IOP,−0.93±4.37 μm (range:−9.50to 7.64 μm) for CCT. The corrected IOP was significantly higher than the noncorrected IOP (P=0.010.3). The noncorrected IOP significantly correlated with CCT (r=−0.4883,P=0.0006). The corrected IOP showed no significant correlation with CCT (r=−0.0285,P=0.8509).Conclusions. NT-530P offered repeatability and reproducibility in both IOP and CCT measurements. The corrected IOP calculated using the NT-530P was independent of the CCT, suggesting that this IOP may be less influenced by the central corneal thickness.

Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects of the central nervous system. The complex multigenic causation of human NTDs, together with the large number of possible candidate genes, has hampered efforts to delineate their molecular basis. Function of folate one-carbon metabolism (FOCM) has been implicated as a key determinant of susceptibility to NTDs. The glycine cleavage system (GCS) is a multi-enzyme component of mitochondrial folate metabolism, and GCS-encoding genes therefore represent candidates for involvement in NTDs. To investigate this possibility, we sequenced the coding regions of the GCS genes: AMT, GCSH and GLDC in NTD patients and controls. Two unique non-synonymous changes were identified in the AMT gene that were absent from controls. We also identified a splice acceptor site mutation and five different non-synonymous variants in GLDC, which were found to significantly impair enzymatic activity and represent putative causative mutations. In order to functionally test the requirement for GCS activity in neural tube closure, we generated mice that lack GCS activity, through mutation of AMT. Homozygous Amt(-/-) mice developed NTDs at high frequency. Although these NTDs were not preventable by supplemental folic acid, there was a partial rescue by methionine. Overall, our findings suggest that loss-of-function mutations in GCS genes predispose to NTDs in mice and humans. These data highlight the importance of adequate function of mitochondrial folate metabolism in neural tube closure.

Purpose Recently a combination of paclitaxel and carboplatin (TC) (without an anthracycline) has begun to be used as an adjuvant or remission induction therapy, without any critical supportive evidence of its efficacy relative to a combination chemotherapy of taxane, platinum and anthracycline such as TEC (paclitaxel, epirubicin and carboplatin). The aim of our present study was to conduct the required clinical evaluations of the relative effectiveness of TC compared to TEC. Methods A retrospective comparison between the efficacy of TEC and TC regimens used for endometrial carcinoma at the Osaka University Hospital and the Osaka Medical Center for Cancer and Cardiovascular Diseases in Osaka, Japan, respectively, from 1999 to 2009 was performed. The clinical characteristics of the patients who received either TEC or TC were not significantly different, and TEC and TC therapies were initiated based on similar indications for chemotherapy. TEC regimen was paclitaxel (150 mg/m2), epirubicin (50 mg/m2) and carboplatin (AUC 4). TC regimen consisted of paclitaxel (175 mg/m2) and carboplatin (AUC 5). Results TEC was demonstrated to provide significantly better survival than TC as an adjuvant therapy for resected Stage III/IV diseases (p = 0.017 for progression-free survival and p = 0.014 for overall survival, by the log-rank test). However, in recurrent or more advanced cases, TC and TEC demonstrated similar effects on survival (p = 0.55 for progression-free survival and p = 0.63 for overall survival). Conclusions TEC should be offered as an adjuvant therapy to Stage III/IV patients. TC may be considered for recurrent or unresectable cases as a remission induction therapy.

Aminomethyltransferase, a component of the glycine cleavage system termed T-protein, reversibly catalyzes the degradation of the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein, resulting in the production of ammonia, 5,10-methylenetetrahydrofolate, and dihydrolipoate-bearing H-protein in the presence of tetrahydrofolate. Several mutations in the human T-protein gene are known to cause nonketotic hyperglycinemia. Here, we report the crystal structure of Escherichia coli T-protein in complex with dihydrolipoate-bearing H-protein and 5-methyltetrahydrofolate, a complex mimicking the ternary complex in the reverse reaction. The structure of the complex shows a highly interacting intermolecular interface limited to a small area and the protein-bound dihydrolipoyllysine arm inserted into the active site cavity of the T-protein. Invariant Arg292 of the T-protein is essential for complex assembly. The structure also provides novel insights in understanding the disease-causing mutations, in addition to the disease-related impairment in the cofactor-enzyme interactions reported previously. Furthermore, structural and mutational analyses suggest that the reversible transfer of the methylene group between the lipoate and tetrahydrofolate should proceed through the electron relay-assisted iminium intermediate formation.

Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of α-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 A resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.

This research was originally published in Journal of Biological Chemistry. Kazuko Fujiwara, Sachiko Toma, Kazuko Okamura-Ikeda, Yutaro Motokawa, Atsushi Nakagawa and Hisaaki Taniguchi. Crystal structure of lipoate-protein ligase A from Escherichia coli : Determination of the lipoic acid-binding site. Journal of Biological Chemistry. 2005; 280, 33645-33651. © the American Society for Biochemistry and Molecular Biology., Lipoate-protein ligase A (LplA) catalyzes the formation of lipoyl-AMP from lipoate and ATP and then transfers the lipoyl moiety to a specific lysine residue on the acyltransferase subunit of α-ketoacid dehydrogenase complexes and on H-protein of the glycine cleavage system. The lypoyllysine arm plays a pivotal role in the complexes by shuttling the reaction intermediate and reducing equivalents between the active sites of the components of the complexes. We have determined the X-ray crystal structures of Escherichia coli LplA alone and in a complex with lipoic acid at 2.4 and 2.9 Å resolution, respectively. The structure of LplA consists of a large N-terminal domain and a small C-terminal domain. The structure identifies the substrate binding pocket at the interface between the two domains. Lipoic acid is bound in a hydrophobic cavity in the N-terminal domain through hydrophobic interactions and a weak hydrogen bond between carboxyl group of lipoic acid and the Ser-72 or Arg-140 residue of LplA. No large conformational change was observed in the main chain structure upon the binding of lipoic acid.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the specific lysine residue of the lipoate-dependent enzymes. We have isolated lipoyltransferase I (LipTI) and II (LipTII) from bovine liver (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605–16609). N-terminal amino acid sequences of LipTI and LipTII were identical except that LipTI had an additional Asn residue on the N terminus. We cloned LipTII cDNA from a bovine liver cDNA library. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids including a mitochondrial targeting signal of 26 amino acids. The calculated molecular mass of the mature protein is 39,137 Da. The predicted amino acid sequence showed 35% identity with that of Escherichia coli lipoate-protein ligase A. Northern and Southern blot analyses showed a single band, and a single species of mRNA for lipoyltransferase was found by reverse transcription-polymerase chain reaction. Recombinant LipTII was expressed in E. coli and purified to apparent homogeneity. The K m app values of the recombinant enzyme for lipoyl-AMP and apoH-protein were comparable with those of native LipTII. An antibody raised against recombinant enzyme cross-reacted with LipTI and LipTII in a similar manner. The results suggest that LipTI and LipTII are derived from the same translated product but processed differently.

This research was originally published in Journal of Biological Chemistry. Kazuko Fujiwara, Nobuo Maita, Harumi Hosaka, Kazuko Okamura-Ikeda, Atsushi Nakagawa Hisaaki Taniguchi. Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A. Journal of Biological Chemistry. 2010; 285, 9971-9980. © the American Society for Biochemistry and Molecular Biology., Lipoate-protein ligase A (LplA) catalyzes the attachment of lipoic acid to lipoate-dependent enzymes by a two-step reaction: first the lipoate adenylation reaction and, second, the lipoate transfer reaction. We previously determined the crystal structure of Escherichia coli LplA in its unliganded form and a binary complex with lipoic acid (Fujiwara, K., Toma, S., Okamura- Ikeda, K., Motokawa, Y., Nakagawa, A., and Taniguchi, H. (2005) J Biol. Chem. 280, 33645–33651). Here, we report two new LplA structures, LplAlipoyl-5-AMP and LplAoctyl-5-AMPapoHprotein complexes, which represent the post-lipoate adenylation intermediate state and the pre-lipoate transfer intermediate state, respectively. These structures demonstrate three large scale conformational changes upon completion of the lipoate adenylation reaction: movements of the adenylate-binding and lipoate-binding loops to maintain the lipoyl-5-AMP reaction intermediate and rotation of the C-terminal domain by about 180°. These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys133 residue plays essential roles in both lipoate adenylation and lipoate transfer reactions. Based on structural and kinetic data, we propose a reaction mechanism driven by conformational changes.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.

H-protein of the glycine cleavage system has lipoic acid on the Lys59 residue. Comparison of amino acid sequences around the lipoate attachment site of H-proteins from various sources and acyltransferases of α-keto acid dehydrogenase complexes indicated that Gly43, Glu56, Glu63 and Gly70 of bovine H-protein are highly conserved among these proteins. Modification of these conserved residues by site-directed mutagenesis indicated that Glu56 and Gly70 are important for the lipoylation of H-protein and suggested that the proper conformation around the lipoic acid attachment site is required for the association of H-protein to the enzyme responsible for the lipoylation.

Four new coumarins, named peroxytamarin (1), cis-casegravol (5), citrusarin-A (7), and citrusarin-B (8), were isolated from root of Citrus plants and their structures were elucidated by chemical and spectrometric methods. Citrusarin-B (8), a coumarin having both a dimethylpyran ring and a dihydrofuran ring in the molecule, was also synthesized.

To better understand the molecular mechanisms of carcinogenesis induced in uterine endometrium by therapeutic anti-estrogenic Tamoxifen (TAM) exposure, 27 uterine tumors (4 benign endometrial polyps and 23 carcinomas) associated with TAM exposure were analyzed for the presence and spectrum of p53 and K-ras mutations. Although there was no significant difference between TAM-associated endometrial carcinomas and sporadic endometrial tumors in the frequency of these mutations, the spectrum of p53 mutations was characteristically unique to the TAM-associated tumors. The median duration of TAM exposure was significantly longer in patients with p53 mutations than those without p53 mutations (62 vs. 30 months, p=0.028). Our observation suggests that prolonged TAM exposure may directly inactivate the p53 gene by acting as a mutagen in a significant fraction of TAM-associated endometrial carcinomas.

The first isolation of the unique homoacridone alkaloids, citropone-A (1), -B (2), and -C (3) from the root or stem bark of some cultivated Citrus plants is described. The structure of citropone-A (1) was established unequivocally by spectrometric and single-crystal X-ray analyses, while citropone-B, which has the same skeleton as 1, was assigned structure 2 by comparison of the 1H- and 13C-nuclear magnetic resonance spectra with those of 1. The structure of citropone-C was proposed as formula 3 on the basis of spectrometric analyses using H-C long-range two-dimensional correlation spectroscopy and heteronuclear multiple bond connectivity spectrometries.

In mammals, lipoate-activating enzyme (LAE) catalyzes the activation of lipoate to lipoyl-nucleoside monophosphate. The lipoyl moiety is then transferred to the specific lysine residue of lipoate-dependent enzymes by the action of lipoyltransferase. We purified LAE from bovine liver mitochondria to apparent homogeneity. LAE activated lipoate with GTP at a 1000-fold higher rate than with ATP. The reaction absolutely required lipoate, GTP, and Mg(2+) ion, and the reaction product was lipoyl-GMP. LAE activated both (R)- and (S)-lipoate to the respective lipoyl-GMP, although a preference for (R)-lipoate was observed. Similarly, lipoyltransferase equally transferred both the (R)- and (S)-lipoyl moieties from the respectively activated lipoates to apoH-protein. Interestingly, however, only H-protein carrying (R)-lipoate was active in the glycine cleavage reaction. cDNA clones encoding a precursor LAE with a mitochondrial presequence were isolated. The predicted amino acid sequence of LAE is identical with that of xenobiotic-metabolizing/medium-chain fatty acid:CoA ligase-III, but an amino acid substitution due to a single nucleotide polymorphism was found. These results indicate that the medium-chain acyl-CoA synthetase in mitochondria has a novel function, the activation of lipoate with GTP.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. Our previous work on Escherichia coli T-protein (ET) showed that the lack of the N-terminal 16 residues caused a loss of catalytic activity [Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K. and Motokawa, Y. (1993) Eur. J. Biochem. 216, 539-548]. To define the role of the N-terminal region of ET, a series of deletion mutants were constructed by site-directed mutagenesis and expressed in E. coli. Deletions of the N-terminal 4, 7 and 11 residues led to reduction in the activity to 42, 9 and 4%, respectively, relative to the wild-type enzyme (wtET). The mutant with 7-residue deletion (ETDelta7) was purified and analyzed. ETDelta7 exhibited a marked increase in Km (25-fold) for E. coli H-protein (EH) accompanied by a 10-fold decrease in kcat compared with wtET, indicating the importance of the N-terminal region in the interaction with EH. The role of this region in the ET-EH interaction was investigated by cross-linking of wtET-EH or ETDelta7-EH complex with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker, in the presence of folate substrates. The resulting tripartite cross-linked products were cleaved with lysylendopeptidase and V8 protease. After purification by reversed-phase HPLC, the cross-linked peptides were subjected to Edman sequencing. An intramolecular cross-linking between Asp34 and Lys216 of wtET which was not observed in wtET alone and an intermolecular cross-linking between Lys288 of wtET and Asp-43 of EH were identified. In contrast, no such cross-linking was detected from the cross-linked product of ETDelta7. These results suggest that EH, when it interacts with ET, causes a change in conformation of ET and that the N-terminal region of ET is essential for the conformational change leading to the proper interaction with EH.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.

OBJECTIVE: This study was conducted to ascertain the correlation between preserved pelvic nerve networks and bladder function after laparoscopic nerve-sparing radical hysterectomy. METHODS: Between 2009 and 2011, 53 patients underwent total laparoscopic radical hysterectomies. They were categorized into groups A, B, and C based on the status of preserved pelvic nerve networks: complete preservation of the pelvic nerve plexus (group A, 27 cases); partial preservation (group B, 13 cases); and complete sacrifice (group C, 13 cases). To evaluate bladder function, urodynamic studies were conducted preoperatively and postoperatively at 1, 3, 6, and 12 months after surgery. RESULTS: No significant difference in sensory function was found between groups A and B. However, the sensory function of group C was significantly lower than that of the other groups. Group A had significantly better motor function than groups B and C. No significant difference in motor function was found between groups B and C. Results showed that the sensory nerve is distributed predominantly at the dorsal half of the pelvic nerve networks, but the motor nerve is predominantly distributed at the ventral half. CONCLUSION: Various types of total laparoscopic nerve-sparing radical hysterectomies can be tailored to patients with cervical carcinomas.

H-protein of the glycine cleavage system has a lipoic acid prosthetic group. Selenolipoic acid is a lipoic acid analog in which both sulfur atoms are replaced by selenium atoms. Two isoforms of bovine lipoyltransferase that are responsible for the attachment of lipoic acid to H-protein had an affinity for selenolipoyl-AMP and transferred the selenolipoyl moiety to bovine apoH-protein comparable to lipoyl-AMP. Selenolipoylated H-protein was overexpressed inEscherichia coli and purified. Selenolipoylated H-protein was 26% as effective as lipoylated H-protein in the glycine decarboxylation reaction, in which reduction of the diselenide bond of selenolipoylated H-protein is catalyzed by P-protein. The diselenide form of selenolipoylated H-protein was a poor substrate for L-protein, and the rate of reduction was 0.5% of that of lipoylated H-protein. The rate of the overall glycine cleavage reaction with selenolipoylated H-protein was

Lipoic acid is a prosthetic group of the acyltransferase components of the pyruvate, alpha-ketoglutarate, and branched chain alpha-ketoacid dehydrogenase complexes, protein X of the eukaryotic pyruvate dehydrogenase complex, and H-protein of the glycine cleavage system. We have purified lipoyl-AMP:Nepsilon-lysine lipoyltransferase I and II from bovine liver mitochondria employing apoH-protein as an acceptor of lipoic acid (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1994) J. Biol. Chem. 269, 16605-16609). In this study, we demonstrated the lipoylation of the lipoyl domains of the mammalian pyruvate (LE2p), alpha-ketoglutarate (LE2k), and branched chain alpha-keto acid (LE2b) dehydrogenase complexes using the purified lipoyltransferase I and II. Lipoyltransferase I and II lipoylated LE2p and LE2k as efficiently as H-protein, but the lipoylation rate of LE2b was extremely low. Comparison of amino acid sequences surrounding the lipoylation site of these proteins shows that the conserved glutamic acid residue situated 3 residues to the N-terminal side of the lipoylation site is replaced by glutamine (Gln-41) in LE2b. When Gln-41 of LE2b was changed to Glu, the rate of lipoylation increased about 100-fold and became comparable to that of LE2p and LE2k. The replacement of the glutamic acid residue of LE2p (Glu-169) and LE2k (Glu-40) by glutamine resulted in decrease in the lipoylation rate more than 100-fold. These results suggest that the glutamic acid residue plays an important role in the lipoylation reaction possibly functioning as a recognition signal. Gly-27 and Gly-54 of LE2k are also well conserved among the lipoyl domains of the alpha-ketoacid dehydrogenase complexes and H-protein. The mutagenesis experiments of these residues indicated that the glycine residue situated 11 residues to the C-terminal side of the lipoylation site (Gly-54 of LE2k) is important for the folding of lipoyl domain, and that existence of a small residue such as Gly or Cys at the position is essential for the lipoylation of these proteins.

Fibrinogen-related peptides, monomeric cyclic peptides through a disulfide bond [cyclo(H-Cys-Arg-Gly-Asp-Phe-Cys-NH2), cyclo(H-Cys-Arg-Gly-Asp-Phe-Cys-Gly-NH2), cyclo(H-Cys-Arg-Gly-Asp-Cys-NH2) and cyclo(H-Cys-Arg-Gly-Asp-Cys-Gly-NH2)], were prepared by the solid phase method with disulfide bond formation on the solid support. The acetamidomethyl group was used for protection of the thiol group of Cys and synthetic peptide-resins were treated with iodine to give the disulfide bond. Monomeric cyclic peptides were obtained as main products. Purified S-acetamidomethylated peptides were also oxidized with iodine, but the desired materials could not be isolated by HPLC. The disulfide formation from S-acetamidomethylcysteine-containing peptide resin by iodine treatment on the solid support was more effective than that from S-acetamidomethylcysteine-containing peptide. The inhibitory effect of the cyclic peptides on platelet aggregation were much more potent than that of H-Arg-Gly-Asp-NH2.

Lipoyl-AMP:N epsilon-lysine lipolytransferase (lipolytransferase) catalyzes the transfer of the lipoyl group from lipoyl-AMP to a lysine residue of the specific enzyme proteins. We have shown previously that the lipoyltransferase activities locate in mitochondria using apoH-protein of the glycine cleavage system as an acceptor of the lipoyl group (Fujiwara, K., Okamura-Ikeda, K., and Motokawa, Y. (1990) J. Biol. Chem. 265, 17463-17467). Here we describe the purification and the characterization of two isoforms of lipolytransferase termed lipoyltransferase I and lipoyltransferase II from bovine liver mitochondria. Lipoyltransferase II was purified to apparent homogeneity, whereas the final product of lipoyltransferase I still contained a minor contaminant. Although the two forms could be resolved on a hydroxylapatite column chromatography, they were indistinguishable, as judged by: (a) behavior during purification on ion exchange, hydrophobic, or affinity columns; (b) molecular mass determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography (40 kDa); and (c) catalytic properties (substrate specificity, kinetic constants, and optimal pH). Both lipoyltransferase I and II could not use lipoic acid plus MgATP as a substrate in place of lipoyl-AMP. Surprisingly, the lipoyltransferases transferred not only the lipoyl group but also the acyl groups from hexanoyl-, octanoyl-, and decanoyl-AMP to apoH-protein to a similar extent.

P-protein, H-protein and T-protein of the glycine cleavage system have been purified from Escherichia coli. Their N-terminal amino acid sequences were determined, and a set of oligonucleotide probes was designed for gene cloning. The nucleotide sequence of a fragment of DNA around the 62-min region of the E. coli chromosome, containing genes for the components of the glycine-cleavage system has been determined. The sequence includes three structural genes encoding T-protein (363 amino acids, 40013 Da), H-protein (128 amino acids, 13679 Da) and P-protein (956 amino acids, 104240 Da). These genes are named gcvT, gcvH and gcvP, respectively. They are organized in the above-mentioned order on the same strand of DNA with short intercistronic sequences. The presence of a potential promoter preceding gcvT and a typical rho-independent terminator sequence following gcvP indicated that the three genes constitute a single operon. Each component of the E. coli glycine-cleavage system exhibits considerable amino acid sequence similarity with the animal and plant counterparts. When the plasmid containing the gcv operon was transfected in E. coli cells, the gene products of gcvT, gcvH and gcvP were overexpressed under the direction of the promoter of the gcv operon. However, bacteria harboring the plasmid that contained the gcv operon without the promoter region and the 5' terminal portion of gcvT failed to overexpress any of the three components.

We analyzed the clonality and human papillomavirus (HPV) infection status of concurrent glandular and squamous lesions and adenosquamous carcinomas of the uterine cervix to clarify their histogenesis. The glandular and squamous components were clonally different from each other in 7 informative concurrent lesions. HPV was episomal in 2 polyclonal glandular dysplasias (GDs). HPV was in a mixed integrated-episomal form in a monoclonal GD, an adenocarcinoma in situ, and an adenocarcinoma. Both tumor components were monoclonal in origin in 6 adenosquamous carcinomas, with identical patterns of X-chromosomal inactivation and types and physical status of HPV. These results imply that the concurrent glandular and squamous lesions are formed separately, whereas adenosquamous carcinoma is more likely to be a combination tumor of monoclonal origin, and that integration of HPV has an important role in the progression from polyclonal GD through monoclonal expansion to adenocarcinoma in situ and adenocarcinoma.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. A partial cDNA for chicken T-protein was isolated by screening a chicken liver cDNA library with oligonucleotide probes based on amino acid sequences. The clone (CT5C) contains a 675-base pair insert encoding the C-terminal half of the T-protein. Screening of a bovine liver lambda gt10 cDNA library with the insert of CT5C as a probe detected a clone, BT5A, with an insert of about 2 kilobase pairs. The 1191-base pair coding region encodes a precursor protein of 397 amino acids (Mr 42,852) comprised of a 22-residue mitochondrial targeting sequence and a 375-residue mature protein (Mr 40,534). The 33 amino acids immediately following the targeting sequence correspond exactly to those determined by sequence analysis of the amino terminus of the purified bovine T-protein. The mature protein contains several hydrophilic segments with a cluster of arginine and lysine. The T-protein cDNA probe hybridized to an mRNA species of about 2 kilobases in bovine brain, lung, heart, and liver. A probe for H-protein hybridized with two species of mRNA in these tissues and weak signals were also found in spleen. Although the enzymatic activities of T-protein and H-protein were found in these tissues where transcripts were found, activity of P-protein was detected only in liver and brain. Southern blot analysis of genomic DNA suggested that T-protein is a single copy gene.

H-protein is a component of the glycine cleavage system loosely associated with the mitochondrial inner membrane and has lipoic acid as a prosthetic group. cDNA clones encoding H-protein were isolated from a bovine liver cDNA library with an oligonucleotide probe from the amino acid sequence of the NH2-terminal region. DNA sequence analysis and deduced amino acid sequence showed that the cDNAs encoded an H-protein precursor of 173 amino acids including a 48-amino acid presequence. Calculated molecular mass of the precursor and mature protein without lipoic acid were 18,790 and 13,846, respectively. Northern blot analysis indicated a major mRNA component of 1.1 kilobases and a minor component of 0.6 kilobase. The result is consistent with the presence of two adenylation signals in the 3'-untranslated region of the cDNA. In vitro transcription and translation of the H-protein cDNA produced a 19-kDa protein recognized by antibody raised to chicken H-protein. Bovine liver H-protein precursor has no lipoic acid prosthetic group. When incubated with isolated bovine liver mitochondria, the precursor was imported into mitochondria, processed to its mature form with a molecular mass of 14 kDa, and lipoylated at lysine 59. These results indicate that lipoylation is not required for the import of H-protein precursor into mitochondria and H-protein is lipoylated in mitochondria which probably contain the physiologically active form of lipoic acid as well as the enzyme(s) responsible for the attachment.

The complete amino acid sequence of H-protein from chicken liver was determined by aligning peptides obtained by cyanogen bromide, endoproteinase Lys-C, Staphylococcus aureus V8 protease, and chymotrypsin cleavage together with the partial NH2- and COOH-terminal sequence of the intact protein. H-protein consists of 125 amino acids and a lipoic acid moiety linked to lysine 59. The sequence is: (sequence in text). The lysyl residue involved in lipoic acid attachment is indicated with an asterisk. The molecular weight including lipoic acid is calculated to be 13,883. From the secondary structure predicted by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148) the lipoic acid binding region shows alpha-helical structure and is predicted to be an interior portion of the protein from the hydropathic profile according to Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132).

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.

T-protein, a component of the glycine cleavage system, has been purified to apparent homogeneity from chicken liver mitochondria. The molecular weight was 37,000 by sedimentation equilibrium centrifugation and 38,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a molecular weight of 41,000, indicating that the protein is composed of a single polypeptide. A partial specific volume of 0.73 ml/g was obtained from the amino acid composition. The absorbance coefficient, A1%280nm, is 6.63 in 50 mM potassium phosphate buffer, pH 7.0. T-protein is a basic protein having a pI value of 9.8 and relatively rich in arginine rather than lysine. The protein catalyzed the degradation of the protein-bound intermediate (-CH2NH2 moiety of glycine) to a 1-carbon unit and NH3. The reaction is dependent on tetrahydrofolate (H4-folate). Apparent Km values for the intermediate and H4-folate were 3.7 microM and 0.17 mM, respectively. T-protein associated with H-protein forming a complex which was composed of one molecule each of T-protein and H-protein. Formation of the complex was not affected by the modification of the cysteinyl residues on H-protein with N-ethylmaleimide.

Glycine is converted to carbon dioxide and an intermediate attached to H-protein in the P-protein-catalyzed partial reaction of the glycine cleavage reaction (Fujiwara, K., and Motokawa, Y. (1983) J. Biol. Chem. 258, 8156-8162). The studies presented in this communication indicate that the amino group of glycine is retained in the intermediate and released as ammonia in the second partial reaction catalyzed by T-protein. The formation of ammonia accompanies the stoichiometric formation of 5,10-methylenetetrahydrofolate from the methylene carbon of glycine and tetrahydrofolate. Kinetic studies show that the reaction proceeds through a sequential mechanism. Km values for the intermediate complex and tetrahydrofolate are 2.2 and 50 microM, respectively. In the absence of tetrahydrofolate, T-protein catalyzes the stoichiometric formation of ammonia and formaldehyde from the intermediate although the velocity is extremely low. Km value for the intermediate complex in the absence of tetrahydrofolate is 10.3 microM, about 4-fold higher than the value in the presence of tetrahydrofolate. The addition of tetrahydrofolate increased the rate about 2400-fold. The modification of the free lipoyl sulfhydryl group with N-ethylmaleimide caused the intermediate complex inactive. The lipoyl sulfhydryl group seems to be essential for both P-protein- and T-protein-catalyzed partial reactions.