Total laparoscopic nerve-sparing radical parametrectomy for occult early-stage cervical cancer: surgical technique and postoperative bladder function.

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.

Various types of total laparoscopic nerve-sparing radical hysterectomies and their effects on bladder function.

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.

Repeatability and reproducibility of measurements using a NT-530P noncontact tono/pachymeter and correlation of central corneal thickness with intraocular pressure.

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: -2.95 to 2.61 mmHg) for IOP, -0.93 ± 4.37 µ m (range: -9.50 to 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.

Obstetric outcomes of patients undergoing total laparoscopic radical trachelectomy for early stage cervical cancer.

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 31.9 years (range 22-42 years). 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 22-28 weeks 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.

Effect of fermented bilberry extracts on visual outcomes in eyes with myopia: a prospective, randomized, placebo-controlled study.

PURPOSE: To investigate clinically the effects of yeast-fermented bilberry extract on visual outcomes in myopic eyes. METHODS: 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. RESULTS: 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 (P>0.05), or any significant changes in any other parameters in either group (P>0.05). CONCLUSIONS: The present data show that fermented bilberry extract is effective in causing increases in subjective accommodation and in mesopic CS in myopic eyes.

Total laparoscopic hysterectomy in 1253 patients using an early ureteral identification technique.

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.

Mutations in genes encoding the glycine cleavage system predispose to neural tube defects in mice and humans.

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.

Investigating the relative efficacies of combination chemotherapy of paclitaxel/carboplatin, with or without anthracycline, for endometrial carcinoma.

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/m(2)), epirubicin (50 mg/m(2)) and carboplatin (AUC 4). TC regimen consisted of paclitaxel (175 mg/m(2)) 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.

High-resolution X-ray crystal structure of bovine H-protein at 0.88 A resolution.

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 A 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 and R factors 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.

Crystal structure of aminomethyltransferase in complex with dihydrolipoyl-H-protein of the glycine cleavage system: implications for recognition of lipoyl protein substrate, disease-related mutations, and reaction mechanism.

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 Arg(292) 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.

Global conformational change associated with the two-step reaction catalyzed by Escherichia coli lipoate-protein ligase A.

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, LplA.lipoyl-5'-AMP and LplA.octyl-5'-AMP.apoH-protein 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 degrees . These changes are prerequisites for LplA to accommodate apoprotein for the second reaction. The Lys(133) 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.

Postpartum outcome of cervical intraepithelial neoplasia in pregnant women determined by route of delivery.

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).

Safety and efficacy of lower-dose unfractionated heparin for prophylaxis of deep vein thrombosis and pulmonary embolism in an Asian population.

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.

Clonality and HPV infection analysis of concurrent glandular and squamous lesions and adenosquamous carcinomas of the uterine cervix.

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.

Frequent inactivation of RUNX3 in endometrial carcinoma.

OBJECTIVE: Our objective was to determine whether RUNX3 tumor suppressor is inactivated in endometrial carcinoma. METHODS: 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. RESULTS: 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 (p<0.01). 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 (p<0.01). 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 (p<0.01). CONCLUSION: These findings indicate that RUNX3 inactivation may play an important role in carcinogenesis of the endometrium, especially in high-grade endometrial carcinoma.

Alterations of the K-ras and p53 genes in Tamoxifen-associated endometrial carcinoma.

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.

PIK3CA gene mutations and amplifications in uterine cancers, identified by methods that avoid confounding by PIK3CA pseudogene sequences.

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.

Crystal structure of bovine lipoyltransferase in complex with lipoyl-AMP.

Lipoic acid is an essential cofactor of the alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. It is covalently attached to a specific lysine residue of the subunit of the complexes. The bovine lipoyltransferase (bLT) catalyzes the lipoic acid attachment reaction using lipoyl-AMP as a substrate, forming a lipoylated protein and AMP. To gain insights into the reaction mechanism at the atomic level, we have determined the crystal structure of bLT at 2.10 A resolution. Unexpectedly, the purified recombinant bLT contains endogenous lipoyl-AMP. The structure of bLT consists of N-terminal and C-terminal domains, and lipoyl-AMP is bound to the active site in the N-terminal domain, adopting a U-shaped conformation. The lipoyl moiety is buried in the hydrophobic pocket, forming van der Waals interactions, and the AMP moiety forms numerous hydrogen bonds with bLT in another tunnel-like cavity. These interactions work together to expose the C10 atom of lipoyl-AMP to the surface of the bLT molecule. The carbonyl oxygen atom of lipoyl-AMP interacts with the invariant Lys135. The interaction might stimulate the positive charge of the C10 atom of lipoyl-AMP, and consequently facilitate the nucleophilic attack by the lysine residue of the lipoate-acceptor protein, accompanying the bond cleavage between the carbonyl group and the phosphate group. We discuss the structural differences between bLT and the lipoate-protein ligase A from Escherichia coli and Thermoplasma acidophilum. We further demonstrate that bLT in mitochondria also contains endogenous lipoylmononucleotide, being ready for the lipoylation of apoproteins.

Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.