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In this study, we report a tumor that resembled previously reported uncommon tumors histologically similar to ameloblastoma or adenomatoid odontogenic tumor (AOT), showing the formation of hard tissue. We evaluated the histological characteristics by reviewing the literature. The patient was a 19-year old male. The lesion was located from the canine to third molar in the right mandible and was unicystic with a comparatively clear demarcation. The tumor tissue was cystic overall, showing multiple formation of small and large cysts. The tumor tissue resembled a variant form of plexiform ameloblastoma. Formation of dentin and dentinoid was observed in the tumor stroma, whereas formation of enamel was not observed. Very few cases of a variant form of ameloblastoma that shows formation of dentinoid have been reported, and the histological picture in this study closely resembled previously reported "adenoid ameloblastoma with dentinoid". [ABSTRACT FROM AUTHOR]

This paper proposes a gravity-based system capable of generating high-resolution pressure for precise cell manipulation or evaluation in a microfluidic channel. While the pressure resolution of conventional pumps for microfluidic applications is usually about hundreds of pascals as the resolution of their feedback sensors, precise cell manipulation at the pascal level cannot be done. The proposed system successfully achieves a resolution of 100 millipascals using water head pressure with an in-phase noise cancelation mechanism. The in-phase mechanism aims to suppress the noises from ambient vibrations to the system. The proposed pressure system is tested with a microfluidic platform for pressure validation. The experimental results show that the in-phase mechanism effectively reduces the pressure turbulence, and the pressure-driven cell movement matches the theoretical simulations. Preliminary experiments on deformability evaluation with red blood cells under incremental pressures of one pascal are successfully performed. Different deformation patterns are observed from cell to cell under precise pressure control.

FD-594 is an unique pyrano[4',3':6,7]naphtho[1,2-b]xanthene polyketide with a trisaccharide of 2,6-dideoxysugars. In this study, we cloned the FD-594 biosynthetic gene cluster from the producer strain Streptomyces sp. TA-0256 to investigate its biosynthesis. The identified pnx gene cluster was 38143 bp, consisting of 40 open reading frames, including a minimal PKS gene, TDP-olivose biosynthetic genes, two glycosyltransferase genes, two methyltransferase genes and many oxygenase/reductase genes. Most of these enzymes coded in the pnx cluster were reasonably assigned to a plausible biosynthetic pathway for FD-594, in which an unique ring opening process via Baeyer-Villiger-type oxidation catalyzed by a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase, is speculated to lead to the unique xanthene structure. To clarify the involvement of pnx genes in the FD-594 biosynthesis, a glycosyltransferase, PnxGT2, and a methyltransferase, PnxMT2, were characterized enzymatically with the recombinant proteins expressed in Escherichia coli. As a result, PnxGT2 catalyzed the triple olivose transfers to the FD-594 aglycon with TDP-olivose as the glycosyl donor to afford triolivoside. Surprisingly, in the PnxGT2 enzymatic reaction, tetraolivoside and pentaolivoside were significantly detected along with the expected triolivoside. To our knowledge, PnxGT2 is the first contiguous oligosaccharide-forming glycosyltransferase in secondary metabolism. Furthermore, addition of PnxMT2 and S-adenosyl-L-methionine into the PnxGT2 reaction mixture afforded natural FD-594 to confirm that the PnxGT2 reaction product was the expected regiospecifically glycosylated compound. Consequently, the identified pnx gene cluster appears to be involved in FD-594 biosynthesis.

FD-891 is a 16-membered cytotoxic antibiotic macrolide that is especially active against human leukemia such as HL-60 and Jurkat cells. We identified the FD-891 biosynthetic (gfs) gene cluster from the producer Streptomyces graminofaciens A-8890 by using typical modular type I polyketide synthase (PKS) genes as probes. The gfs gene cluster contained five typical modular type I PKS genes (gfsA, B, C, D, and E), a cytochrome P450 gene (gfsF), a methyltransferase gene (gfsG), and a regulator gene (gfsR). The gene organization of PKSs agreed well with the basic polyketide skeleton of FD-891 including the oxidation states and alpha-alkyl substituent determined by the substrate specificities of the acyltransferase (AT) domains. To clarify the involvement of the gfs genes in the FD-891 biosynthesis, the P450 gfsF gene was inactivated; this resulted in the loss of FD-891 production. Instead, the gfsF gene-disrupted mutant accumulated a novel FD-891 analogue 25-O-methyl-FD-892, which lacked the epoxide and the hydroxyl group of FD-891. Furthermore, the recombinant GfsF enzyme coexpressed with putidaredoxin and putidaredoxin reductase converted 25-O-methyl-FD-892 into FD-891. In the course of the GfsF reaction, 10-deoxy-FD-891 was isolated as an enzymatic reaction intermediate, which was also converted into FD-891 by GfsF. Therefore, it was clearly found that the cytochrome P450 GfsF catalyzes epoxidation and hydroxylation in a stepwise manner in the FD-891 biosynthesis. These results clearly confirmed that the identified gfs genes are responsible for the biosynthesis of FD-891 in S. graminofaciens.

The 16-membered macrolide FD-891 exerts cytotoxicity toward several cancer cell lines. In this study, we showed that FD-891 induces apoptosis in various human cancer cell lines. Human leukemia Jurkat cells were highly sensitive to FD-891, exhibiting caspase activation and mitochondrial release of cytochrome c into the cytosol at early time points after exposure to FD-891. By contrast, Jurkat cells deficient in caspase-8 were resistant to FD-891-induced apoptosis and manifested little induction of cytochrome c release as well as caspase-9 processing. Consistent with these results, the overexpression of the Bcl-2 family member Bcl-x(L) or the caspase-8 modulator c-FLIP(L) markedly prevented FD-891-induced apoptosis. These results clearly demonstrate that FD-891 triggers caspase-8-dependent mitochondrial release of cytochrome c and subsequent apoptosis in Jurkat cells.

The first asymmetric total synthesis of FD-838, a naturally occurring azaspirobicyclic product, has been accomplished allowing determination of its absolute stereochemistry.

A new neuritogenic compound NGA0187 was isolated from the fermentation broth of Acremonium sp. TF-0356. The structure of NGA0187 was determined by means of spectroscopic analysis and X-Ray diffraction. NGA0187 induced significant neurite outgrowth in PC12 cells. However, survival effect of NGA0187 on the primary culture of cerebral cortical neurons was not observed.

Background: Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA --> vRNA synthesis. The protease related activity of PA has been discussed ever since protease-inducing activity was demonstrated in transfection experiments., Results: PA protein was highly purified from insect cells infected with the recombinant baculovirus carrying PA cDNA, and a novel chymotrypsin-type serine protease activity was identified with the synthetic peptide, Suc-LLVY-MCA, in the PA protein. [3H]DFP was crosslinked with PA and a mutational analysis revealed that serine624 was as an active site for the protease activity., Conclusions: These results constitute the demonstration of protease activity in PA subunit of the influenza virus RNA polymerase complexes.

A novel potentiator of nerve growth factor (NGF), NG-061, which had been isolated from the fermentation broth of Penicillium minioluteum F-4627, was synthesized from methoxybenzoquinone and phenylacetylhydrazine in a single step. A series of acyl hydrazone derivatives were also synthesized and their potentiator activity of neurotrophic effect of NGF on neurite outgrowth was evaluated by assay with a rat pheochromocytoma cell line PC12.

The absolute stereochemistry of FD-594 1, a new cytotoxic antibiotic, was determined by X-ray diffraction, and its conformation was studied by CD and NMR spectroscopy. The aglycon part of 1 was found to have (3R,6S,7S) configuration. Particularly interesting was the solvent-dependent atropisomerism of 1 and related compounds. The CD spectra of 1 exhibited in two solvent systems almost opposite mirror-image curves depending on the solvent. While a large negative Cotton effect (Deltaepsilon = -33.9, 279 nm) was observed in CHCl(3), a similar positive Cotton effect (Deltaepsilon = 38.9, 279 nm) appeared in methanol most probably due to dramatic conformational changes. Similar chiroptical reversal was observed in aglycon 2 and aglycon methyl ether 4. These results can be best described in terms of solvent-dependent atropisomerism. This constitutes the first observation of solvent-dependent atropisomerism of a natural product. The crucial factor that perturbs the stable conformation in different solvents is discussed on the basis of molecular mechanics calculations.

During the course of our screening program for low molecular natural products with their ability to potentiate and/or mimic neurotrophic effect of NGF, a novel fungal metabolite, phenylacetic acid hydrazide derivative NG-061 was isolated from the fermentation broth of Penicillium minioluteum F-4627. NG-061 enhanced and mimicked neurotrophic effect of NGF on neurite outgrowth in a rat pheochromocytoma cell line PC12.

The structure of NG-061, a new potentiator of nerve growth factor (NGF) isolated from Penicillium minioluteum F-4627, was determined by spectroscopic analysis and X-ray diffraction method to be phenylacetic acid 2-(2-methoxy-4-oxocyclohexa-2,4-dienylidene)-hydrazide.

This paper focuses on the inhibitory mechanism of E-64 and its derivatives (epoxysuccinyl-based inhibitors) with some cysteine proteases, based on the binding modes observed in the x-ray crystal structures of their enzyme-inhibitor complexes. E-64 is a potent irreversible inhibitor against general cysteine proteases, and its binding modes with papain, actinidin, cathepsin L, and cathepsin K have been reviewed at the atomic level. E-64 interacts with the Sn subsites of cysteine proteases. Although the Sn-Pn (n = 1-3) interactions of the inhibitor with the main chains of the active site residues are similar in respective complexes, the significant difference is observed in the side-chain interactions of S2-P2 and S3-P3 pairs because of different residues constituting the respective subsites. E-64-c and CA074 are representative derivatives developed from E-64 as a clinical usable and a cathepsin B-specific inhibitors, respectively. In contrast with similar binding/inhibitory modes of E-64-c and E-64 for cysteine proteases, the inhibitory mechanism of cathepsin B-specific CA074 results from the binding to the Sn' subsite.

The Ile-Pro sequence of CA074, potent covalent-type inhibitor, is necessary to exhibit the specificity for cathepsin B, but not for papain. In order to elucidate how its sequence binds to papain and why such binding does not exhibit the specificity for papain at the atomic level, two CA074-related compounds, 1 (N-(L-3-carboxyloxirane-2-carbonyl)-L-isoleucyl-L-proline) and 2 (N-(L-3-carboxyloxirane-2-carbonyl)-L-isoleucyl-diethylamide), were designed and their structure--inhibitory activity relationship was investigated by the X-ray crystal analyses of the complexes with papain. The Ile-Pro moiety of 1 was located at the S2 and S3 subsites consisting of Val-133, Val-157, and Asp-158 and of Tyr-61, Gly-66, and Tyr-67 residues of papain, respectively, which is in contrast with the binding of CA074 to S'n (n = 1 approximately 2) subsites in the complex with cathepsin B. Although 2 in the complex with papain showed the similar binding pattern to 1, its inhibitory activity was about two-fold higher than of 1, suggesting the importance of tight S3-P3 hydrophobic interaction for the activity. The difference of the substrate specificity between papain and cathepsin B has also been discussed based on the X-ray results of the present and cathepsin B-inhibitor complexes.

During our screening of microbial metabolites for effective drugs against tumor cell lines, we discovered a new pyrano[4',3':6,7]naphtho[1,2-b]xanthene derivative, FD-594 from the fermentation broth of Streptomyces sp. TA-0256. FD-594 shows moderate activity against tumor cell lines, comparative to that of adriamycin, as well as antibacterial activity against some Gram-positive bacteria.

The structure of a novel antitumor antibiotics FD-594 (1), produced by Streptomyces sp. TA-0256, was determined to have a glycosylated pyrano[4',3':6,7]naphtho[1,2-b]-xanthene skeleton by means of spectral data. The biosynthetic studies of the chromophore of 1 was also carried out by feeding experiments with [1-13C]-, [2-13C]-, and [1,2-13C2]sodium acetate. The labeling pattern was determined by 13C NMR including 2D INADEQUATE experiments, which allowed us to elucidate that the chromophore of 1 is derived from 14 acetate, followed by the loss of one carbon atom.

Stachybotrin C and parvisporin, novel neuritogenic compounds, were isolated from the culture broth of Stachybotrys parvispora F4708. Stachybotrin C induced significant neurite outgrowth in PC12 cells and showed cell survival activity in the primary culture of cerebral cortical neurons. Parvisporin demonstrated only weak neuritogenic activity.

The structures of stachybotrin C and parvisporin have been determined by spectroscopic analyses and chemical derivatization. Stachybotrin C contains a unique pyrano-isoindolinone ring system, while parvisporin has a hydroxyl farnesyl phenol structure.

In the course of our microbial transformation study on erythromycin derivatives, Streptomyces hygroscopicus ATCC 31080, which produces a polyether antibiotic carriomycin, was found to transform erythromycin derivatives to their inactivated derivatives. The structures of inactivated derivatives prepared by enzyme reaction using the cell extract, UDP-glucose (or UDP-galactose) and Mg2+ (or Mn2+) were elucidated on the basis of analysis of thei spectral data to be the compounds glycosylated at C-2' of a desosamine moiety, indicating that the enzyme is a macrolide glycosyl transferase (MGT). The MGT activity of cell extract from S. antibioticus ATCC 11891, a producing organism of oleandomycin, could be distinguished from that of ATCC 31080, based on the ability to glycosylate tylosin. We examined 32 actinomycete strains producing such polyketides as macrolide and polyether antibiotics, and found that 15 strains of Streptomyces have macrolide glycosyl transferase activity. It suggests that the MGTs have been distributed among at least polyketide producing Streptomyces strains.

Three cytotoxic cardenolides, acovenosigenin A 3-O-alpha-L-ramnopyranoside (1), euonymoside A (2) and euonymusoside A (3), were isolated from the woods of Euonymus alata (Celastraceae). The chemical structure of a new cardenolide, euonymusoside A (3) has been elucidated on the basis of extensive spectral analysis and enzymic hydrolysis to be acovenosigenin A (1 beta, 3 beta, 14 beta-trihydroxy-5 beta-cardenolide) 3-O-beta-D-glucopyranosyl (1-->6)-beta-D-glucopyranosyl(1-->4)-alpha-L-rhamnopyranoside. All three showed potent cytotoxic activity against some neoplastic cell lines.

During our screening program for natural product drugs effective against multidrug-resistant mammalian cells, we have discovered a new delta lactone FD-211 from the fermantation broth of Myceliophthora lutea TF-0409. FD-211 had a broad spectrum activity against cultured tumor cell lines, including adriamycin-resistant HL-60 cells.

During the course of our screening program for natural product drugs effective against multidrug resistant cells by using adriamycin resistant HL-60 cells, we have discovered a new 12 membered macrolide FD-895 in the fermentation broth of Streptomyces hygroscopicus A-9561 isolated from a soil sample collected at Iriomote Island, Okinawa prefecture, Japan. FD-895 showed stronger cytocidal activities against in vitro tumor cell lines than adriamycin. FD-895 had the same IC50 values against parent and adriamycin resistant HL-60 cells.

New 18-membered macrolides FD-891 and FD-892 were discovered from the fermentation broth of Streptomyces graminofaciens A-8890 isolated from a soil sample collected at Yamanashi prefecture Japan. They induce morphological changes of HL-60 cells at low concentration below IC50s and have cytocidal activity against in vitro tumor cell lines. FD-891 showed 2 approximately 7 times stronger activity than doxorubicin whereas FD-892 was 20 approximately 100 fold weaker than FD-891 and doxorubicin.

Structures of FD-891 and FD-892 were determined by extensive NMR spectral analysis as shown in Fig. 1. They belong to such 18-membered macrolides as concanamycins and virustomycin.

To enzymatically synthesize active metabolites of vitamin D3, we screened about 500 bacterial strains and 450 fungal strains, of which 12 strains were able to convert vitamin D3 to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] via 25-hydroxyvitamin D3 [25(OH)D3]. The conversion activity was only detected in strains belonging to the genus Amycolata among all the organisms tested. A preparative-scale conversion of vitamin D3 to 25(OH)D3 and 1 alpha,25(OH)2D3 in a 200-1 tank fermentor using A. autotrophica FERM BP-1573 was accomplished, yielding 8.3 mg 25(OH)D3/l culture and 0.17 mg 1 alpha,25(OH)2D3/l culture. A related compound, vitamin D2, could be also converted to 25-hydroxyvitamin D2 and 1 alpha,25-dihydroxyvitamin D2 using the same strain. The cytochrome P-450 of FERM BP-1573 was detected by reduced CO difference spectra in whole-cell suspensions. Vitamin D3 in the culture induced cytochrome P-450 and the conversion activity simultaneously, suggesting that the hydroxylation at C-25 of vitamin D3 and at C-1 of 25(OH)D3 originates from cytochrome P-450.

NG-011 and NG-012, novel potentiators of nerve growth factor (NGF) were isolated from the culture broth of Penicillium verruculosum F-4542. The structures of NG-011 and NG-012 were elucidated by their spectral analysis and degradation experiments as shown in Fig. 1.

NG-011 and NG-012, novel potentiators of nerve growth factor (NGF), were isolated from the culture broth of Penicillium verruculosum F-4542, together with 3,4-dihydro-6,8-dihydroxy-3-methylisocoumarin. They potentiated the neurite outgrowth induced by NGF in rat pheochromocytoma cell line (PC12).

Lonomycin B (II), C44H75O14Na, m.p. 181-182 degrees C, and lonomycin C (III), C43H73O14Na, m.p 186-187 degrees C, were isolated as their sodium salts from the fermentation broth of Streptomyces ribosidificus TM-481. Their physicochemical properties demonstrated that II and III are closely related congeners of lonomycin A (I). The identical mass spectra of methyl esters of I and II indicated that II is a stereoisomer of I. On the other hand, the mass spectrum of a methyl ester of III showed a peak at m/e 810 due to M+-H2O which is smaller by 14 mass units than the maximum peak at m/e 824 due to M+-H2O of the methyl esters of I and II. This result together with the elemental analysis strongly suggested that III is a demethyl derivative of I or II. II and III are slightly less active than I in their antimicrobial activities.

All the resonances observed in the 13C-NMR spectra of polyether antibiotics, dianemycin and lenoremycin (Ro 21-6150) have been assigned by the aid of selective proton decoupling experiments, T1 value measurements and biosynthetic methods as well as comparison to model compounds such as monensin, nigericin, etheromycin and carriomycin.

All resonances observed in the 13C-nmr spectra of the polyether antibiotics lonomycin A and mutalomycin have been assigned by the aid of biosynthetic method, selective proton decoupling as well as comparison with structurally related compounds.

Lonomycin A at various concentrations was tested for its inhibitory effect on Toxoplasma multiplication in host cells cultured in vitro. Results indicate that lonomycin A at a concentration of 0.01 micrograms per ml in TC-199 medium demonstrated a high degree of antitoxoplasma activity with complete inhibition of Toxoplasma multiplication in the host cells. Lymphokines, a supernatant produced from spleen cells of mice infected chronically with Toxoplasma gondii, inhibited Toxoplasma multiplication in mice macrophage and kidney cell monolayers. However, lonomycin A inhibited completely Toxoplasma multiplication in non-specific cells, i.e. not only in mice macrophages and kidney cells but also in cells of human and other animal species.