Function of cytochrome P450 enzymes MycCI and MycG in Micromonospora griseorubida, a producer of the macrolide antibiotic mycinamicin.

The cytochrome P450 enzymes MycCI and MycG are encoded within the mycinamicin biosynthetic gene cluster and are involved in the biosynthesis of mycinamicin II (a 16-membered macrolide antibiotic produced by Micromonospora griseorubida). Based on recent enzymatic studies, MycCI is characterized as the C-21 methyl hydroxylase of mycinamicin VIII, while MycG is designated multifunctional P450, which catalyzes hydroxylation and also epoxidation at C-14 and C-12/13 on the macrolactone ring of mycinamicin. Here, we confirm the functions of MycCI and MycG in M. griseorubida. Protomycinolide IV and mycinamicin VIII accumulated in the culture broth of the mycCI disruption mutant; moreover, the mycCI gene fragment complemented the production of mycinamicin I and mycinamicin II, which are produced as major mycinamicins by the wild strain M. griseorubida A11725. The mycG disruption mutant did not produce mycinamicin I and mycinamicin II; however, mycinamicin IV accumulated in the culture broth. The mycG gene was located immediately downstream of the self-resistance gene myrB. The mycG gene under the control of mycGp complemented the production of mycinamicin I and mycinamicin II. Furthermore, the amount of mycinamicin II produced by the strain complemented with the mycG gene under the control of myrBp was approximately 2-fold higher than that produced by the wild strain. In M. griseorubida, MycG recognized mycinamicin IV, mycinamicin V, and also mycinamicin III as the substrates. Moreover, it catalyzed hydroxylation and also epoxidation at C-14 and C-12/13 on these intermediates. However, C-14 on mycinamicin I was not hydroxylated.

Enhanced pyrite passivation by carrier-microencapsulation using Fe-catechol and Ti-catechol complexes.

Acid mine drainage (AMD) formation is mainly caused by the oxidation of pyrite. Carrier-microencapsulation (CME) using metal-catecholate complexes has been proposed to passivate sulfide minerals by forming surface-protective coatings on their surfaces. Among the various metal-catecholate complexes, Ti-catecholate formed stable coatings having superior acid-resistance, but a thick enough passivating film required considerable time (ca. 14 days) to grow. Meanwhile, Fe-catecholates can form Fe-oxyhydroxide coatings within 2 days, however, they are less stable than Ti-based coating. To address these drawbacks of using a single metal-complex, this study investigated the concurrent use of Fe-catechol and Ti-catechol complexes for accelerating the formation of stable passivating coating on pyrite. Compared with a single metal-complex system, the coating formation was significantly accelerated in mixed system. Linear sweep voltammetry showed the simultaneous decomposition of [Fe(cat)]+ and [Ti(cat)3]2- as the main reason for improved coating formation. Electrochemical properties of coatings formed by single and mixed complex systems, confirmed by electrochemical impedance spectroscopy and cyclic voltammetry, indicated the coating formed in the mixed system had higher resistance and more electrochemically inert than the other cases. The simultaneous use of Fe-catechol and Ti-catechol complexes enhanced pyrite passivation by accelerating metal-complex decomposition and forming more stable coating composed of Fe2TiO5.

Suppressive effects of ferric-catecholate complexes on pyrite oxidation.

Pyrite, a common gangue mineral in complex sulfide ores and coals, is rapidly oxidized in water by ferric ions and dissolved oxygen to form a very acidic and heavy metal-laden leachate called acid mine drainage (AMD). Carrier-microencapsulation (CME) using Ti4+, Si4+, and Al3+ was reported as a promising new approach to prevent pyrite oxidation by forming a passivating barrier on the pyrite surface. In CME, the presence of Fe3+-catecholate complexes is unavoidable but their effects on pyrite oxidation remain unclear. In this study, the effects of Fe3+-catecholate complexes on pyrite oxidation were investigated. Formations of mono-, bis-, and tris-catecholate complexes of Fe3+ were verified by UV-Vis spectrophotometry and their speciation with pH was consistent with thermodynamic considerations. Linear sweep voltammetry was conducted to evaluate the redox properties of Fe3+-catecholate complexes, and the results indicate that ligands in the three complexes were sequentially oxidized until Fe3+ is released. Coating formation on pyrite was confirmed after treatment with mono- and bis-catecholate complexes. Results of SEM-EDX and ATR-FTIR indicate that the coating is composed primarily of iron oxyhydroxide phases. The results of leaching experiments showed that pyrite oxidation was suppressed by Fe3+-catecholate complexes via two mechanisms: (1) electron donating effects of the complexes, and (2) formation of a protective coating on pyrite. The results provide not only a better understanding of the effects of Fe3+-catecholate complexes on pyrite oxidation but also some possible applications of Fe3+-based CME such as the suppression of pyrite oxidation to prevent AMD formation and depression of pyrite floatability in mineral processing.

Epileptogenic activity induced by histamine H(1) antagonists in amygdala-kindled rats.

The epileptogenic activities induced by histamine H(1) antagonists in amygdala-kindled rats were studied in comparison with activities in nonkindled rats (sham rats). Intraperitoneal injection of pyrilamine, diphenhydramine and ketotifen resulted in behavioral and electroencephalogram (EEG)-detected seizures in amygdala-kindled rats at doses which caused no or negligible seizures in sham rats. On the other hand, loratadine and cetirizine caused no behavioral or EEG seizures in either amygdala-kindled or sham rats even at a dose of 40 mg/kg. In conclusion, first-generation H(1) antagonists likely elicit epileptogenic activity in amygdala-kindled rats more potent than that in sham rats.

Inhibitory effect of iodophenpropit, a selective histamine H3 antagonist, on amygdaloid kindled seizures.

The effect of histamine H(3) antagonist, iodophenpropit on amygdaloid kindled seizures in rats was studied in comparison with those of other H(3) antagonists. Under pentobarbital anesthesia, the rats were fixed to a stereotaxic apparatus and bipolar electrodes were implanted into the amygdala. Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. To cause kindled seizures, electrical stimulation was applied to the amygdala bipolarly every day by a constant current stimulator, and electroencephalogram and convulsive behavior were observed. Drug effects were estimated in rats showing generalized kindled seizures. Intraperitoneal injection of H(3) antagonists, iodophenpropit, thioperamide, AQ0145 and clobenpropit, resulted in a dose-related inhibition of amygdaloid kindled seizures. The effect of iodophenpropit on amygdaloid kindled seizures was more potent than those of thioperamide, AQ0145 and clobenpropit. In conclusion, iodophenpropit may be useful for the treatment of partial epilepsy and/or secondary generalized seizures in humans.

Involvement of the Wnt-ß-catenin pathway in invasion and migration of oral squamous carcinoma cells.

The Wnt/ß-catenin pathway plays a critical role in cell proliferation and oncogenesis. To clarify the role of cytoplasmic accumulation of ß-catenin in oral squamous cell carcinoma (SCC), the cDNA of a mutant form of ß-catenin that lacks the entire region with the glycogen synthase kinase-3 ß (GSK-3ß)-specific phosphorylation site was transfected into Ca9-22 cells whose ß-catenin had been expressed predominantly at the membrane, and permanent cell lines expressing aberrant ß-catenin in the cytoplasm and nucleus were produced. These transfectants, C1 and C5, proliferated at similar rates to the parental Ca9-22 cells, but the cell morphology changed from polygonal to spindle-shaped and close cell-cell interaction was lost. These mutant ß-catenin-expressing cells exhibited a significantly higher invasion/migration capacity than wild-type Ca9-22 cells. The transcriptional activities of this mutant ß-catenin form was enhanced in these cells which could be demonstrated by an elevated level of the transcription factor T-cell factor (Tcf)/lymphoid enhancer factor (Lef)-dependent reporter gene activity as well as by the up-regulation of Wnt/ß-catenin target gene matrix metalloproteinase (MMP)-7. Moreover, we observed the redistribution of E-cadherin, the rearrangement of actin filaments, and the elevation of active Rho family members, Cdc42 and Rac. These results suggest that aberrant cytoplasmic accumulation of ß-catenin can induce Tcf/Lef-mediated transcriptional activity, up-regulate MMP-7, and induce epithelial and mesenchymal transition (EMT). This would enhance the invasion and migration of oral SCC cells.

Latent hypoparathyroidism in an osteoporotic patient with multiple endocrinopathies and secondary hemochromatosis due to multiple blood transfusions, unmasked by alendronate and glucocorticoid at adrenal crisis.

A 30-year-old normocalcemic man with hypopituitarism, hypogonadism, diabetes mellitus, and secondary hemochromatosis due to multiple blood transfusions was admitted because of adrenal crisis. After intravenous administration of saline and cortisol, the corrected serum level of calcium decreased to 7.3 mg/dl. This osteoporotic patient had been prescribed alendronate for radial bone fracture. Since the increase in intact PTH (68 pg/ml) was impaired compared to that seen in hypocalcemic patients with secondary hyperparathyroidism, we presume that the patient has had latent hypoparathyroidism, which was unmasked by the administration of glucocorticoid and bisphosphonate. With a supplemented dose of 1alpha-OHD3, the patient has been eucalcemic.

Expression of type 5 somatostatin receptor in TSH-secreting pituitary adenomas: a possible marker for predicting long-term response to octreotide therapy.

In TSH-secreting pituitary adenomas (TSHoma), octreotide (OCT) therapy reduces tumor size and TSH secretion in some cases but not in others. As OCT acts through various types of somatostatin receptors (SSTRs), the different responses of TSHoma to OCT might be explained by the differences of SSTR expression. We therefore studied the expression of subtype-specific SSTR mRNA transcripts in tumor tissues by RT-PCR. Type 2 (SSTR2) mRNA transcripts were detected in all 8 tumors but those of SSTR3 and SSTR5 were demonstrated only in 5 of them. Serum TSH levels were decreased by OCT administration test in all patients but OCT therapy was effective in two patients out of three. SSTR5 mRNA was detected in two tumors from the responder, but not in one tumor that was resistant to OCT. These observations suggest that the temporal decrease of TSH by OCT may be mediated by SSTR2, and that the long term response to OCT therapy may be related with the expression of SSTR5. Therefore, the expression of SSTR5 in TSHoma may be a useful marker for predicting the outcome of the therapy, but further studies with larger numbers of patients are necessary.

Development of amygdaloid kindling in histidine decarboxylase-deficient and histamine H1 receptor-deficient mice.

PURPOSE: This study attempted to clarify the role of histamine or histamine H1 receptors in the development of amygdaloid kindling by using histidine decarboxylase (HDC)-deficient and histamine H1 receptor (H1R)-deficient mice. METHODS: Under pentobarbital anesthesia, mice were fixed to a stereotaxic apparatus, and bipolar electrodes were implanted into the right amygdala. Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. A bipolar electroencephalogram was recorded; bipolar stimulation of the amygdala was applied every day with a constant-current stimulator and continued until a generalized convulsion was obtained. RESULTS: The development of amygdaloid kindling in HDC-deficient and H1R-deficient mice was significantly accelerated compared with that in their respective wild-type mice. In addition, the afterdischarge (AD) duration and generalized seizure duration in HDC-deficient and H1R-deficient mice were prolonged. Intraperitoneal injection of histidine resulted in an inhibition of amygdaloid kindled seizures in wild-type mice at doses that caused an increase in the histamine contents of the brain. However, no significant effect was observed with histidine in H1R-deficient mice at the same dose. CONCLUSIONS: These findings suggest that histaminergic mechanisms through H1 receptors play a crucial role not only in amygdaloid kindled seizures but also in the development of amygdaloid kindling.