Unveiling dynamic hepatocyte plasticity in HepaRG cells with a dual CYP reporter system.

Primary hepatocytes are widely utilized for investigating drug efficacy and toxicity, yet variations between batches and limited proliferation capacity present significant challenges. HepaRG cells are versatile cells, capable of maintaining an undifferentiated state and differentiating through dimethyl sulfoxide treatment, allowing for molecular analysis of hepatocyte plasticity. To elucidate the underlying molecular mechanisms of HepaRG cell plasticity, we used CYP3A4G/7R HepaRG cells engineered to express DsRed under the control of the fetus-specific CYP3A7 gene and EGFP under the adult-specific CYP3A4 gene promoter.

[Evaluation of the Stability and Antibacterial Activity of Burow's and Neo-Burow's Solutions was Prepared Using Different Methods].

Burow's solution is a 13% aluminum acetate solution used for treating chronic suppurative otitis media. However, multiple formulations for Burow's and neo-Burow's solutions are used as in-hospital preparations. Each formulation uses different types and amounts of reagents, and takes a different time to prepare.

Actinoplanes kirromycinicus sp. nov., isolated from soil.

A novel actinomycete, designated as TPMA0078, was isolated from a soil sample collected in Shinjuku, Tokyo, Japan. 16S rRNA gene sequence analysis indicated that strain TPMA0078 belongs to the genus Actinoplanes and is closely related to Actinoplanes regularis IFO 12514 (99.86% 16S rRNA gene sequence similarity).

Isolation of Hydrazide-alkenes with Different Amino Acid Origins from an Azoxy-alkene-Producing Mutant of 7434AN4.

A triple mutant (strain KA57) of 7434AN4 produces an azoxy-alkene compound, KA57A, which was not detected in a parent strain or other single and double mutants. This strain accumulated several additional minor components, whose structures were elucidated. HPLC analysis of strain KA57 indicated the presence of two UV active components (KA57D1 and KA57D2) as minor components.

Isolation, Biosynthetic Investigation, and Biological Evaluation of Maniwamycin G, an Azoxyalkene Compound from sp. TOHO-M025.

A new maniwamycin analogue, maniwamycin G, was isolated from sp. TOHO-M025 as a major product. Maniwamycin G has a molecular formula of CHNO, and its extensive NMR analysis revealed that maniwamycin G contains a methoxycarbonyl group instead of an amide as found in maniwamycin F.

Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450.

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules.

sp. nov., an endophytic actinomycete isolated from aerial parts of .

A novel actinomycete, designated NUM-2625, was isolated as an endophytic bacterium in aerial parts of , an endemic species in the Altai, Himalaya mountain chain area, collected from Khasagt Khairkhan Mountain in Mongolia. The 16S rRNA gene sequence of strain NUM-2625 showed the highest similarity to TT2-10 (99.4 %), KV-744 (99.

An overview of the cytochrome P450 enzymes that catalyze the same-site multistep oxidation reactions in biotechnologically relevant selected actinomycete strains.

Cytochrome P450 enzymes (P450s) are one of the major factors responsible for the diversity of metabolites produced through many biosynthetic and biodegradative processes in actinomycetes. P450s typically catalyze a single oxidative modification; however, several P450s have been identified with the unique ability to iteratively oxidize the same-site of the substrate. These P450s are capable of forming diverse compounds that affect biological processes, including alcohols, ketones, aldehydes, and carboxylic acids.

Computational-Based Mechanistic Study and Engineering of Cytochrome P450 MycG for Selective Oxidation of 16-Membered Macrolide Antibiotics.

MycG is a cytochrome P450 that performs two sequential oxidation reactions on the 16-membered ring macrolide . The enzyme evolved to oxidize preferentially over and , which differ only by the presence of methoxy vs free hydroxyl groups on one of the macrolide sugar moieties. We utilized a two-pronged computational approach to study both the chemoselective reactivity and substrate specificity of MycG.

Artificial control of the multistep oxidation reactions catalyzed by the cytochrome P450 enzyme RosC.

The cytochrome P450 monooxygenase RosC catalyzes the three-step oxidation reactions, which leads to the formation of a hydroxy, formyl, and carboxy group at C-20 during rosamicin biosynthesis in Micromonospora rosaria IFO13697. To determine if amino acid substitutions in RosC could allow for the control of the multistep oxidation reactions, we screened RosC random mutants. The RosC mutant RM30, with five amino acid substitutions (P107S, L176Q, S254N, V277A, and I319N), catalyzed only the first step of the oxidation reaction.

Quorum Sensing Inhibitors against Chromobacterium violaceum CV026 Derived from an Actinomycete Metabolite Library.

Quorum sensing (QS) is a microbial signaling system that regulates the expression of many virulence genes. Herein, we studied five compounds-No. 1: (E)-2-methyl-3- (4-nitro-phenyl)-acrylaldehyde; No.

Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation.

Cytochrome P450 enzyme RosC catalyzes a multistep oxidation reaction to form the non-active compound 20-carboxyrosamicin.

The cytochrome P450 enzyme RosC catalyzes a two-step, hydroxylation and alcohol oxidation, oxidation reaction to form the C-20 formyl group in the biosynthesis of a 16-membered macrolide antibiotic rosamicin produced by Micromonospora rosaria IFO13697. RosC is presumed to be involved in the formation of 20-carboxyrosamicin because it has been isolated from the culture broth of M. rosaria.

Maniwamycins: new quorum-sensing inhibitors against Chromobacterium violaceum CV026 were isolated from Streptomyces sp. TOHO-M025.

Quorum sensing is an important microbial signaling system that controls the expression of many virulence genes. Maniwamycins C-F, new compounds and quorum-sensing inhibitors, were isolated from the culture broth of Streptomyces sp. TOHO-M025 using a silica gel column and preparative HPLC.

Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG.

A new mycinosyl rosamicin derivative produced by an engineered Micromonospora rosaria mutant with a cytochrome P450 gene disruption introducing the D-mycinose biosynthetic gene.

Genetic engineering of post-polyketide synthase-tailoring genes can be used to generate new macrolide analogs through manipulation of the genes involved in their biosynthesis. Rosamicin, a 16-member macrolide antibiotic produced by Micromonospora rosaria IFO13697, contains a formyl group and an epoxide at C-20 and C-12/13 positions which are formed by the cytochrome P450 enzymes RosC and RosD, respectively. The D-mycinose biosynthesis genes in mycinamicin II biosynthesis gene cluster of Micomonospora guriseorubida A11725 were introduced into the rosC and rosD disruption mutants of M.

New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate.

Isolation and structural elucidation of novel cholestane glycosides and spirostane saponins from Polygonatum odoratum.

Much attention has been paid to cholestane-type steroidal glycosides because of their importance from the perspectives of both chemical diversity and significant biological activities. A phytochemical investigation of the rhizomes of Polygonatum odoratum (Liliaceae) resulted in the isolation of three novel cholestane-type steroidal glycosides (1-3) with unique Δ(14,16)-unsaturated D-ring structures as well as two novel spirostane-type steroidal saponins (4 and 5) and three known steroidal glycosides (6-8). Their structures were determined by various spectroscopic methods and chemical reactions.

Function of cytochrome P450 enzymes RosC and RosD in the biosynthesis of rosamicin macrolide antibiotic produced by Micromonospora rosaria.

The cytochrome P450 enzyme-encoding genes rosC and rosD were cloned from the rosamicin biosynthetic gene cluster of Micromonospora rosaria IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with M. rosaria and bioconversion of rosamicin biosynthetic intermediates with Escherichia coli expressing RosC and RosD.

Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin hydroxylation and epoxidation reactions.

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida.

Production of a hybrid 16-membered macrolide antibiotic by genetic engineering of Micromonospora sp. TPMA0041.

Some polyketide-derived bioactive compounds contain sugars attached to the aglycone core, and these sugars often enhance or impart specific biological activity to the molecule. Mycinamicin II, a 16-member macrolide antibiotic produced by Micromonospora griseorubida A11725, contains a branched lactone and two different deoxyhexose sugars, D-desosamine and D-mycinose, at the C-5 and C-21 positions, respectively. We previously engineered an expression plasmid pSETmycinose containing the D-mycinose biosynthesis genes from M.

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.

A new structural form in the SAM/metal-dependent o‑methyltransferase family: MycE from the mycinamicin biosynthetic pathway.

O-linked methylation of sugar substituents is a common modification in the biosynthesis of many natural products and is catalyzed by multiple families of S-adenosyl-L-methionine (SAM or AdoMet)-dependent methyltransferases (MTs). Mycinamicins, potent antibiotics from Micromonospora griseorubida, can be methylated at two positions on a 6-deoxyallose substituent. The first methylation is catalyzed by MycE, a SAM- and metal-dependent MT.