Two cyclic electron flows around photosystem I differentially participate in C4 photosynthesis.

C4 plants assimilate CO2 more efficiently than C3 plants because of their C4 cycle that concentrates CO2. However, the C4 cycle requires additional ATP molecules, which may be supplied by cyclic electron flow (CEF) around photosystem I. One CEF route, which depends on a chloroplast NADH dehydrogenase-like (NDH) complex, is suggested to be crucial for C4 plants despite the low activity in C3 plants. The other route depends on proton gradient regulation 5 (PGR5) and PGR5-like photosynthetic phenotype 1 (PGRL1) and is considered a major CEF route to generate the proton gradient across the thylakoid membrane in C3 plants. However, its contribution to C4 photosynthesis is still unclear. In this study, we investigated the contribution of the two CEF routes to the NADP-malic enzyme subtype of C4 photosynthesis in Flaveria bidentis. We observed that suppressing the NDH-dependent route drastically delayed growth and decreased the CO2 assimilation rate to approximately 30% of the wild-type rate. On the other hand, suppressing the PGR5/PGRL1-dependent route did not affect plant growth and resulted in a CO2 assimilation rate that was approximately 80% of the wild-type rate. Our data indicate that the NDH-dependent CEF substantially contributes to the NADP-malic enzyme subtype of C4 photosynthesis and that the PGR5/PGRL1-dependent route cannot complement the NDH-dependent route in F. bidentis. These findings support the fact that during C4 evolution, photosynthetic electron flow may have been optimized to provide the energy required for C4 photosynthesis.

Involvement of an Oct4-related PouV gene, pou5f3/pou2, in neurogenesis in the early neural plate of zebrafish embryos.

In early vertebrate embryos, the dorsal ectoderm is induced by the axial mesendoderm to form the neural plate, which is given competence to form neural cells by soxB1 genes. Subsequently, neurogenesis proceeds in proneural clusters that are generated by a gene network involving proneural genes and Notch signaling. However, what occurs between early neural induction and the later initiation of neurogenesis has not been fully revealed. In the present study, we demonstrated that during gastrulation, the expression of the Oct4-related PouV gene pou5f3 (also called pou2), which is widely observed at earlier stages, was rapidly localized to an array of isolated spotted domains, each of which coincided with individual proneural clusters. Two-color in situ hybridization confirmed that each pou5f3-expressing domain included a proneural cluster. Further analysis demonstrated that anterior pou5f3 domains straddled the boundaries between rhombomere 1 (r1) and r2, whereas posterior domains were included in r4. The effects of forced expression of an inducible negative dominant-interfering pou5f3 gene suggested that pou5f3 activated early proneural genes, such as neurog1 and ebf2, and also soxB1, but repressed the late proneural genes atoh1a and ascl1b. Furthermore, pou5f3 was considered to repress her4.1, a Notch-dependent Hairy/E(spl) gene involved in lateral inhibition in proneural clusters. These results suggest that pou5f3 promotes early neurogenesis in proneural clusters, but negatively regulates later neurogenesis. Suppression of pou5f3 also altered the expression of other her genes, including her3, her5, and her9, further supporting a role for pou5f3 in neurogenesis. In vitro reporter assays in P19 cells showed that pou5f3 was repressed by neurog1, but activated by Notch signaling. These findings together demonstrate the importance of the pou5f3-mediated gene regulatory network in neural development in vertebrate embryos.

[Recurrent High-grade Astroblastoma Treated with Stereotactic Radiotherapy:A Case Report].

INTRODUCTION: Astroblastoma is a rare, supratentorial glial tumor, occurring predominantly in children and young adults. However, treatment strategies have not yet been established for this rare disease. CASE PRESENTATION: A 6-year-old boy presented with headache and nausea. CT and MRI revealed a left frontal mass lesion with slight edema and macrocalcifications. Gross tumor resection was performed. Histological examination found neoplastic cells with astroblastic characteristics, and a striking perivascular array of pseudorosettes. The final diagnosis was high-grade astroblastoma. MRI 13 months after surgery suggested local recurrence, and an enlargement was found 3 months later. Stereotactic radiotherapy(SRT)was performed. MRI after SRT showed enhanced cyst formation around the tumor bed, suggesting tumor recurrence. However, 11C-methionine positron emission tomography(PET)revealed radiation necrosis. The last follow-up MRI 15 months after SRT showed no further recurrence. CONCLUSION: Astroblastoma is rare, therefore, no optimal management is known. SRT may be effective to treat recurrent astroblastomas. 11C-methionine PET/CT was useful to differentiate metastatic disease from radiation necrosis.

In vitro analysis of the transcriptional regulatory mechanism of zebrafish pou5f3.

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development.

[A Case of Stage IV Breast Cancer with Long-Term Survival by Chemotherapy Developing Pulmonary Tumor Thrombotic Microangiopathy during the Treatment Course].

Pulmonary tumor thrombotic microangiopathy(PTTM)caused by pulmonary artery microscopic tumor emboli and fibrocellular and/or fibromuscular proliferation leads to progressive pulmonary hypertension and respiratory failure.The prognosis is extremely poor and most patients die shortly after onset.We report a patient with Stage IV breast cancer and long-term survival who developed PTTM during chemotherapy treatment.A 63-year-old woman with multiple metastases in her cerebellum, bone, lung, and lymph node after left breast conserving surgery started to experience dyspnea and malaise 7 years after the surgery.Two months later, she was urgently admitted to hospital because of respiratory failure and was diagnosed with pulmonary hypertension.However, pulmonary thrombosis and tumor thrombus were not observed.We clinically diagnosed her with PTTM and administered chemotherapy in addition to treatment for pulmonary hypertension.Her medical condition improved gradually and she survived for the subsequent 2 years.When observing progressive hypoxia and pulmonary hypertension without obvious pulmonary embolism findings on imaging, PTTM should be considered.Early diagnosis and immediate induction of chemotherapy for primary disease can improve the survival of patients with PTTM.

Uric acid transporter ABCG2 is increased in the intestine of the 5/6 nephrectomy rat model of chronic kidney disease.

BACKGROUND: Uric acid (UA) remains a risk factor of chronic kidney disease (CKD). Therefore, it is important to clarify the mechanism of UA excretion in CKD. The specific mechanisms of extrarenal excretion from the intestine are unknown. We evaluated the expression of the UA transporter in the intestinal tract--the ATP-binding cassette transporter G2 (ABCG2)--in a 5/6 nephrectomy rat model of CKD. METHODS: Male Wistar rats (6 weeks old) were randomly assigned to the 5/6 nephrectomized (Nx) group or the sham-operated control group. Urine and blood samples were collected every 4 weeks. All the rats were killed at 8 weeks to obtain liver, duodenum, jejunum, ileum, and transverse colon tissues. Uricase activity was measured in the liver. Expression of ABCG2 in intestinal mucosa was measured with real time polymerase chain reaction (PCR). RESULTS: The Nx group showed significantly decreased urine UA excretion/body weight and UA clearance compared to the control group at 4 and 8 weeks after nephrectomy. In contrast, serum UA and uricase activity were not significant. The expression of ABCG2 in the ileum of the Nx group showed significantly increased upregulation, while no changes were seen in the intestines of the control group. CONCLUSIONS: The Nx rats exhibited lower excretion of urine UA and over-expression of ABCG2 in the ileum. The fact that serum UA did not increase despite the decrease in UA excretion suggests that an excretory pathway other than the kidney, probably the intestine, may operate in a complementary role that corroborates the increase in ABCG2 expression in the ileum.

Assessment of permanent brachytherapy combined with androgen deprivation therapy in an intermediate-risk prostate cancer group without a Gleason score of 4 + 3: a single Japanese institutional experience.

OBJECTIVES: To evaluate the outcome of low-dose-rate permanent brachytherapy combined with anti-androgen deprivation therapy for intermediate-risk prostate cancer excluding biopsy Gleason score 4 + 3. METHODS: Patients included in the intermediate-risk group were those presenting clinical stage T1c to T2c (by magnetic resonance imaging staging), Gleason score 3 + 4 or lower and/or prostate-specific antigen less than 20 ng/mL, whereas those with clinical stage T1c to T2a, Gleason score 3 + 3 and prostate-specific antigen less than 10 ng/mL represented the low-risk group, and were used as controls. In the intermediate-risk group, therapy with a luteinizing hormone-releasing hormone analog was continued for at least 6 months before and after permanent brachytherapy. RESULTS: A total of 147 low-risk group patients and 139 intermediate-risk group patients were included in the study. The median follow up was 51 and 52 months for the intermediate-risk group and low-risk group, respectively. The 5-year overall, cause-specific and distant-metastasis-free survival rates in the low-risk group and intermediate-risk group were 97.6/99.2, 100/100 and 100/100%, respectively. The 5-year biochemical disease-free survival in these groups were 95.9 and 92.5%, respectively (P = 0.18). There was no sexual activity and desire for erection before treatment in 50%, and in 46% of the patients in the low-risk group and intermediate-risk group, respectively. Overall satisfaction score at 2 years after permanent brachytherapy significantly improved, compared with pretreatment (P = 0.0399). CONCLUSIONS: In intermediate-risk prostate cancer, excluding biopsy Gleason score 4 + 3, permanent brachytherapy combined with androgen deprivation therapy for 6 months or more represents an effective treatment option in Japanese patients, based on a favorable prognosis, adverse event profile and quality of life analysis.

Assessment of metabolic status in young Japanese females using postprandial glucose and insulin levels.

Lifestyle-related diseases develop through the accumulation of undesirable lifestyle habits both prior to the onset of disease as well as during normal healthy life. Accordingly, early detection of, and intervention in, metabolic disorders is desirable, but is hampered by the lack of an established evaluation index for young individuals. The purpose of this study was to investigate the utility of a biomarker of health in young female subjects. The subjects were young healthy Japanese females in whom energy expenditure was measured for a period of 210 min after a test meal. In addition, Δplasma glucose and Δserum insulin were calculated from the fasting and 30 min values. ΔPlasma glucose and Δserum insulin levels varied widely compared to fasting levels. Both the area under the curve of carbohydrate oxidation rate and serum free fatty acid levels were higher in individuals in the high Δplasma glucose group. Moreover, Δplasma glucose was higher in individuals in the high Δserum insulin group than in the low Δserum insulin group. We conclude that nutritional balanced liquid loading test using Δplasma glucose and Δserum insulin as the evaluation index is useful for the detection of primary metabolic disorders in young females.

A Phase I Trial of Weekly Nab-paclitaxel Plus Carboplatin With Thoracic Radiotherapy for Non-small Cell Lung Cancer.

BACKGROUND/AIM: This study aimed to evaluate the safety and recommended dose of nab-paclitaxel in combination with carboplatin and thoracic radiotherapy for locally advanced non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Nab-paclitaxel was administered weekly with escalating doses, combined with carboplatin area under the curve (AUC) 2 and concurrent standard thoracic radiotherapy. Escalating doses of nab-paclitaxel were as follows: level 0, 30 mg/m2; level 1, 35 mg/m2; level 2, 40 mg/m2; level 3, 45 mg/m2 Results: Twelve patients were enrolled and received the treatment according to the protocol; seven patients (58%) had squamous cell carcinoma and all cases had stage III disease. At level 1, none of the three patients experienced dose limiting toxicity (DLT). At level 2, one of the first three patients experienced a fatal DLT of bronchopulmonary hemorrhage. None of the three more additional patients experienced DLT. At level 3, two of the three patients experienced a DLT of grade 3 febrile neutropenia and grade 4 neutropenia, respectively. Consolidation chemotherapy was provided to 10 of 12 patients. Radiation pneumonitis developed in five of 12 patients (42%). Eight patients (66.7%) showed partial response, and four (33.3%) showed stable disease. For the above reasons, level 2 (40 mg/m2) was considered the recommended dose in this study. CONCLUSION: Concurrent chemoradiotherapy with weekly nab-paclitaxel (40 mg/m2) and carboplatin (AUC 2) is a feasible and well-tolerated regimen in patients with previously untreated locally advanced NSCLC. A phase II trial with this regimen is warranted.

Spin dynamics phenomena of a cerium(III) double-decker complex induced by intramolecular electron transfer.

Switchable spin dynamic properties in single-molecule magnets (SMMs) via an applied stimulus have applications in single-molecule devices. Many SMMs containing heavy lanthanoid ions with strong uniaxial magnetic anisotropy have been reported to exhibit SMM characteristics in the absence of an external magnetic field. On the other hand, SMMs containing light lanthanoid cerium(III) (Ce3+) ions exhibit field-induced slow magnetic relaxation. We investigated the chemical conversion of a diamagnetic Ce4+ ion (4f0) to a paramagnetic Ce3+ ion (4f1) in Ce-phthalocyaninato double-decker complexes (TBA+[Ce(obPc)2]- (1) and TBA+[Ce(Pc)2]- (2)) which exhibit field-induced SMM behaviour due to a 4f1 system. The phthalocyaninato ligands with electron-donating substituents (obPc2- = 2,3,9,10,16,17,23,24-octabutoxyphthalocyaninato) in 1 have a significant effect on the valence state of the Ce ion, which is reflected in its magnetic properties due to the mixed valence state of the Ce ion. Given that Ce double-decker complexes with π-conjugated ligands undergo intramolecular electron transfer (IET) to the Ce ion mixed valence state, characterised by a mixture of 4f0 and 4f1 configurations, we examined the dynamic disorder inherent in IET influencing magnetic relaxation.

Inter-fractional error and intra-fractional motion of prostate and dosimetry comparisons of patient position registrations with versus without fiducial markers during treatment with carbon-ion radiotherapy.

A few reports have discussed the influence of inter-fractional position error and intra-fractional motion on dose distribution, particularly regarding a spread-out Bragg peak. We investigated inter-fractional and intra-fractional prostate position error by monitoring fiducial marker positions. In 2020, data from 15 patients with prostate cancer who received carbon-ion beam radiotherapy (CIRT) with gold markers were investigated. We checked marker positions before and during irradiation to calculate the inter-fractional positioning and intra-fractional movement and evaluated the CIRT dose distribution by adjusting the planning beam isocenter and clinical target volume (CTV) position. We compared the CTV dose coverages (CTV receiving 95% [V95%] or 98% [V98%] of the prescribed dose) between skeletal and fiducial matching irradiation on the treatment planning system. For inter-fractional error, the mean distance between the marker position in the planning images and that in a patient starting irradiation with skeletal matching was 1.49 ± 1.11 mm (95th percentile = 1.85 mm). The 95th percentile (maximum) values of the intra-fractional movement were 0.79 mm (2.31 mm), 1.17 mm (2.48 mm), 1.88 mm (4.01 mm), 1.23 mm (3.00 mm), and 2.09 mm (8.46 mm) along the lateral, inferior, superior, dorsal, and ventral axes, respectively. The mean V95% and V98% were 98.2% and 96.2% for the skeletal matching plan and 99.5% and 96.8% for the fiducial matching plan, respectively. Fiducial matching irradiation improved the CTV dose coverage compared with skeletal matching irradiation for CIRT for prostate cancer.

Oct-3/4 promotes migration and invasion of glioblastoma cells.

As a result of increased glioblastoma migration and invasion into normal brain parenchyma, treatment of local tumor recurrence following initial treatment in glioblastoma patients remains challenging. Recent studies have demonstrated increased Oct-3/4 expression, a self-renewal regulator in stem cells, in glioblastomas. However, little is known regarding the influence of Oct-3/4 in glioblastoma cell invasiveness. The present study established Oct-3/4-overexpressing glioblastoma cells, which were prepared from human glioblastoma patients, to assess migration, invasion, and mRNA expression profiles of integrins and matrix metalloproteinases (MMPs). Compared with control cells, Oct-3/4 expressing-glioblastoma cells exhibited increased migration and invasion in wound healing and Matrigel invasion assays. Oct-3/4 overexpression resulted in upregulated FAK and c-Src expression, which mediate integrin signals. Vinculin accumulated along the leading edges of Oct-3/4 expressing-glioblastoma cells and associated with membrane ruffles during cell migration. Oct-3/4 expressing-cells exhibited increased MMP-13 mRNA expression and MMP-13 knockdown by shRNA suppressed cell invasion into Matrigel and organotypic brain slices. These results suggested that Oct-3/4 enhanced degradation of surrounding extracellular matrix by increasing MMP-13 expression and altering integrin signaling. Therefore, Oct-3/4 might contribute to tumor promoting activity in glioblastomas.

[Renin-angiotensin-aldosterone system inhibitors and electrolyte disturbances].

Recent numerous clinical studies have suggested that the renin-angiotensin-aldosterone system (RAAS) inhibitors have beneficial effects on hypertension, cardiovascular and chronic kidney diseases (CKD). Electrolyte disorder, especially hyperkalemia, is observed after the administration of RAAS inhibitors. This review focuses on the prevalence and severity of hyperkalemia with the use of RAAS inhibitors. Clinical evidences suggest that hypertensive patients with heart failure and CKD, and patients with combination treatment of RAAS inhibition are at higher risk of hyperkalemia, and serum potassium levels should be carefully monitored.

Rh-catalyzed carbonylation of arylzinc compounds yielding symmetrical diaryl ketones by the assistance of oxidizing agents.

Carbonylative homocoupling of arylzinc compounds 1 using 1 atm of CO and 1,2-dibromoethane as an oxidant was achieved in the presence of Rh-dppf catalyst, affording symmetrical diaryl ketones in good yields. Under similar conditions, Pd or Ni catalysts induced oxidative homocoupling of 1 to yield biaryls instead. The beneficial catalysis by Rh in the carbonylation was presumed to stem from the facility by which the migration of the aryl ligand to CO at the Rh(3+) intermediate occurred.

Optimal duration of androgen deprivation in combination with radiation therapy for Japanese men with high-risk prostate cancer.

OBJECTIVES: To evaluate the efficacy and toxicity of external beam radiation therapy (EBRT) combined with androgen deprivation therapy (ADT) for Japanese high-risk prostate cancer (PCa) patients in a single institution. METHODS: Seventy-five high-risk PCa patients were treated by three-dimensional conformal radiotherapy of 70 Gy combined with neoadjuvant, concurrent and adjuvant ADT. RESULTS: Median age was 72 (59-82) years. Median initial serum prostate-specific antigen (PSA) was 19.0 (4.7-200) ng/ml. Median duration of the entire ADT was 27 (8-63) months. Median follow-up after initiating ADT and after completing EBRT was 66 (41-105) and 59 (36-94) months, respectively. Five-year overall, clinical progression-free, and biochemical progression-free survival rates were 98.3, 97.2, and 87.4%; 2 (2.7%) cancer deaths, 3 (4.0%) clinical progressions, and 11 (14.7%) biochemical progressions. Multivariate analysis suggested a total duration of ADT shorter than 24 months as an independent risk factor of biochemical progression (p = 0.01). Grade 3 toxicities related to EBRT were observed: 1 patient with proctitis and rectal bleeding and 1 patient with rectal bleeding. CONCLUSIONS: It is suggested that 70 Gy EBRT combined with ADT confers disease-free survival benefit with tolerable adverse events for Japanese high-risk PCa patients. ADT of 24 months or longer might be recommended to minimize biochemical progression.

Computational study of the three-dimensional structure of N-acetyltransferase 2-acetyl coenzyme a complex.

N-Acetyltransferase 2 (NAT2) is one of the most important polymorphic drug-metabolizing enzymes and plays a significant role in individual differences of drug efficacies and/or side effects. Coenzyme A (CoA) is a cofactor in the experimentally determined crystal structure of NAT2, although the acetyl source of acetylation reactions catalyzed by NAT is not CoA, but rather acetyl CoA. In this study, the three-dimensional structure of NAT2, including acetyl CoA, was calculated using molecular dynamics simulation. By substituting acetyl CoA for CoA the amino acid residue Gly286, which is known to transform into a glutamate residue by NAT2*7A and NAT2*7B, comes close to the cofactor binding site. In addition, the binding pocket around the sulfur atom of acetyl CoA expanded in the NAT2-acetyl CoA complex.

Modeling the enolization of succinimide derivatives, a key step of racemization of aspartic acid residues: importance of a two-H2O mechanism.

Racemization of aspartic acid residues in peptides and proteins is assumed to proceed via succinimide intermediates. An enolization of the succinimide intermediate is required for the racemization to occur. In this study, we modeled the enolization step by density-functional theory (DFT) calculations (B3LYP/6-31+G**), using two model compounds, N-methylsuccinimide (1) and its formylamino derivative 2. Three mechanisms were investigated for 1, i.e., the direct mechanism without active participation of H(2)O molecules, and one-H(2)O and two-H(2)O mechanisms, in which one or two H(2)O molecules actively participate in the reaction. We found that the two-H(2)O mechanism was the most favorable with an activation barrier of 37 kcal mol(-1). In the two-H(2)O mechanism, a concerted bond reorganization involving a triple H-atom transfer occurred in an eight-membered cyclic structure formed between the imide and two H(2)O molecules. For 2, we investigated only the two-H(2)O mechanism and found that the activation barrier was lowered to 31 kcal mol(-1) due to an H-bond between the CO O-atom of the formylamino group ('the neighboring residue') and one of the H(2)O molecules. Our results suggest that, in proteins, the Asp racemization is severely controlled by the accessibility of H(2)O molecules to the reaction site of the succinimide intermediate.

Molecular-dynamics simulations for amyloid beta 1-42 monomer with D-aspartic acid residues using continuous solvent.

Molecular-dynamics simulations of amyloid-beta(1-42) peptides including D-aspartic acid residues were performed, and their three-dimensional structures were compared. The simulations were performed in an aqueous environment using a continuous solvent model. In the structures obtained from simulations, the occurrence ratio of beta-extended structures for the peptide that included D-Asp23 was larger than that for the wild-type peptide. These beta-extended structures appeared in the C-terminal region of the peptide, and the alpha-helix structures of the region were lost. On the other hand, for the peptide that included the stereo-inverted form of Asp1 as well as D-Asp23, the occurrence ratio of beta-extended structures in the C-terminal region was lower than that of the peptide including only D-Asp23.

Computational insight into the mechanism of serine residue racemization.

Despite the known racemization of serine (Ser) residues in proteins from aged or diseased human brains, the mechanism of Ser racemization in peptides and proteins has not been studied. This is in contrast to the case of the rapid racemization of aspartic acid (Asp) residues, for which a succinimide-mediated mechanism is established. In a possible mechanism for Ser racemization, the enolization involving the H(alpha)-atom and the CO(alpha) group occurs by active participation of two H(2)O molecules. In this study, we computationally modeled this two-H(2)O mechanism of enolization for Ser and alanine (Ala) residues using model compounds, and the results were compared with each other and with the case of succinimide analogues reported in a separated paper. Our results suggest that Ser residues are much more racemization-prone than Ala residues, but to a lesser extent than Asp residues. The side-chain C-O bond of the Ser residue stabilizes the enolization transition state hyperconjugatively.

Computational modeling of the enolization in a direct mechanism of racemization of the aspartic acid residue.

The rapid racemization of aspartic acid (Asp) residues in peptides and proteins is due mainly to the succinimide intermediate. However, there should be another mechanism for Asp racemization without intermediacy of the succinimide. The direct H-atom abstraction from the C(alpha)-atom that leads to the enol form of the Asp residue is one possibility. In another study, we have computationally predicted that the corresponding enolization in the succinimide intermediate occurs by assistance of two H(2)O molecules. In the present study, we, therefore, investigated the similar two-H(2)O-assisted enolization for an Asp-containing model compound by the same computational method as before (B3LYP/6-31+G**). Rather surprisingly, the activation barrier for the two-H(2)O-assisted enolization of the Asp residue (protonated form) was calculated to be almost equal to that for the corresponding succinimide. Therefore, an Asp residue is expected to be prone to enolization to almost the same degree as the corresponding succinimide form, and the 'direct' (i.e., non-succinimide-mediated) mechanism of Asp racemization may compete with the succinimide-mediated mechanism.