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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.

In order to reinforce the composite consisting of isoprene rubber (IR) and calcium carbonate (CaCO3) particles, the surface treatment of CaCO3 particles with a mixture of amino- and mercapto-functi...

The interfacial layer structure of a model incompatible polymer blend system was analyzed using 1H pulse nuclear magnetic resonance (pulse NMR) spectroscopy. Non-crosslinked and crosslinked poly(n-butyl methacrylate) particles with a mean size of ca. 0.9 μm were prepared by seeded emulsion polymerization, and the degree of crosslinking was varied. The particles were powdered using a freeze-dry method and dispersed in poly(vinyl chloride) by melt blending. Dynamic mechanical analysis indicated that the non-crosslinked particles were completely compatible. In contrast, mutual diffusion of the polymer chains in the crosslinked particles was restricted within the particle/matrix interfacial layer. As a result, an incompatible phase structure in which the crosslinked particles were dispersed in the continuous phase was formed. Pulse NMR analysis indicated that the interfacial layer thickness was in the range of 17–98 nm. The thickness decreased with an increase in the degree of crosslinking in the part...

Colloidal aqueous dispersions of poly(n-butyl acrylate) (PBA)-silica (SiO2) nanocomposite particles were synthesized through free radical polymerization in the presence of SiO2 nanoparticles and poly(N-vinyl pyrrolidone) colloidal stabilizer using a cationic radical initiator. The nanocomposite particles were stable colloids with a reasonably narrow size distribution and a SiO2 content of 40%. Morphology of the nanocomposite particles was confirmed to involve a PBA-core/SiO2 nanoparticle-shell using electron microscopies. Pressure-sensitive adhesive (PSA) films were prepared from blends of the nanocomposite particles and PBA homopolymer particles at various weight ratios. Probe tack tests at the standard probe rate, which is close to the actual peeling speed for PSAs, confirmed that maximum stress of tack adhesion improved 2.36 times and adhesion energy improved 1.39 times upon addition of nanocomposite particles compared to that of PBA homopolymer film. The nano-scale mechanical deformation and energy dissipation ability of the ‘soft-hard’ nanocomposite particles was expected to increase adhesion energy.

Objective To assess the obstetric outcomes of our total laparoscopic radical trachelectomy (TLRT) cases for early stage cervical cancer. Materials and methods A total of 56 patients who underwent TLRT between December 2001 and August 2012 were reviewed retrospectively using clinicopathological, surgical, and follow-up data from patients' medical records. Results We performed this operation on 56 patients during the study period. The mean age of these 56 patients was 31.9years (range 22–42years). Fifty-three patients' fertility was preserved without requiring post-operative adjuvant treatment. Twenty-five women attempted to conceive, of whom 13 succeeded for a total of 21 pregnancies (52% pregnancy rate). Ten of these 21 pregnancies were the result of assisted reproductive technologies. Of those, 5 resulted in first trimester miscarriages, 2 in second trimester miscarriages, and 13 in live births. Ten pregnancies reached the third trimester. Preterm premature rupture of membranes (8/13, 61.5%) was the most common complication during pregnancy. The rate of preterm delivery was 47.6%. Three patients delivered at 22–28weeks of gestational age. Two of these babies showed permanent damage: one has cerebral palsy; the other has developmental retardation. One pregnancy is ongoing. Conclusion TLRT is a useful technique associated with an excellent pregnancy rate in fertility-preserving surgery to treat early stage cervical cancer.

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.

The influence of diblock copolymer addition on the tack properties of a polyacrylic triblock copolymer/tackifier system was investigated. For this purpose, poly(methyl methacrylate)-block-poly(n-butyl acrylate)-block-poly(methyl methacrylate) triblock copolymer (MAM) and a 1/1 blend with a diblock copolymer consisting of the same components (MA) were used as base polymers, and a tackifier was added in amounts ranging from 10 to 30 wt %. The temperature dependence of tack was measured by a probe tack test. The tack of MAM/MA at room temperature was significantly higher than that of MAM, and the improvement of MAM/MA upon the addition of the tackifier was higher than that of MAM. The peeling process at the probe/adhesive interface during the probe tack test was observed using a high-speed microscope. It was found that for MAM/MA, cavitation was caused in the entire adhesive layer, and peeling initiation was delayed by the absorption of strain energy due to deformation of the adhesive layer. In contrast, for MAM, peeling progressed linearly from the edge to the center of the probe. The greater flexibility of the soft block chain in the diblock copolymer resulted in improved interfacial adhesion. 1H pulse nuclear magnetic resonance analysis showed that the addition of the tackifier improved the cohesive strength of the adhesive. Adhesion strength is affected by two factors: the development of interfacial adhesion and cohesive strength. In the MAM/MA/tackifier system, the presence of MA and the tackifier improved the interfacial adhesion and cohesive strength, respectively. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Aim: The aim of this study was to determine the incidence of perioperative complications and evaluate risk factors for the major complications of total laparoscopic hysterectomy (TLH) using an early ureteral identification technique. We describe the technique we standardized and used for TLH, without exclusion criteria. Material and Methods: A retrospective study was carried out at Kurashiki Medical Center, Japan, based on 1253 TLH procedures performed from January 2005 to March 2009. We reviewed records to identify the major perioperative complications, including bladder, ureteral, and intestinal injuries, and incidences of reoperation. Risk factors for major complications were analyzed using multivariate logistic regression models. Results: A total of 24 patients encountered major complications (1.91%). Complications included 10 intraoperative urologic injuries, five cases of postoperative hydronephrosis, five cases of vaginal dehiscence, one bowel injury, one postoperative hemorrhage, one bowel obstruction, and one ureterovaginal fistula. All 11 cases of intraoperative visceral injury were recognized during the surgery and repaired during the same laparoscopic surgical procedure. Of the risk factors analyzed, a history of abdominal surgery was the only one associated with the occurrence of major complications, with an odds ratio of 2.48 (95% confidence interval 1.23–6.49). Conclusion: While complications are inevitable, even in the hands of the most skilled surgeon, they can be minimized without conversion to laparotomy by a sufficiently developed suturing technique and a precise knowledge of pelvic anatomy. The presented data indicate that our method allows for safe TLH and minimization of ureteral injury, without the use of stringent exclusion criteria.

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.

PIK3CA codes for a Class IA p110-alpha catalytic subunit of the PI3Ks (phosphatidylinositol 3-kinases) that regulate various signaling pathways important for neoplasia, including cell proliferation, motility, adhesion, and survival. Pro-oncogenic mutations in exons 9 and 20 of the PIK3CA gene have been frequently observed in numerous types of human malignancies. Amplification of the PIK3CA gene has been reported in uterine cervical cancers. In this study, we have done in depth analysis of uterine cervical and endometrial cancers for PIK3CA gene mutations and amplifications. In uterine cervical cancers, PIK3CA mutations were found in 3 of 22 cases (14%), all of them in exon 9. In endometrial cancers, a similar incidence of mutations was found, in 3 of 29 cases (10%), however they were all within exon 20. Amplification of the PIK3CA gene was also detected in 2 out of 22 (9%) cervical cancers and 3 out of 29 (10%) endometrial cancers. In this study, we were unable to find a clear association between PIK3CA mutations and gene amplifications, nor with tumor histological subtypes or staging. Mutations and amplifications of the PIK3CA gene are relatively infrequent in human cervical and endometrial cancers; however, PIK3CA gene alteration may still play a role in some subset of uterine cancers.

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.

Surgery and radiotherapy are both regarded as standard treatments for occult cervical cancers. Surgery has several theoretical advantages over radiotherapy; therefore, such cancers, especially in their early stages, are commonly treated with radical parametrectomy. However, postoperative bladder dysfunction is an important potential complication of this type of surgery. This is a case report of total laparoscopic nerve-sparing radical parametrectomy for an occult cervical cancer using our original surgical concept based on detailed anatomical investigation of pelvic nerve networks in a fresh cadaver. We evaluated the validity of our nerve-sparing technique by assessing postoperative bladder function using urodynamic studies.

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.

Endometriosis has been reported as a major cause of pelvic pain. Most notably, deep infiltrating endometriosis is a very active disease that occurs in 20 % of women with endometriosis. We have been actively dissecting deep infiltrating diseased areas within the sacral ligaments around the uterus in order to improve dysmenorrhea and chronic pelvic pain caused by deep infiltrating endometriosis. Laparoscopic surgery is an ideal option to treat deep infiltrating endometriosis involving complete cul-de-sac obliteration due to its minimal invasiveness and ability to achieve an appropriate depth of surgical field. It is important to prevent pain recurrence by providing systematic surgery and removing the deep infiltrating endometriosis safely and widely. To reduce recurrence, it is ideal to provide postoperative education to maintain the effect of surgery.

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.

To investigate clinically the effects of yeast-fermented bilberry extract on visual outcomes in myopic eyes.In a prospective, randomized, placebo-controlled, cross-over study, we examined 30 eyes of 30 middle-aged healthy volunteers (mean age±standard deviation, 39.5±7.2 years) with myopia [manifest spherical equivalent, -2.40±1.88 diopters (D)], who were randomly assigned to 1 of 2 oral regimens: fermented bilberry extract (400 mg/day) or placebo. We quantitatively assessed visual acuity, refraction, pupil constriction rate, accommodation, and mesopic contrast sensitivity (CS), before and 1 month after treatment. Only the right eyes were tested. The amplitude of accommodation and CS were measured with an accommodometer (D'ACOMO; WOC) and a CS unit (VCTS-6500; Vistech), respectively. From the CS, the area under the log contrast sensitivity function (AULCSF) was calculated.The mean amplitude of accommodation increased significantly, from 4.62±1.88 D before treatment, to 5.33±2.03 D after treatment in the study group (Wilcoxon signed-rank test, P=0.002). Moreover, the mesopic AULCSF was significantly increased, from 1.04±0.16 before, to 1.13±0.17 after, treatment (P=0.009). However, we found no significant changes in accommodation or AULCSF in the control group (P0.05), or any significant changes in any other parameters in either group (P0.05).The present data show that fermented bilberry extract is effective in causing increases in subjective accommodation and in mesopic CS in myopic eyes.

The aim of this study was to determine the incidence of perioperative complications and evaluate risk factors for the major complications of total laparoscopic hysterectomy (TLH) using an early ureteral identification technique. We describe the technique we standardized and used for TLH, without exclusion criteria.A retrospective study was carried out at Kurashiki Medical Center, Japan, based on 1253 TLH procedures performed from January 2005 to March 2009. We reviewed records to identify the major perioperative complications, including bladder, ureteral, and intestinal injuries, and incidences of reoperation. Risk factors for major complications were analyzed using multivariate logistic regression models.A total of 24 patients encountered major complications (1.91%). Complications included 10 intraoperative urologic injuries, five cases of postoperative hydronephrosis, five cases of vaginal dehiscence, one bowel injury, one postoperative hemorrhage, one bowel obstruction, and one ureterovaginal fistula. All 11 cases of intraoperative visceral injury were recognized during the surgery and repaired during the same laparoscopic surgical procedure. Of the risk factors analyzed, a history of abdominal surgery was the only one associated with the occurrence of major complications, with an odds ratio of 2.48 (95% confidence interval 1.23-6.49).While complications are inevitable, even in the hands of the most skilled surgeon, they can be minimized without conversion to laparotomy by a sufficiently developed suturing technique and a precise knowledge of pelvic anatomy. The presented data indicate that our method allows for safe TLH and minimization of ureteral injury, without the use of stringent exclusion criteria.

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.

Recent technical improvements in macromolecular X-ray crystallography have significantly improved the resolution limit of protein structures. However, examples of high-resolution structure determination are still limited. In this study, the X-ray crystal structure of bovine H-protein, a component of the glycine cleavage system, was determined at 0.88 Å resolution. This is the first ultrahigh-resolution structure of an H-protein. The data were collected using synchrotron radiation. Because of limitations of the hardware, especially the dynamic range of the CCD detector, three data sets (high-, medium- and low-resolution data sets) were measured in order to obtain a complete set of data. To improve the quality of the merged data, the reference data set was optimized for merging and the merged data were assessed by comparing merging statistics andRfactors against the final model and the number of visualized H atoms. In addition, the advantages of merging three data sets were evaluated. The omission of low-resolution reflections had an adverse effect on visualization of H atoms in hydrogen-omit maps. Visualization of hydrogen electron density is a good indicator for assessing the quality of high-resolution X-ray diffraction data.

Cervical intraepithelial neoplasia (CIN) has its highest incidence during women’s reproductive years. During 2 sequential 7-year periods, 1994 to 2000 and 2001 to 2007, 3695 and 3894 deliveries were performed, respectively, at Osaka University Hospital. CIN was detected in 21 cases (0.57%) during 1994–2000 and in 43 cases (1.1%) during 2001–2007. By comparison, cervical intraepithelial neoplasia—complicated pregnancies increased significantly in the latter period (P = .015 by Fisher exact test, Odds ratio = 1.95; 95% CI: 1.16–3.30). We observed CIN regression in 34 (76%) of 45 cases of vaginal delivery and in 6 (50%) of 12 cases of cesarean delivery, indicating that the outcome of an initially diagnosed CIN and the delivery routes appeared not to be significantly related. However, a different result was obtained when only those patients whose CIN lesions persisted until the delivery were analyzed. Among the 35 such cases in the vaginal delivery group, 24 cases (69%) regressed after the delivery; in 8 such cases from the cesarean delivery group, only 2 cases (25%) regressed afterward. Our study clearly shows that pregnancy complicated with CIN is increasing rapidly in Japan. We also find that there is a significantly more frequent postpartum regression of biopsy-proven CIN lesions following a vaginal delivery compared to cesarean section (P = .042 by Fisher exact test, Odds ratio = 6.55; 95% CI: 1.13–37.8).

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.

The objective of this study is to analyze the tolerance and efficacy of the subcutaneous administration of a reduced 2,500-unit low-dose unfractionated heparin given for an efficacious, yet Asian population-sensitive, prophylaxis for deep vein thrombosis and fatal pulmonary embolism. Eighty-seven Japanese patients were operated on either for abdominal or pelvic complications or both, as well as for gynecologic conditions including ovarian, cervical, and corpus cancers. Thirty-two of the patients were administered the experimental low dose of unfractionated calcium heparin for prophylaxis. The 2,500 units of low-dose unfractionated heparin were given subcutaneously 2 h preoperatively and again 12 h postoperatively. Other standard methods of mechanical prophylaxis, including graduated compression stockings and intermittent pneumatic compression, were performed. Fifty-five of the patients were not administered heparin, but did receive the same standard mechanical graduated compression stockings and intermittent pneumatic compression prophylaxis. We compared the surgical and postsurgical complications noted for low-dose unfractionated heparin patients with the results of those who received no heparin prophylaxis and analyzed this data using the Mann-Whitney U-test. There was no significant difference in the mean of the blood loss volumes. There were also no significant differences found in the perioperative bleeding complications between the two groups. However, three (3/55; 6%) of the patients in the no-heparin group suffered a symptomatic pulmonary embolism, although none were fatal. There were no pulmonary embolism onsets in the heparin prophylaxis group. We feel that we have provided evidence that several serious complications, such as perisurgical hemorrhage, deep vein thrombosis, fatal pulmonary embolism, and increased postoperative recovery times, can be prevented by prophylaxis with 2,500-unit low-dose unfractionated heparin.

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.

Our objective was to determine whether RUNX3 tumor suppressor is inactivated in endometrial carcinoma.We have investigated 24 endometrial carcinomas, 3 endometrial carcinoma cell lines, and 9 normal endometria for genetic and epigenetic alterations of RUNX3. Reverse-transcription PCR (RT-PCR), methylation-specific PCR (MS-PCR) analysis, and loss of heterozygosity (LOH) analysis were performed. We also tested RUNX3 protein expression by immunohistochemistry.Using RT-PCR technique, we observed a significant loss of RUNX3 mRNA expression in nine of 24 endometrial carcinomas (38%) and in all 3-cell lines (100%). In contrast, all nine of the normal endometria showed an abundant expression of RUNX3 mRNA. Methylation-specific PCR (MS-PCR) analysis of the CpG islands of RUNX3 showed the promoter region to be hypermethylated in 18 of 21 analyzed carcinomas (86%), whereas only two of nine normal endometria (22%) were methylated (p0.01). By using two polymorphic microsatellite markers, D1S199 and D1S1676, we detected 1p36 LOH in 7 of 21 carcinomas (33%). We observed a significant relationship between the loss of RUNX3 mRNA expression and this regional LOH (p0.01). Immunohistochemical staining showed that RUNX3 protein expression was lost in 12 of 21 endometrial carcinomas (57%). We observed a significantly more frequent loss of RUNX3 protein expression in the histologically higher-grade tumors (Grade 3) than in Grade 1 or 2 tumors (p0.01).These findings indicate that RUNX3 inactivation may play an important role in carcinogenesis of the endometrium, especially in high-grade endometrial carcinoma.