Subcellular localization of aphidicolin biosynthetic enzymes heterologously expressed in Aspergillus oryzae.

The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.

Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae.

In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker.

Clinical experiences of bixalomer usage at our hospital.

In 2012, bixalomer was launched as new non-calcium (Ca) containing phosphorus (P) binder, increasing the choices available for the treatment of hyperphosphatemia. In this study, among the maintenance dialysis patients at our hospital, we newly administered bixalomer to 21 patients who were not receiving any P binders, and switched to bixalomer for 13 patients who had been receiving sevelamer hydrochloride and 23 patients who had been receiving lanthanum carbonate. The initial dosage of bixalomer was set as 1500 mg/day for new administration patients and dosage equivalent to that of the previously-used P binder for patients who were switched to bixalomer. The dosage of bixalomer was increased if the effects were insufficient. The serum P, Ca and intact parathyroid hormone concentrations as well as serum pH, HCO3 concentration and base excess were evaluated prior to administering bixalomer, 3 months and 6 months after administering bixalomer. For the group who were newly administered bixalomer, significant reductions in serum P concentrations were seen (P<0.01) and no significant changes were seen in clinical test items that serve as indices for acidosis. For the group who were switched from sevelamer hydrochloride to bixalomer, significant reductions in serum P concentrations were seen (P<0.01) together with significant improvements in acidosis (P<0.01). For the group who were switched from lanthanum carbonate to bixalomer, by increasing the dosage of bixalomer to approximately three times the dosage of lanthanum carbonate, it was possible to maintain post-switch serum P concentrations at almost the same levels as before the switch. Furthermore, there were minor, yet significant improvements in acidosis (P<0.01). From these results, it was shown that bixalomer can be useful treatment alternative in dialysis patients for whom it is necessary to change the P binder due to insufficient management of serum P concentrations or development of acidosis.

Time-course of health status in patients with rheumatoid arthritis during the first year of treatment with infliximab.

OBJECTIVES: A longitudinal study was performed to examine changes in health status in comparison with rheumatoid arthritis (RA) inflammation in patients with RA during the first 54 weeks of infliximab (IFX) treatment. METHODS: Health status in active RA patients (n=13) was assessed monthly using the Arthritis Impact Measurement Scale 2 (AIMS2) and the VAS-GH during the first year of IFX treatment. Simultaneously, RA activity was assessed using inflammation markers, MMP-3 and the Disease Activity Score in 28 joints (DAS-28) based on CRP [DAS-28(CRP)] and ESR[DAS-28(ESR)]. RESULTS: Serum CRP and ESR decreased significantly from 2.14+/-0.52mg/dL and 56.9+/-6.96mm/h, respectively, at baseline to 0.24+/-0.11mg/dL and 31.6+/-4.39mm/h, respectively, at 2 weeks after initiation of IFX. Other inflammatory markers and MMP-3 were also suppressed significantly after 2 weeks of IFX treatment. DAS-28(CRP) and DAS-28(ESR) were also significantly decreased after 2 weeks and suppression of both DAS values remained significant until 54 weeks of IFX treatment. After initiation of IFX, patient-reported general health also showed a significant improvement based on the changes in the six summary component scores on the AIMS2 (physical, affect, symptom, role, social interaction, and patient satisfaction). These scores all improved progressively until 14-18 weeks after initiation of IFX treatment, and then exhibited a temporary but insignificant exacerbation. The six components of the physical score also improved in a time-dependent manner until 14-18 weeks, but the scores for walking and bending, hand and finger function, arm function, self-care, and household tasks showed significant exacerbation at 22-30 weeks. The score for mobility level did not show this change. CONCLUSION: IFX treatment significantly improved both RA disease activity and health status in active RA patients. Time-dependent improvement of ADL until 14-18 weeks after initiation of IFX treatment, as reflected in the six components of the physical score, might have contributed to the temporary exacerbation of health status thereafter in these patients.

Beneficial effect of risedronate on arterial thickening and stiffening with a reciprocal relationship to its effect on bone mass in female osteoporosis patients: a longitudinal study.

AIMS: A longitudinal study was performed to examine the effect of risedronate on arterial thickening and stiffening in postmenopausal female osteoporosis patients. MAIN METHODS: Patients treated with risedronate (2.5mg/day) (n=33) and those that did not receive risedronate (n=30, control group) were monitored over a 1-year period. Bone metabolic markers, bone mineral density (BMD) of the femur neck (FN), brachial-ankle pulse wave velocity (baPWV), and intima-media thickness at the carotid artery (CA-IMT) were measured. KEY FINDINGS: At baseline, there was no significant difference in blood pressure, serum lipid profiles, FN BMD, baPWV and CA-IMT between the risedronate-treated patients and the controls. Baseline levels of FN BMD were significantly negatively correlated with those of CA-IMT and baPWV. During the study, FN BMD increased significantly in the risedronate group (p=0.0097), but decreased significantly in the control group (p=0.0013). BaPWV and CA-IMT did not change significantly in the risedronate group, but both increased significantly in the control group. The percentage change in FN BMD over the study period showed a significant negative correlation with those for baPWV (r=-0.294, p=0.0262) and CA-IMT (r=-0.305, p=0.0234) in all subjects (risedronate-treated patients and controls). SIGNIFICANCE: In addition to increasing BMD, risedronate significantly suppressed the progression of arterial thickening and stiffening in postmenopausal osteoporotic patients over one year. These changes may indirectly be due to the effect of risedronate on bone.