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Anti-thymocyte globulin (ATG) is an important prophylactic drug against acute graft-versus-host disease (aGVHD) after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). This study analyzed the pharmacokinetics of rabbit ATG 2.5 mg/kg and its effect against aGVHD in 24 patients undergoing unmanipulated haplo-HSCT. All patients had hematological malignancies not in remission. The median absolute lymphocyte count (ALC) before rabbit ATG administration was 9.5/µL (range 0-41/µL). The grade ≥ II aGVHD group had a significantly lower median rabbit ATG concentration on days 0 (C

OBJECTIVES To determine the impact of the use of hydroxyethyl starch (HES) in granulocyte apheresis using Spectra Optia. BACKGROUND Granulocyte transfusion (GT) is a therapeutic option for neutropenic patients with severe bacterial or fungal infections. Recent studies in emergency medicine have shown the potential risk of using HES, which is routinely used in granulocyte apheresis to increase yield by sedimenting red blood cells. We hypothesized that the use of a newer device (Spectra Optia) would spare the need for HES. METHODS We retrospectively compared granulocyte apheresis with HES (HES group, n = 89) and without HES (non-HES group, n = 36) using Spectra Optia. RESULTS The granulocyte yield was significantly higher in the HES group (7.3 × 1010 vs. 2.0 × 10, p

HLA haploidentical hematopoietic stem cell transplantation (HSCT), i.e., HSCT from a 1-HLA-haplotype-mismatched family donor, has been successfully performed even as a second transplantation for posttransplant relapse. Is the haploidentical the limit of HLA mismatches in HSCT? In order to explore the possibility of HLA-mismatched HSCT from family donors beyond haploidentical relatives, we conducted a prospective phase I/II study of 2-HLA-haplotype-mismatched HSCT (2-haplo-mismatch HSCT). We enrolled 30 patients with posttransplant relapse (acute myeloid leukemia: 18, acute lymphoblastic leukemia: 11, non-Hodgkin lymphoma: 1). 2-haplo-mismatch HSCT was performed as the second to sixth transplantations. The donors were siblings (n = 12), cousins (n = 16), and second cousins (n = 2). The conditioning regimen consisted of fludarabine, cytarabine, melphalan, low-dose anti-thymocyte globulin, and 3 Gy of total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus, methylprednisolone, and mycophenolate mofetil. All patients achieved neutrophil engraftment, except for a case of early death. The cumulative incidences of grades II–IV and III–IV acute GVHD were 36.7% and 16.7%, respectively. The overall survival at 1 year, relapse, and non-relapse mortality rates was 30.1%, 38.9%, and 44.3%, respectively. Considering the poor prognosis of posttransplant relapse, 2-haplo-mismatch HSCT can be an alternative option in a second or third transplantation.

Letermovir (LMV) is a new antiviral drug used to prevent cytomegalovirus infection in hematopoietic stem cell transplantation (HSCT) recipients. It has been reported to increase tacrolimus (TAC) exposure and decrease voriconazole (VRCZ) exposure in healthy participants. However, VRCZ inhibits the metabolism of TAC. Thus, the effects of LMV on TAC exposure in patients receiving VRCZ are unknown. This retrospective, observational, single-center study was conducted between May 2018 and April 2019. The TAC concentration/dose (C/D) ratio, VRCZ concentration, and VRCZ C/D ratio for 7 days before and for the first and second 7-day periods after the initiation of LMV administration were evaluated. Fourteen HSCT recipients receiving VRCZ were enrolled. There was no significant difference in the TAC C/D ratio for 7 days before and for the first and second 7-day periods after initiating LMV administration (median: 866 [IQR: 653-953], 842 [IQR: 636-1031], and 906 [IQR: 824-1210] [ng/mL]/[mg/kg], respectively). In contrast, the VRCZ C/D ratio and concentration for the first and second 7-day periods after LMV initiation were significantly lower than those before initiating LMV administration (mean 1.11 ± 0.07, 0.12 ± 0.08, and 0.22 ± 0.12 [μg/mL]/[mg/kg] and 0.7 ± 0.5, 0.8 ± 0.5, and 1.3 ± 0.7 μg/mL, respectively; n = 12). This can be explained by the increase in TAC concentration caused by CYP3A4 inhibition due to LMV and by the decrease in TAC concentration ascribed to the decrease in VRCZ concentration by CYP2C19 induction due to LMV. These results suggest that it is unnecessary to adjust the dose of TAC based on LMV initiation; however, it is necessary to adjust the dose of TAC based on conventional TAC concentration measurements.

Letermovir is used to prevent cytomegalovirus infection in hematopoietic stem cell transplantation (HSCT) recipients. Although this agent decreases voriconazole exposure in healthy individuals, the effect of coadministration of letermovir and voriconazole in HSCT recipients is unknown. This retrospective, observational, single-center study was conducted between January 2016 and July 2019 to examine the voriconazole concentration-to-dose ratio over three periods: (A) (days -7 to -1 [day 0: day of HCST]), (B) (days 4-10), and (C) (days 11-17). Forty-two HSCT recipients administered voriconazole were divided into the following two groups based on letermovir coadministration: letermovir (n = 15) and control (n = 27). The percent change (-33.2%, p < 0.05) in the voriconazole concentration-to-dose ratio from periods A to C in the letermovir group was significantly lower than that in the control group. Therefore, frequent therapeutic drug monitoring of voriconazole concentrations and subsequent dose adjustments should be performed regularly in HSCT recipients.

Summary Background Hepatotoxicity and visual symptoms are common adverse effects (AEs) of voriconazole therapy. Objective To retrospectively evaluate the effects of treatment modification based on therapeutic drug monitoring on AEs in patients undergoing voriconazole therapy. Methods The target voriconazole trough concentration (C min) was 1‐5 µg/mL. Receiver operating characteristic curves were used to determine C min cut‐offs for AEs. Results A total of 401 patients were included. Among 108 patients with high initial C min, voriconazole was discontinued in 32 and the dose was reduced in 71. Among 44 patients with low initial C min, voriconazole was discontinued in 4 and the dose was increased in 19. Hepatotoxicity occurred in 6.0% of patients, after a median of 10 days. Visual symptoms were evident in 9.5% of patients after a median of 4 days. Initial C min was significantly associated with visual symptoms but not hepatotoxicity, which suggested the effect of treatment modification on hepatotoxicity. However, both hepatotoxicity and visual symptoms were significantly correlated with C min at the onset of AEs, and the C min cut‐offs were 3.5 μg/mL for hepatotoxicity and 4.2 μg/mL for visual symptoms. Voriconazole was discontinued after the occurrence of AEs in 62.5% of patients with hepatotoxicity but only 26.3% of patients with visual symptoms. With dose adjustment, treatment was completed in 8/9 patients with hepatotoxicity and 27/28 patients with visual symptoms. Conclusions A significant preventive effect was demonstrated on hepatotoxicity, but not on visual symptoms because of earlier occurrence. With treatment modification after the occurrence of AEs, most patients completed therapy.

We evaluated the impact of FLT3-ITD, NPM1 mutations, and double mutant CEBPa (dmCEBPa) on overall survival (OS) after relapse in patients with cytogenetically intermediate-risk acute myeloid leukemia (AML) who were treated with chemotherapy alone in the first remission (CR1). Patients aged 16–65 years diagnosed with cytogenetically intermediate-risk AML, and who achieved CR1 were included. We retrospectively analyzed FLT3-ITD, NPM1 mutations and CEBPa using samples obtained at diagnosis, which therefore did not affect the therapeutic decisions. Among 235 patients who had achieved CR1, 152 relapsed, and 52% of them achieved second CR. The rate of achieving second CR was significantly higher (85%) in those with dmCEBPa. Patients with FLT3-ITD had significantly worse OS after relapse than those without (19% vs 41%, p = 0.002), while OS was comparable between patients with and without NPM1 mutations (37% vs 34%, p = 0.309). Patients with dmCEBPa had improved OS than those without (61% vs 32%, p = 0.006). By multivariate analysis, FLT3-ITD was independently associated with worse OS after relapse [hazard ratio (HR) 1.99, 95% CI 1.27–3.12, p = 0.003], and dmCEBPa with improved OS (HR 0.40, 95% CI 0.17–0.93, p = 0.033). Our data show that screening for these mutations at diagnosis is useful for facilitating effective therapeutic decision-making even after relapse.

Acute lymphoblastic leukemias (ALL) positive for KMT2A/AFF1 (MLL/AF4) translocation, which constitute 60% of all infant ALL cases, have a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This poor prognosis is due to one of two factors, either resistance to TNFα, which mediates a graft-versus-leukemia (GVL) response after allo-HSCT, or immune resistance due to upregulated expression of the immune escape factor S100A6. Here, we report an immune stimulatory effect against KMT2A/AFF1-positive ALL cells by treatment with the anti-allergy drug amlexanox, which we found to inhibit S100A6 expression in the presence of TNF-α. In KMT2A/AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1-positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of KMT2A/AFF1-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of KMT2A/AFF1-positive ALL. Cancer Res; 77(16); 4426–33. ©2017 AACR.

The clinical impact of KIT mutations in core binding factor acute myeloid leukemia (CBF-AML) is still unclear. In the present study, we analyzed the prognostic significance of each KIT mutation (D816, N822K, and other mutations) in Japanese patients with CBF-AML. We retrospectively analyzed 136 cases of CBF-AML that had gone into complete remission (CR). KIT mutations were found in 61 (45%) of the patients with CBF-AML. D816, N822K, D816 and N822K, and other mutations of the KIT gene were detected in 29 cases (21%), 20 cases (15%), 7 cases (5%), and 5 cases (4%), respectively. The rate of relapse-free survival (RFS) and overall survival (OS) in patients with D816 and with both D816 and N822K mutations was significantly lower than in patients with other or with no KIT mutations (RFS: p

HLA haploidentical hematopoietic stem cell transplantation (HSCT), i.e., HSCT from a 1-HLA-haplotype-mismatched family donor, has been successfully performed even as a second transplantation for posttransplant relapse. Is the haploidentical the limit of HLA mismatches in HSCT? In order to explore the possibility of HLA-mismatched HSCT from family donors beyond haploidentical relatives, we conducted a prospective phase I/II study of 2-HLA-haplotype-mismatched HSCT (2-haplo-mismatch HSCT). We enrolled 30 patients with posttransplant relapse (acute myeloid leukemia: 18, acute lymphoblastic leukemia: 11, non-Hodgkin lymphoma: 1). 2-haplo-mismatch HSCT was performed as the second to sixth transplantations. The donors were siblings (n = 12), cousins (n = 16), and second cousins (n = 2). The conditioning regimen consisted of fludarabine, cytarabine, melphalan, low-dose anti-thymocyte globulin, and 3 Gy of total body irradiation. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus, methylprednisolone, and mycophenolate mofetil. All patients achieved neutrophil engraftment, except for a case of early death. The cumulative incidences of grades II-IV and III-IV acute GVHD were 36.7% and 16.7%, respectively. The overall survival at 1 year, relapse, and non-relapse mortality rates was 30.1%, 38.9%, and 44.3%, respectively. Considering the poor prognosis of posttransplant relapse, 2-haplo-mismatch HSCT can be an alternative option in a second or third transplantation.

Background: Acute kidney injury (AKI) and chronic kidney disease (CKD) are considered common complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Objectives and Method: In this study, 114 patients who had undergone allo-HSCT were retrospectively analyzed to investigate the risk factors for onset of posttransplant AKI and CKD as defined by the new Kidney Disease Improving Global Outcomes criteria. Results: Seventy-four patients (64.9%) developed AKI and 25 (21.9%) developed CKD. The multivariate analysis showed that the risk factors for developing stage 1 or higher AKI were age ≥46 years at the time of transplant (p = 0.001) and use of ≥3 nephrotoxic drugs (p = 0.036). For CKD, the associated risk factors were disease status other than complete remission at the time of transplantation (p = 0.018) and onset of AKI after transplant (p = 0.035). The 5-year overall survival (OS) was significantly reduced by development of AKI (p < 0.001), but not CKD. Posttransplant AKI significantly increased the 5-year nonrelapse mortality (p < 0.001), whereas posttransplant CKD showed an increasing tendency, but the difference was not significant. Conclusions: Posttransplant AKI impacts OS, significantly increases the risk of CKD, and is significantly associated with disseminated intravascular coagulation and use of ˃3 nephrotoxic drugs.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P

Nafuredin-γ (2), converted from nafuredin (1) under mild basic conditions, demonstrates potent and selective inhibitory activity against helminth complex I. However, 2 is unstable in air because the conjugated dienes are oxygen-labile. To address this, we designed and synthesized air-stable nafuredin-γ analogs. Although the complex I inhibitory activities of all the new nafuredin-γ analogs were lower than that of 2, all were in the high nM range (IC50: 300–820 nM).

Background TDM of VRCZ might be useful, especially in Asian people because of CYP2C19 genetic polymorphisms. However, limited data are available because of the small sample size. Methods Patients who received VRCZ and had TDM were reviewed retrospectively at five institutions. Adequate VRCZ dosage was defined as a loading dose of 5–6 ± 0.5 mg/kg twice daily followed by a maintenance dose of 3–4 ± 0.5 mg/kg twice daily. For prophylaxis, the loading dose was left to the physician’s discretion. Optimal timing of TDM was defined as 4–7 days after starting therapy. Patients with adequate dosing and optimal timing of TDM were evaluated for analysis of trough levels (Cmin). Target Cmin was set at 1–5 µg/mL. Results The study included 584 patients (treatment: 402; prophylaxis: 182). TDM was conducted on days 4–7 in 66.5% of patients (>7, 30.2%). A low adequate dosage (44.5%) was observed for treatment mainly because of a low performance of the loading dose (46.8%). Achievement of target Cmin was obtained in 62.7% (>5 µg/mL, 32.2%) in the treatment group and in 67.6% (11.0%) in the prophylaxis group. Seventy-one of 81 (81.7%) patients who required a dose reduction reached target Cmin by the second TDM. In 38 patients whose dose was not altered at oral switching, Cmin was significantly reduced from 2.5 ± 1.6 to 1.2 ± 1.3 μg/mL (P = 0.002), which indicated the necessity of TDM after oral switching. Hepatotoxicity occurred in 4.6% and visual symptoms in 7.9% of patients. Visual symptoms resolved without discontinuation of VRCZ in 73.9% of patients. Because of dosage adjustment based on TDM, high Cmin did not cause hepatotoxicity. However, the incidence of visual symptoms was significantly higher in patients with a high Cmin (12.7% vs. 5.4%, P = 0.002). Conclusion One-third of Japanese patients who underwent VRCZ treatment with a loading dose showed high Cmin. Occurrence of hepatotoxicity was prevented with alteration of dosage in these patients (AMED, JP18fk0108045). Disclosures All authors: No reported disclosures.

Objective In hematological malignancy patients, the complication of acute respiratory failure often reaches a degree of severity that necessitates mechanical ventilation. The objective of the present study was to investigate the therapeutic outcomes of mechanical ventilation in hematological malignancy patients with respiratory failure and to analyze the factors that are associated with successful treatment in order to identify the issues that should be addressed in the future. Methods The present study was a retrospective analysis of 71 hematological malignancy patients with non-cardiogenic acute respiratory failure who were treated with mechanical ventilation at Nippon Medical School Hospital between 2003 and 2014. Results Twenty-six patients (36.6%) were treated with mechanical ventilation in an intensive care unit (ICU). Non-invasive positive pressure ventilation (NPPV) was applied in 29 cases (40.8%). The rate of successful mechanical ventilation treatment with NPPV alone was 13.8%. The rate of endotracheal extubation was 17.7%. A univariate analysis revealed that the following factors were associated with the successful extubation of patients who received invasive mechanical ventilation: respiratory management in an ICU (p=0.012); remission of the hematological disease (p=0.011); female gender (p=0.048); low levels of accompanying non-respiratory organ failure (p=0.041); and the non-use of extracorporeal circulation (p=0.005). A subsequent multivariate analysis revealed that respiratory management in an ICU was the only variable associated with successful extubation (p=0.030). Conclusion The outcomes of hematological malignancy patients who receive mechanical ventilation treatment for respiratory failure are very poor. Respiratory management in an ICU environment may be useful in improving the therapeutic outcomes of such patients.

In recent years, it has been reported that DNA-methylation regulatory gene mutation - mutation of the genes that regulate gene expression through DNA methylation - is observed with high frequency in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. The subjects were 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutation was observed in 135 of the 308 cases (43.8%). acute myeloid leukemia associated with DNA-methylation regulatory gene mutation was more frequent in older patients ( p

Hyperbaric air (HBA) is used to improve healing of wounds including diabetic ulcer. The aim of this study was to clarify the effects of HBA exposure on lipid and glu- cose metabolism in db/db mice. HBA did not influence the weight of db/db mice. Serum levels of free fatty acid and triglyceride, but not glucose and insulin, were significantly decreased after 6 weeks of treatment with HBA. The mRNA expressions of CPT-1, PPARα and PGC-1α genes, which are related to lipid metabolism, were significantly up-regulated in the muscle and liver. Increases in TNF α and MCP1 mRNA, which impaired lipid me- tabolism, were also attenuated by HBA treatment. These results suggest that exposure of HBA could have beneficial effects on lipid metabolism in patients with type 2 diabetes mellitus. J. Med. Invest. 57 : 224-231, August, 2010

We performed a decision analysis comparing allogeneic hematopoietic cell transplantation (allo-HCT) versus chemotherapy in first complete remission for patients with cytogenetically intermediate-risk acute myeloid leukemia, depending on the presence or absence of FLT3-internal tandem duplication (ITD), nucleophosmin (NPM1), and CCAAT/enhancer binding protein alpha (CEBPA) mutations. Adjusted means of the patient-reported EQ-5D index were used as quality-of-life (QOL) estimates. In 332 patients for which FLT3-ITD status was available, FLT3-ITD was present in 60. In 272 patients without FLT3-ITD, NPM1 mutations were present in 83. CEBPA biallelic mutations were detected in 53 patients. For patients harboring FLT3-ITD, allo-HCT improved life expectancy (LE) (52 versus 32 months during 10-year observation) and QOL-adjusted life expectancy (QALE, 36 versus 21). Monte-Carlo simulation identified allo-HCT as the favored strategy in 100% of simulations. In patients without FLT3-ITD, allo-HCT improved LE/QALE with or without NPM1 mutations. However, sensitivity analyses showed that the results were not robust enough. For patients harboring CEBPA biallelic mutations, chemotherapy was favored (LE, 53 versus 84; QALE, 37 versus 59), whereas, for patients with monoallelic mutations or wild-type CEBPA, allo-HCT was favored (LE, 68 versus 54; QALE, 48 versus 37). Sensitivity analyses did not change the results in either group. In conclusion, based on a Markov decision analysis, allo-HCT was a favored postremission strategy in patients with FLT3-ITD, and chemotherapy was favored in patients with biallelic CEBPA mutations. A prospective study is warranted to determine the value of allo-HCT, especially in FLT3-ITD–negative patients.

We have developed a simple and highly sensitive semimicro high-performance liquid chromatographic method for the simultaneous determination of free and N-acetylated polyamines in urine. Polyamines and N-acetylated polyamines were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 50 degrees C for 15 min at pH 9. The fluorescent derivatives were separated on a reversed-phase column with a gradient elution using water-acetonitrile-methanol at 50 degrees C and detected by fluorescence measurement at 318 nm (excitation) and 406 nm (emission). The detection limits (signal-to-noise ratio=3) of the polyamines and N-acetylated polyamines were 0.7-4.5 fmol/injection. The within-day and day-to-day relative standard deviations were 3.2-7.9 and 3.0-7.7%, respectively. Significant differences were found in the urinary excretion of polyamines between cancer patients and normal subjects.

A simple and highly sensitive high-performance liquid chromatography (HPLC) for the determination of pipecolic acid (PA) in serum was developed. Pipecolic acid and nipecotic acid (internal standard (IS)) were derivatised with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride (DMS-Cl) to produce fluorescent sulfonamides. The labelling reaction was carried out at 70 °C for 15 min at pH 9.0. The fluorescent derivatives were separated on a reversed-phase column (45 °C) with a stepwise elution using methanol/acetonitrile/10 mmol l−1 acetic acid (42:5:53) and methanol at a flow rate of 1.0 ml/min and detected at excitation and emission wavelengths of 316 and 403 nm, respectively. The labelling yield was 100.8%. The detection limit of pipecolic acid was 4 fmol at signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 3.3–8.1 and 1.4–6.4%, respectively. The concentration of pipecolic acid in normal human serum was 1.09±0.37 μmol l−1.

Gene-modified dendritic cells (DCs) are promising targets for cancer immunotherapy. In this study, we demonstrated that lentiviral transduction of DCs with the gp34/OX40L gene, one of the costimulatory molecules, facilitates alloreactive CD4+ T-cell response in vitro. We achieved a 20% to 40% efficiency of gp34/OX40L gene transfer into DCs by the lentiviral vector, and lentiviral gp34/OX40L gene transfer did not alter the surface phenotype of either immature or mature DCs, suggesting that expression of gp34/OX40L did not induce the maturation of immature DCs and that gp34/OX40L-transduced DCs could fully differentiate into mature DCs. gp34/OX40L gene transfer facilitated an allogeneic CD4+ T-cell response in vitro by mature DCs but not by immature DCs. Dose escalation of the transgene induced an increasing amount of gp34/OX40L expression, leading to an increasing level of up-regulation of the allogeneic CD4+ T-cell response. The addition of anti-gp34 monoclonal antibody totally abrogated this up-regulation. These results suggest that this facilitation of allogeneic CD4+ T-cell response is specifically dependent on gp34/OX40L expressed on transduced DCs. Taken together, our findings show that gp34/ OX40L plays an important role in allogeneic CD4+ T-cell activation by DCs and that lentiviral gp34/OX40L gene transfer into DCs may be a useful strategy for cancer immunotherapy.

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed. Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 degrees C for 20 min at pH 7.5. The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and 392 nm, respectively. The detection limit (signal-to-noise ratio=3) of taurine was 3 fmol per injection. The within-day and day-to-day relative standard deviations were 3.0-4.8 and 2.5-4.7%, respectively. The concentration (means) of taurine in normal human plasma was 48.9+/-7.5 microM.

OX40 is a member of the tumor necrosis factor receptor (TNF-R) superfamily. We observed that overexpression of OX40 activated NF-kappaB, which was inhibited by dominant negative forms of TRAF2, NF-kappaB-inducing kinase (NIK), and IkappaB kinase (IKK) alpha. This indicates that OX40 signaling leads to NF-kappaB activation through the same cascade as TNF-R2. We then investigated the negative regulatory function of TRAF3 on OX40-induced NF-kappaB activation. TRAF3 blocked OX40-, TRAF2-induced NF-kappaB activation, but not NIK- and IKKalpha-induced NF-kappaB activation, indicating that TRAF3 blocks the pathway between TRAF2 and NIK. C-terminal deletion mutants as well as the N-terminal deletion mutant of TRAF3 inhibited NF-kappaB activation induced by OX40 or TRAF2. Since TRAF3 bound to OX40 through the C-terminal TRAF domain, the C-terminal domain is likely to work as a dominant negative mutant to compete the recruitment of TRAF2 to the receptor, which transmits the signal from OX40 to the downstream, NIK kinase. On the other hand, the N-terminal domain of TRAF3 seems to affect the downstream of TRAF2 binding. Thus, it is suggested that TRAF3 actively inhibits NF-kappaB activation induced by OX40.

We describe 3 unrelated Japanese patients with familial Mediterranean fever (FMF) due to a compound heterozygous E148Q/M694I mutation in the MEFV gene. The first patient is a 38-year-old man who also has chronic myelogenous leukemia (CML). Because genomic DNA analysis of the patient's nail revealed the E148Q/M694I mutation, we concluded that the individual mutations were obtained congenitally. Interferon alpha therapy was effective against not only the CML but also the FMF. The second patient is a 42-year-old man with consanguineous parents and a 14-year history of recurrent lower abdominal and back pain associated with fever. He successfully responded to colchicine treatment. The third patient is a 23-year-old woman who has a family history of FMF and since the age of 11 years has had recurrent chest and abdominal pain with fever. The onset of FMF was at an early age in this case, in contrast with the late onset of the disease in the first 2 cases. This patient's mother also has a heterozygous M694I mutation and experienced the same symptoms until 30 years of age. Our data suggest that it should be recognized that there are more FMF patients in Japan than previously expected and that the frequency of the E148Q/M694I mutation may be significant in Japanese FMF patients.

Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells. Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL. Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet. Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox. Disclosures No relevant conflicts of interest to declare.

Background: Core binding factor acute myeloid leukemia (CBF-AML) is a form of AML associated with the chromosomal abnormalities t(8;21)(q22;q22) (t(8;21)AML) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) (inv16 AML). It accounts for approximately 8% of AML cases and is considered to be a karyotype with a favorable prognosis. Mutations of c-kit gene which constitute type III tyrosine kinase receptor were found in approximately 30% of patients with CBF-AML. In CBF-AML, KIT mutation is observed mainly in two domains: the extracellular domain (exon8), and the tyrosine kinase domain (exon17), and has frequently been reported to be an unfavorable prognostic factor for early relapse and for shorter survival. On the other hand, some reports conclude that KIT mutation has no prognostic impact in CBF-AML, and in 2016's guidelines of NCCN, CBF-AML with KIT gene mutation was not assigned to the intermediate prognosis group. One potential reason why the reported prognostic impact of KIT mutation in CBF-AML differs between studies is that CBF-AML cases with t(8;21) and those with inv(16) have differing clinical profiles. Another reason is that KIT mutation occurs at various locations on the gene. We have previously reported that KIT D816 in CBF-AML is associated with a higher relapse rate than KIT N822K and has unfavorable prognosis. The aim of the present study was to clarify that these two types of KIT mutations have differing prognostic impacts in CBF-AML. Methods: We analyzed 138 cases of CBF-AML who achieved complete remission (CR), retrospectively. Mutation analyses were performed by direct sequence analysis Mutation Biased PCR, direct sequence analysis, and the next-generation sequencer Ion PGMTM. Results: Average age was 45 years (15-80 years). The inv16 AMLand t(8;21) AMLwere28 cases and 110 cases, respectively. KIT mutations were found in 62 (45%) cases of patients with CBF-AML. Patients with KIT mutations were significantly more frequently associated with male gender (p=0.029) and higher white blood cell count (>1×104/μl) (p=0.002) compared to KIT wild type cases. No significant differences were found in other clinical characteristics between those with and without KIT mutations. Analyzing all CBF-AML patients and inv16 AML, rates of relapse free survival (RFS) and overall survival (OS) in those with KIT mutations were significantly lower than those in patients without c-kit mutations (All patients: RFS, 38% vs 58% at 3 years after CR1, p=0.040; OS, 61% vs 77%, p=0.033; inv16 AML: RFS, 46% vs 82%, p=0.044; OS, 75% vs 100%, p=0.045). However, there was no significant difference between t(8;21)AML with and without KIT mutations (RFS, 35% vs 51%, p=0.219, OS, 58% vs 70%, p=0.161). Next, we analyzed prognosis of CBF-AML according to the types of c-kit mutations. D816, N822K, co-expression of D816 and N822K, and other mutations of KIT gene were detected in 29 cases (21%, D816A:1 case, D816Y:2 cases, D816V:26 cases), 20 (14%), 7 (5%, D816H:1 case, D816V:6 cases), and 6 cases (4%), respectively. (RFS, 58.2% vs 21.6% vs 58.9% vs 14.3% vs 60.0%, p=0.005; OS,77.2% vs 40.0% vs 78.9% vs 60.0% vs 100.0%, p Conclusions: In ASH meeting of 2014, we showed that the D816V confers higher proliferation activity by JAK-STAT and Src family kinase compared to N822K by in vitro assay. In this study, we showed that patients with D816 had a significantly poorer prognosis than those with N822K or other mutations of KIT in CBF-AML. Going forward, a study is needed in which a trial of autologous stem cell transplantation in first CR or conventional chemotherapy with dasatinib or novel molecular target drug such as PKC 412 for CBF-AML with D816. Disclosures No relevant conflicts of interest to declare.

Backgrounds: Acute myeloid leukemia (AML) is a heterogeneous disease whose onset involves a variety of chromosomal abnormalities and genetic mutations. To improve the outcome of AML treatment, it is very important to establish a prognosis from cytogenetic and genetic analysis and provide accordingly differentiated treatment. However, the treatment strategy has not been clear in elderly patients with AML due to intolerance of treatment and its adverse characteristics such as concomitant comorbidity and poor performance status. To clarify the prognostic impact of genetic abnormalities in elderly AML, we conducted a comprehensive analysis of recurrently mutated 28 genes in AML. Method: We retrospectively analyzed newly diagnosed 128 AML patients excluding M3 who were more than 65 years old at Nippon Medical School Hospital or its associated facilities between 1990 and 2014. Mutation analyses were performed by direct sequence analysis, Mutation Biased PCR, direct sequence analysis, and the next-generation sequencer Ion PGMTM. Results: Average age was 70.5 years (65-91years). The patients from 65 years old to 74 years old (early elderly) were 92 (71.9%)cases, the patients 75 years or older (late elderly) were 36 (28.1%)cases. Chromosomal analysis according to the prognostic risk classification of the Eastern Cooperative Oncology Group (ECOG) classified into 14 of the favorable cytogenetic risk group (10.9%), 80 of the intermediate cytogenetic risk group (62.5%), and 24 of the poor cytogenetic risk group (18.8%). Gene mutations were observed in 112 cases (87.5%). The most frequent gene mutations were NPM1 (89 cases/28.9%), FLT3/ITD (25 cases/19.5%), TET2 (20 cases/15.6%), and DNMT3A (19 cases/14.8%). CEBPA double mutation was detected at low frequency in elderly patients (younger patients: 15/311 (4.8%) vs elderly patients: 1/128 (0.8%)). There were no significant differences in gene mutations between early elderly and late elderly patients). Of 102 patients who received induction therapy, 52 patients (51.0%) achieved first hematological complete remission (CR), but primary refractory and early death were observed in 50 patients (49.0%). There was no significant difference in CR rate between the two groups in the early elderly patients and late elderly patients (early elderly: 53.8% vs late elderly: 41.7%. p= 0.354). In addition, there was no difference in CR rate in the standard chemotherapy and low dose chemotherapy group (standard chemotherapy: 48.7% vs low dose chemotherapy: 38.9%. p= 0.601). Median overall survival (OS) was 287days, OS at 3years was 35.9%. Prognostic stratification was possible for CR rate on the chromosomal classification (favorable cytogenetic risk 92.9%, intermediate cytogenetic risk 50.0%, poor cytogenetic risk 12.5%, p< 0.001). For total cohort of patients, rates of relapse free survival (RFS) at 3 years are significantly lower in patients with FLT3ITD (p=0.006) and TP53 abnormality (p< 0.001) compared to without it. Rates of overall survival (OS) at 3 years are significantly lower in patients with DNMT3A mutation (p=0.004), FLT3ITD (p=0.003), and TP53 abnormality compared to without it. Also late elderly patients (p=0.010) and high white blood cell count (≥ 20,000/μl) (p=0.023) were powerful factors for unfavorable prognosis. In stratified analysis of FLT3ITD-negative cases aged below 75 years with intermediate cytogenetic prognosis, TP53 abnormality was associated with unfavorable prognosis (RFS: p=0.062, OS: p< 0.001). Finally multivariate analysis demonstrated that FLT3ITD (p=0.014) and TP53 abnormality (p=0.015) were an independent poor prognostic factor for RFS, and poor cytogenetic risk (p=0.002), Flt3ITD (p=0.014), TP53 abnormality (p=0.015), and DNMT3A mutation (p=0.001) were an independent poor prognostic factor for OS. Conclusions: Our results suggest that the genetic abnormalities have a prognostic importance in elderly patients, as well as younger patients with AML. FLT3ITD mutation is an important factor for unfavorable prognosis, but going forward TP53 mutation may also serve as a clinically important gene mutation marker in elderly patients with AML. Disclosures No relevant conflicts of interest to declare.

Summary APOBEC3G (A3G) is an antiretroviral host factor that functions by deaminating dC to dU in retroviral cDNA [1–5]. HIV-1 Vif protein counteracts A3G via a ubiquitin-proteasome pathway [6–12]. In the case of a simple retrovirus such as the murine leukemia virus (MLV), it remains unclear why it can replicate in cells expressing APOBEC3 (A3) even though it doesn't possess any accessory proteins such as Vif [2, 13]. In this study, we demonstrate that MLV escapes from murine A3 (mA3) via two distinct novel mechanisms. First, viral RNA (vRNA) blocks the binding of mA3 to Gag, resulting in the exclusion of mA3 from MLV virions. Second, viral protease (vPR) cleaves mA3 after maturation of virions. Here, we suggest that each virus has its own strategy to escape from A3 proteins and that these mechanisms might be used by other viruses that do not possess Vif-like protein. On the other hand, mice possess another form of mA3, Δexon5, that escapes from the cleavage by vPR to show more antiviral activity than the wild type mA3. This also suggests that battles between host intrinsic immunity and viruses have led to the evolution of proteins on both sides.

APOBEC3 proteins are antiviral host factors for a wide variety of retroviruses. HIV-1 Vif overcomes the antiviral activity of APOBEC3G by ubiquitinating the protein. In this study, we examined the ability of Vif to antagonize other family members of APOBEC3 proteins, together with its mechanism. Using HIV infectivity, virion incorporation, immunoprecipitation, and in vitro ubiquitin conjugation assays, we show that the ability of Vif to inhibit antiviral activity of APOBEC3 proteins positively correlates with its ability to bind and ubiquitinate these proteins by a Vif-Cullin5-ElonginB-ElonginC (Vif-BC-Cul5) complex. These results suggest that Vif exhibits its anti-APOBEC3 activity by the ubiquitin ligase activity of the Vif-BC-Cul5 complex.

A highly sensitive method for the determination of bisphenol-A in water with semi-micro column high-performance liquid chromatography using 2-methoxy-4-(2-phthalimidinyl)phenylsulfonyl chloride as a fluorescent labeling reagent has been developed. The labeling reaction was carried out at 70 degrees C for 20 min in borate buffer (pH 9.5). The derivative eluted at 11.6 min on a reversed-phase column with methanol-water (78:22, v/v) at a flow-rate of 0.2 ml/min. The fluorescence was monitored at 308 nm for excitation and 410 nm for emission. The detection limit (S/N = 3) was 10 fmol per injection. The labeling yield was about 95%.

Human APOBEC3G (huAPOBEC3G), also known as CEM15, is a broad antiretroviral host factor that deaminates dC to dU in the minus strand DNA of human immunodeficiency virus type 1 (HIV-1), other lentiviruses, and murine leukemia virus (MLV), thereby creating G-to-A hypermutation in the plus strand DNA to inhibit the infectivity of these viruses. In this study, we examined the antiretroviral function of a murine homologue of APOBEC3G (muAPOBEC3G) on several retrovirus systems with different producer cells. MuAPOBEC3G did not suppress the infectivity of murine retroviral vectors produced from human or murine cells, whereas it showed antiviral activity on both wild-type and Δvif virions of HIV-1 in human cells. In contrast, huAPOBEC3G showed broad antiviral activity on HIV-1 and murine retroviral vectors produced from human cells as well as murine cells. These data suggested that muAPOBEC3G does not possess antiretroviral activity on murine retroviruses and has a different target specificity from that of huAPOBEC3G and that huAPOBEC3G works as a broad antiviral factor not only in human cells but also in murine cells. A functional interaction study between human and murine APOBEC3G supported the former hypothesis. Furthermore, studies on the expression of APOBEC3G in producer cells and its incorporation into virions revealed that muAPOBEC3G is incorporated into HIV-1 virions but not into MLV virions. Thus, muAPOBEC3G cannot suppress the infectivity of murine retrovirus because it is not incorporated into virions. We suggest that murine retroviruses can replicate in murine target cells expressing muAPOBEC3G because they are not targets for this enzyme.

All plants examined to date possess non-symbiotic hemoglobin whose physiological role remains unclear. The present study explored the catalytic function of three representative classes of the plant hemoglobin from Arabidopsis thaliana: AtGLB1, AtGLB2, and AtGLB3. Purified recombinant proteins of these hemoglobins displayed hydrogen peroxide-dependent oxidation of several peroxidase substrates that was sensitive to cyanide, revealing intrinsic peroxidase-like activity. In the presence of nitrite and hydrogen peroxide, AtGLB1 was the most efficient at mediating tyrosine nitration of its own and other proteins via the formation of reactive nitrogen species as a result of nitrite oxidation. AtGLB1 mRNA significantly accumulated in Arabidopsis seedlings exposed to nitrite, supporting the physiological relevance of its function to nitrite and nitrite-derived reactive nitrogen species.

Human immunodeficiency virus, type 1 (HIV-1) Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion. Vif functions to counteract an anti-HIV-1 cellular factor in non-permissive cells, CEM15/Apobec-3G, which shares a cytidine deaminase motif. CEM15/Apobec-3G deaminates dC to dU in the minus strand DNA of HIV-1, resulting in G to A hypermutation in the plus strand DNA. In this study, we have done the mutagenesis analysis on two cytidine deaminase motifs in CEM15/Apobec-3G and examined their antiviral functions as well as the DNA editing activity. Point mutations in the C-terminal active site such as E259Q and C291A almost completely abrogated the antiviral function, while those in the N-terminal active site such as E67Q and C100A retained this activity to a lesser extent as compared with that of the wild type. The DNA editing activities of E67Q and E259Q mutants were both retained but impaired to the same extent. This indicates that the enzymatic activity of this protein is essential but not a sole determinant of the antiviral activity. Furthermore, all the deletion mutants tested in this study lost the antiviral activity because of the loss of the activity for dimerization, suggesting that the entire protein structure is necessary for the antiviral function.

Background Recent studies have shown that mutations in internal tandem duplication (ITD) of the FLT3 gene (FLT3-ITD) influence prognosis of cytogenetically normal AML. Our previous decision analysis (Kurosawa et al, Blood 2011) showed that, for patients with cytogenetically standard-risk AML in first complete remission (CR1), allogeneic hematopoietic cell transplantation (HCT) provided better survival with and without a quality-of-life (QOL) adjustment. In this study, we undertook a decision analysis to determine a favored treatment strategy for patients with cytogenetically standard-risk AML in CR1 depending on FLT3-ITD profile. Patients and Methods Inclusion criteria comprised patients aged 16 to 70 years who were diagnosed with intermediate- or unknown-risk AML according to the Southwest Oncology Group cytogenetic classification, and achieved CR1. We collected bone marrow or peripheral blood samples obtained at diagnosis for patients registered, and retrospectively analyzed mutations of FLT3-ITD using DNA extracted from the specimens. We constructed a Markov decision model to reflect outcomes of HCT and chemotherapy in CR1 with three Markov health states; (1) alive after HCT, (2) alive after chemotherapy, or (3) dead. After the decision node of HCT, we added a branch to consider patients who had relapse before receiving HCT in CR1. Cycle length was 3 months and the analyses were performed for 40 cycles, 10 years. Transition probabilities between health states were calculated from the underlying survival rate of the cohort registered. Results were obtained as life expectancy (LE) and QOL-adjusted life expectancy (QALE). We conducted another cross-sectional study to investigate patient-reported QOL from those treated for acute leukemia. Adjusted means of EQ-5D index were adapted from the study as utility estimates that reflect QOL after HCT or chemotherapy (overall mean: post HCT overall, 0.74; post HCT with GVHD, 0.67; post chemotherapy overall, 0.70), and were allowed to change over time ( Results Among 541 patients registered in the database, mutations in FLT3-ITD were successfully tested in 332 patients (61%). The median age of patients was 52 years and the median follow up was 37 months among survivors. FLT3-ITD mutations were found in 60 patients (18%). Eighty-five patients received allogeneic HCT in CR1 and the other 247 patients were treated with chemotherapy alone during the period of CR1. Patients who received HCT were significantly younger (median age, 41 vs 54 years), more frequently received 2 courses of induction therapy and had dysplasia at diagnosis compared to those who were treated with chemotherapy. In FLT3-positive and FLT3-negative patients, similar proportions of patients received HCT in CR1 (23%, N=14 vs 26%, N=71). In 10-year observation, LE and QALE were 5.9 and 5.1 years, respectively, for total patients. For FLT3-positive patients, allogeneic HCT in CR1 improved LE and QALE by 22 and 17 months, respectively (Table). QALE benefit of HCT was still apparent when we used utility estimates for post HCT with GVHD. Sensitivity analyses across the range of plausible utility estimates (HCT, 0.63-0.83; chemotherapy, 0.64-0.75) did not change the conclusion. For FLT-3 negative patients, HCT benefit was less than 1 year for LE and QALE. Sensitivity analyses showed that chemotherapy would be the preferred strategy when utility estimates for chemotherapy was higher than 0.7. An analysis of FLT3-negative patients stratified by age indicated that HCT in CR1 provided better LE and QALE than chemotherapy alone for patients aged 55 years or older (LE: 77 vs 57; QALE: 54 vs 38 months). In contrast, HCT did not improve LE (74 vs 79 months) or QALE (52 vs 54 months) compared to chemotherapy alone in younger patients with FLT3-negative AML. Conclusion For patients with FLT3-ITD-positive, cytogenetically standard-risk AML, allogeneic HCT in CR1 offered overall and QOL-adjusted survival benefit. For patients without FLT3-ITD, survival benefit of HCT in CR1 was less apparent. Disclosures No relevant conflicts of interest to declare.

Objective The prognosis for elderly patients with acute myeloid leukemia (AML) remains dismal. To explore the potential of immunotherapy for improving clinical outcomes for these patients, we performed a phase I clinical trial of dendritic cell (DC)−based immunotherapy for elderly patients with AML. Materials and Methods Autologus monocytes were obtained after reducing tumor burden by chemotherapy. Immature DCs induced with granulocyte-macrophage colony-stimulating factor and interleukin-4 were pulsed with autologous apoptotic leukemic cells as antigens. DCs were administered intradermally to four patients five times at 2-week intervals. To facilitate DC migration to lymph nodes, injection sites were pretreated with killed Streptococcus pyogenes OK-432 one day before. DCs were coinjected with OK-432 to induce maturation and interleukin-12 production in vivo. Results Antileukemic responses were observed by an interferon-γ enzyme-linked immunospot assay or a tetramer assay in two of four patients. In a human leukocyte antigen−A∗2402-positive patient, induction of CD8 + T-cell responses to WT1- and human telomerase reverse transcriptase – derived peptides were observed, indicating cross-priming in vivo. The two patients with antileukemic immunity showed longer periods of disease stabilization than the other two patients. Conclusions This study demonstrates the immunogenicity of autologous DCs that cross-present leukemia-associated antigens from autologous apoptotic leukemic cells in vivo in elderly patients with AML.

1. 1. A new enzyme which catalyses the reversible condensation of pyruvate and glyoxylate to form γ-hydroxy-α-ketoglutarate was purified in excess of 120-fold from rat-liver acetone powder. 2. 2. The enzyme was specific for glyoxylate and pyruvate. Condensation could not be demonstrated between other aldehydic acids and keto acids which were tested, distinguishing this enzyme from the formaldehyde and pyruvate condensing enzyme (2-oxo-4-hydroxybutyrate formaldehyde-lyase, EC 4.1.2.1) and the cleavage enzyme of γ-hydroxyglutamate (4-hydroxyglutamate glyoxylate-lyase). The apparent Km values (at optimal pH, 7.5) for pyruvate, glyoxylate and γ-hydroxy-α-ketoglutarate were 10 mM, 0.43 nM and 1.33 mM, respectively. The equilibrium facors the biosynthesis of γ-hydroxy-α-ketoglutarate. 3. 3. The product of the enzymic condensing reaction was isolated as the Ca salt and identified as γ-hydroxy-α-ketoglutaric acid from its chemical properties. 4. 4. On the basis of the experimental findings, the possible metabolic pathways of pyruvate, glyoxylate and γ-hydroxyglutamate (the main oxidation product of L-hydroxyproline) and the regulating role of glyoxylate for the tricarboxylic acid cycle were discussed.

1. 1. An enzyme which catalyses the transamination between γ-hydroxyglutamate and α-ketoglutarate was extracted from rat-liver acetone powder and purified in excess of 15-fold. Fractionation of the enzyme from a separate aspartate transaminase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) remained incomplete. 2. 2. [2-14C]Pyruvate was incorporated into γ-hydroxy-α-ketoglutarate and γ-hydroxyglutamate in the presence of glyoxylate in rat-liver homogenate. Specific activity values of these three compounds indicated the occurence of the following pathway: Pyruvate + glyoxylate ⇌ γ-hydroxy-α-ketoglutarate ⇌ γ-hydroxyglutamate. 3. 3. A further possible pathway of γ-hydroxy-α-ketoglutarate metabolism leading to malate and of γ-hydroxyglutamate to its decarboxylation product or to L-hydroxyproline were inferred and discussed.