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At least 739 records · Page 41Linked to original sources
PubMed1999

Implication of proteasome in estrogen receptor degradation.

In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.

Cysteine Endopeptidases↗
PubMed1997

Management of nonmatrix interfering peaks in a chiral high-performance liquid chromatographic assay produced by solid-phase extraction of rat plasma.

PD 146923, under evaluation as an alkylating radiosensitizing drug, contains one chiral center and one chemically reactive aziridine ring. A method was developed to evaluate possible in vivo enantiomeric inversion of PD 146923 in rat plasma. Normal-phase chiral HPLC was necessary to separate the enantiomers, but a typical aqueous-based solid-phase extraction (SPE) was needed to isolate the analytes from plasma. SPE at higher analyte concentrations removed all interfering peaks and gave acceptable recoveries. However, peaks (A-G) from seven new components interfering with analyte detection at lower concentrations were produced by SPE. The interfering peaks overlapped each other, so some were not observed until other, more intense interfering peaks had been managed. The low separation efficiency of the chiral column precluded management of interfering peaks by modifying chromatographic parameters. Chemical reactivity of the analytes forced the use of mild conditions for management of interfering peaks. Peaks A-F were: (A) water from the SPE cartridge; (B) SPE sorbent endcapping; (C, E and F) nonvolatile salts of the SPE elution acid reacting with bases from the injection solvent or with unidentified bases from the SPE cartridge; (D and G) analyte degradation products. This study identifies the nonmatrix peaks coeluting with the analytes, and describes how an aqueous-based SPE method was developed for isolating these very polar, highly reactive analytes in plasma for separation in a normal-phase chiral HPLC assay. Additionally, B, C, E or F probably are present in many other solid-phase extractions, but are not observed because of polarity or solubility properties.

Acetic Acid↗
PubMed2000

Evidence of an estrogen receptor form devoid of estrogen binding ability in MCF-7 cells.

In MCF-7 breast cancer cells, hydroxytamoxifen (OH-Tam) up-regulates the estrogen receptor (ER) in a form unable to bind [(3)H]estradiol (E(2)). We show here that this property is not restricted to this antiestrogen. [(3)H]E(2) binding assays (whole cell assays, DCC assays on cell extracts) and enzyme immunoassays (Abbott) performed in parallel, establish the permanent presence of such unusual ERs in the absence of any exposure of the cells to a ligand. E(2) and the pure antiestrogen RU 58 668, which down-regulate ER, also decrease [(3)H]E(2) binding. In control cells, these ERs represent about the half of the whole receptor population; they also display a tendency to stabilize within the cell nucleus. Loss of E(2) binding ability appears irreversible, since we failed to label receptor accumulated under OH-Tam with [(3)H]E(2) or [(3)H]tamoxifen aziridine (TAZ). Cycloheximide (CHX), which blocks E(2)-induced down regulation of ER, failed to stabilize [(3)H]E(2) binding (whole cell assay) after an [(3)H]E(2) pulse (1 h), confirming that regulation of E(2) binding and peptide level are related to different regulatory mechanisms. Loss of binding ability could not be ascribed to any ER cleavage as demonstrated by Western blotting with a panel of ER antibodies raised against its various domains (67 kDa ER solely detected). We propose that loss of E(2) binding ability is related to the aging process of the receptor, i.e. it is progressively converted to a form devoted to degradation after it has accomplished its physiological role. Ligands may favor (E(2), RU 58 668) or impede (OH-Tam) this elimination process.

Antineoplastic Agents, Hormonal↗
PubMed2000

Calcium-induced flexibility changes in the troponin C-troponin I complex.

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.

Animals↗
PubMed2000

Characterization of commercially available and synthesized polyethylenimines for gene delivery.

Five new polyethylenimines (PEI) were synthesized by polymerization of aziridine in aqueous solution and compared to several commercially available PEI used for gene transfer. Polymers were characterized by 13C NMR spectroscopy, capillary viscosimetry, potentiometric titration and Cu(II) complex formation to gain insight into structural and functional properties. 13C NMR analysis revealed differences in the extent of branching based on the ratio of primary, secondary and tertiary amino groups. An amino group ratio 1 degrees :2 degrees :3 degrees =1:2:1 was obtained for the synthesized PEI, whereas commercially available PEI generally showed a higher degree of branching (1:1:1). Capillary viscosimetry of aqueous PEI solutions with a sufficient amount of salt gave Mark-Houwink parameters of alpha=0.26 and K(V)=1.00 cm(3)/g for the commercially available polymers. In case of the synthesized polymers, variation of reaction conditions yielded viscosity average molar masses (M(v)) in the range of 8000-24000 g/mol. PEI solutions were investigated by potentiometric titration analysis showing that their buffer capacity was not significantly influenced by molar mass or polymer structure. The pK(a) values (8.18-9.94) and the buffer capacity beta (0.08-0.014 mol/l) were of comparable magnitude. This study highlights the necessity of more detailed characterization methods for PEI used in gene transfer protocols since physico-chemical properties do not reflect the vast differences found in transfection efficiencies.

Buffers↗
PubMed2003

Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine.

Low-molecular-weight polyethylenimine (LMW-PEI) was synthesized by the acid-catalyzed, ring-opening polymerization of aziridine and compared with commercially available high-molecular-weight PEI (HMW-PEI) of 25 kDa. Molecular weights were determined by size-exclusion chromatography in combination with multi-angle laser light scattering. The weight average molecular weight (M(w)) of synthesized LMW-PEI was determined as 5.4+/-0.5 kDa, whereas commercial HMW-PEI showed a M(w) of 48+/-2 kDa. DNA polyplexes of LMW-PEI and HMW-PEI were characterized with regard to DNA condensation (ethidium bromide fluorescence quenching), size (photon correlation spectroscopy) and surface charge (laser Doppler anemometry). Compared with HMW-PEI, DNA condensation of LMW-PEI was slightly impaired at lower N/P ratios. Complexes with plasmid DNA at a N/P ratio of 6.7 showed significantly increased hydrodynamic diameters (590+/-140 vs. 160+/-10 nm), while the zeta-potential measurements were similar (23+/-2 vs. 30+/-3 mV). The cytotoxicity of LMW-PEI in L929 fibroblasts was reduced by more than one order of magnitude compared with HMW-PEI, as shown by MTT assay. LMW-PEI exhibited increased transfection efficiency in six different cell lines. Reporter gene expression was found to be increased by a factor of 2.1-110. The pharmacokinetics and biodistribution of 125I-PEI in mice were similar for both molecular weights with an AUC of ca. 330+/-100% ID/ml min. Approximately half of the injected dose accumulated in the liver. LMW-PEI proved to be an efficient gene delivery system in a broad range of cell lines. Due to differences in polyplex structure, as well as its relatively low cytotoxicity, which makes the application of high N/P ratios possible, LMW-PEI appears to possess advantageous qualities with regard to transfection efficiency over PEI of higher molecular weight.

Action Potentials↗
PubMed1983

Reusable ultraviolet monitors: design, characteristics, and efficacy.

Reusable ultraviolet dosimetry badges have been developed that provide a visual indication of daily cumulative ultraviolet (UV) exposure. These two-sided, tapelike devices measure UV radiation emitted by sunlight or an artificial UV light source exposure by means of a photochromic aziridine color change reaction that is UV-integrating but optically reversible. UV radiation falling on the exposure side of the badge generates a color change that can be seen from the opposite or readout side. End points are indicated by a visual match of the photochromic with a surrounding reference. This paper describes the construction, component characteristics, and clinical testing of two versions of a new photochromic dosimeter that selectively responds to either UVB (280-320 nm) radiation or UVA (320-400 nm) radiation of the solar spectrum. One version of this monitor, sensitive only to the mid-range UVB, has a peak sensitivity to 300 nm and has four end point markers revealing color changes corresponding to 0.4, 0.8, 2.2, and 6.5 times the minimal erythema dose of an average Caucasian. A second version, sensitive only to UVA, has a peak sensitivity at 355 nm and can monitor exposures ranging from 0.8 to 10 joules/cm2. Outdoor efficacy testing has shown that the UVB monitor is an effective predictor of UV dose-related 24-hour erythema response induced by sunlight. Following a measurement, these monitors can be rezeroed by exposing the readout side to sunlight for a few minutes. They can be reused for eight to ten times. The limitation of the sunlight-calibrated UVB monitor tag is its failure to predict erythema response produced by artificial UVB sources such as FS40 sunlamps.

Calibration↗
PubMed2003

Anti-androgenic properties of Compound A, an analog of a non-steroidal plant compound.

We investigated the interactions between Compound A (CpdA), an analog of a hydroxyphenyl aziridine precursor found in an African shrub, and the androgen receptor (AR). CpdA represses androgen-induced activation of both specific and non-specific androgen DNA response elements. While a similar effect was obtained for the progesterone receptor (PR) via a non-specific hormone response element, CpdA had no effect on the actions of the glucocorticoid and mineralocorticoid receptors. CpdA represses the ligand-dependent interaction between the NH(2)- and COOH-terminal domains of the AR, similar to well-characterised anti-androgens. CpdA also interferes with the interaction of steroid receptor co-activator 1 (SRC1) with the activation domain AF2 but not with AF1. However, CpdA does not compete with androgen for binding to the AR. These results demonstrate that CpdA elicits anti-androgenic actions by a mechanism other than competitive binding for the AR.

Acetates↗
PubMed1998

Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements.

The estrogen receptor (ER) is a ligand-activated transcription factor that binds to specific DNA sequences, estrogen response elements (EREs). Estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned EREs that are surrounded by naturally-occurring AT-rich sequences with a stoichiometry of one E2-ER dimer per ERE. When ER is bound by 4-hydroxytamoxifen (4-OHT), the active metabolite of the widely used therapeutic antiestrogen tamoxifen (TAM), the receptor binds to EREs with high affinity. However, one molecule of 4-OHT ligand dissociates from the ER dimer apparently during the process of binding to DNA, yielding a stoichiometry of one [3H]4-OHT molecule per ERE. To determine whether DNA-binding induced ligand dissociation is a general property of type I antiestrogens that are not covalently attached to the ER, we examined the interaction of ER liganded by tamoxifen (TAM) with EREs. We demonstrate that TAM-ER binds EREs with lower affinity than E2-ER, 4-OHT-ER, or ER liganded by the covalent antiestrogen tamoxifen aziridine. Unlike E2-ER, both TAM and 4-OHT-ER bind EREs non-cooperatively. Like 4-OHT, TAM appears to dissociate from the liganded ER as the receptor binds EREs. Additionally, partial proteolysis of ERE-bound ER by trypsin revealed different cleavage patterns for E2 versus 4-OHT and TAM. These findings indicate that the behavior of the ER liganded by TAM is generally similar to that of the antiestrogen 4-OHT.

Animals↗
PubMed2001

Mitomycin antitumor compounds. Part 1. CD studies on their molecular structure.

The UV-Vis and circular dichroism (CD) spectra of several mitomycin antitumor compounds and some of their derivatives were analyzed in order to attribute the proper assignment to their electronic transitions. The lowest energy pi-->pi* transition was found to depend on the effect of the auxochromic group in the aromatic ring, whereas the three n-->pi* transitions, present at around 240, 400 and 560 nm, are related to the C(9)==O of the carbamoyl group and to the C(8)==O and the C(5)==O of the quinone, respectively. The chirality of the C(9) is responsible for the sign of the Cotton effect (CE) at around 240 nm, whereas the substituents of the chromophore for mitosane derivatives and the conformation of the carbamoyloxymethyl group at C(9) determine the CE sign of the (1)A-->(1)L(b) transition. When the aziridine ring was opened and mitosenes derivatives were obtained, CD spectra did not differ significantly among the compounds and the bands associated to the different transitions had similar Cotton effect. Our findings suggest that the differences in the CD spectra, observed between mitosanes and mitosenes, are probably related to the more rigid molecular structure of the mitosene derivatives and the different conformations in solution of the C(9) side chain.

Antibiotics, Antineoplastic↗
PubMed1996

Stability of the ligand-estrogen receptor interaction depends on estrogen response element flanking sequences and cellular factors.

To determine whether accessory proteins mediate the ligand- and DNA sequence-dependent specificity of estrogen receptor (ER) interaction with DNA, the binding of partly purified vs highly purified bovine ER to various estrogen response elements (EREs) was measured in the presence of different ER ligands. Partly purified estradiol-liganded ER (E2-ER) binds cooperatively to stereoaligned tandem EREs flanked by naturally occurring AT-rich sequences, with a stoichiometry of one E2-ER dimer per ERE. In contrast, highly purified E2-ER binds with a 10-fold lower affinity and non-cooperatively to EREs flanked by the AT-rich region. Moreover, the binding stoichiometry of highly purified E2-ER was 0.5 E2-ER dimer, or one monomer per ERE, independent of the ERE flanking sequence. Interestingly, the binding of ER liganded with the antiestrogen 4-hydroxytamoxifen (4-OHT-ER) was non-cooperative with an apparent stoichiometry of 0.5 4-OHT-ER dimer per ERE, regardless of ER purity or ERE flanking sequence. We recently showed that when 4-OHT-ER binds DNA, one molecule of 4-OHT dissociates from the dimeric 4-OHT-ER-ERE complex, accounting for the reduced apparent binding stoichiometry. In contrast, ER covalently bound by tamoxifen aziridine (TAz) gave an ERE binding stoichiometry of one TAz-ER dimer per ERE, and TAz-ER binds cooperatively to multiple AT-rich EREs, regardless of the purity of the receptor. We have obtained evidence that purification of ER removes an accessory protein(s) that interacts with ER in a sequence- and/or DNA conformational-dependent manner, resulting in stabilization of E2, but not 4-OHT, in the ligand binding domain when the receptor binds to DNA. We postulate that retention of ligand by ER maintains the receptor in a conformation necessary to achieve high-affinity, cooperative ERE binding.

Animals↗
PubMed1997

Protein synthesis is not implicated in the ligand-dependent activation of the estrogen receptor in MCF-7 cells.

In MCF-7 cells, estrogen receptor (ER) elimination occurs rapidly under stimulation with estradiol (E2) at 1 nM ('ER processing'); cycloheximide (CHX) at 50 microM impedes this phenomenon. ER processing is also observed when E2 is removed after the first hour of incubation, indicating that the role of the hormone would be limited to the initiation of this process. When CHX is removed at the same time, receptor processing and, later, the induction of progesterone receptor (PgR) both proceed. The initial estrogenic signal which activates ER is therefore not influenced by CHX. In support of this conclusion, no effect of the drug on E2 binding affinity of residual ER was detected. A similar result was recorded for a series of estrogens and antiestrogens, indicating that CHX exerts no influence on the potential agonistic/antagonistic potency of any ligand. Size-exclusion chromatography (FPLC) revealed that [3H]E2-induced ER activation leads to the cleavage of the native receptor (67 kDa) into low molecular weight isoforms which subsequently become less detectable over time (proteolysis). In the presence of CHX, such ER isoforms persist, confirming the absence of interference of the drug with the activation step. When the cells were prelabelled with [3H]tamoxifen aziridine ([3H]TAZ) before their exposure to E2, ER cleavage could not be detected due to the lack of activation potency of the antiestrogenic ligand. However, the [3H]TAZ-ER complexes were subjected to E2-induced processing; CHX blocked this phenomenon, which is associated with the maintenance of ER synthesis and activation.

Binding, Competitive↗
PubMed1999

Role of estrogen receptor ligand and estrogen response element sequence on interaction with chicken ovalbumin upstream promoter transcription factor (COUP-TF).

Estrogen-responsive genes are regulated by altering the balance of estrogen receptor (ER) interaction with transcription activators and inhibitors. Here we examined the role of ER ligand on ER interaction with the Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) orphan nuclear receptor. COUP-TF binding to half-site estrogen response elements (EREs) was increased by the addition of estradiol (E2) -liganded ER (E2-ER), but not by ER liganded with the antiestrogens 4-hydroxytamoxifen (4-OHT-ER) or tamoxifen aziridine (TAz-ER). ER did not bind to single half-sites. Conversely, COUP-TF enhanced the ERE binding of purified E2-ER, but did not affect TAz-ER-ERE binding. In contrast, only antiestrogens enhanced direct interaction between ER and COUP-TF as assessed by GST pull-down assays. Identical results were obtained using either purified bovine or recombinant human ERalpha. Co-immunoprecipitation assays showed that ER and COUP-TF interact in extracts from MCF-7 and ERalpha-transfected MDA-MB-231 cells. Here we document that ER ligand impacts COUP-TF-ER interaction. COUP-TF interaction is mediated by the DNA binding and ligand-binding domains of ER. We suggest that changes in ER conformation induced by DNA binding reduce ER-COUP-TF interaction. Transient transfection of human MCF-7 breast cancer cells with a COUP-TFI expression vector repressed E2-induced luciferase reporter gene expression from single or multiple tandem copies of a consensus ERE. COUP-TFI stimulated 4-OHT-induced luciferase activity from a minimal ERE. Alone, COUP-TFI increased transcription from ERE half-sites or a single ERE in a sequence-dependent manner. These data provide evidence that the ERE sequence and its immediate flanking regions influence whether COUP-TF enhances, inhibits, or has no effect on ER ligand-induced ERE reporter gene expression and that COUP-TFI activates gene transcription from ERE half-sites. We suggest that COUP-TFI plays a role in mitigating estrogen-responsive gene expression.

Animals↗
PubMed2001

2-Benzyl-3,4-iminobutanoic acid as inhibitor of carboxypeptidase A.

2-Benzyl-3,4-iminobutanoic acid (3) was evaluated as a novel class of inhibitor for carboxypeptidase A (CPA). All four stereoisomers of 3 are found to have competitive inhibitory activity for CPA, although their inhibitory potencies differ widely with (2R,3R)-3 being most potent. The molecular modeling study for CPA(2R,3R)-3 complex suggested that the lone pair electrons on the nitrogen of the aziridine ring in the inhibitor forms a coordinative bond with the active site zinc ion and the proton on the nitrogen is engaged in hydrogen bonding with one of the carboxylate oxygens of Glu-270.

Butyrates↗
PubMed2003

Synthesis, antimicrobial and antifungal activity of a new class of spiro pyrrolidines.

A new class of spiro pyrrolidines, dispiro[oxindole-cyclohexanone]pyrrolidines, dispiro[oxindole-tetrahydronaphthalen-1-one]pyrrolidines, dispiro[oxindole-arylidene-cyclohexanone]pyrrolidines, dispiro[oxindole-hexahydro-indazole]pyrrolidines, and spiro[butenolide]pyrrolidines, have been screened for their antibacterial and antifungal activity against ten human pathogenic bacteria and four dermatophytic fungi. They were found to have antimicrobial and antifungal activity compounds against various pathogens except Bacillus subtilis. The spiro pyrrolidinines were synthesized by the regioselective 1,3-dipolar cycloaddition reaction of azomethine ylides generated either from isatin and sarcosine or from aziridine. The azomethine ylide so generated reacted with various dipolarophiles such as 2-arylidene-cyclohexanones, 2-arylidene-tetrahydronapthalen-1-ones, 2,6-bis(arylmethylidene)cyclohexanones and 3-arylidene-5-phenyl- butenolides.

Anti-Bacterial Agents↗
PubMed2000

Leaving group and gas phase neighboring group effects in the side chain losses from protonated serine and its derivatives.

The gas phase fragmentation reactions of protonated serine and its YNHCH(CH2X)CO2H derivatives, beta-chloroalanine, S-methyl cysteine, O-methyl serine, and O-phosphoserine, as well as the corresponding N-acetyl model peptides have been examined via electrospray ionization tandem mass spectrometry (MS/MS). In particular, the competition between losses from the side chain and the combined loss of H2O and CO from the C-terminal carboxyl group of the amino acids or H2O or CH2CO from the N-acetyl model peptides are compared. In this manner the effect of the leaving group (Y = H or CH3CO, vary X) or of the neighboring group can be examined. It was found that the amount of HX lost from the side chain increases with the proton affinity of X [OP(O)(OH)2 > OCH3 approximately equals OH > Cl]. The ion due to the side chain loss of H2O from the model peptide N-acetyl serine is more abundant than that from protonated serine, suggesting that the N-acetyl group is a better neighboring group than the amino group. Ab initio calculations at the MP2(FC)/6-31G*//HF/6-31G* level of theory suggest that this effect is due to the transition state barrier for water loss from protonated N-acetyl serine being lower than that for protonated serine. The mechanism for side chain loss has been examined using MS3 tandem mass spectrometry, independent synthesis of proposed product ion structures combined with MS/MS, and hydrogen/deuterium exchange. Neighboring group rather than cis 1,2 elimination processes dominate in all cases. In particular, the loss of H3PO4 from O-phosphoserine and N-acetyl O-phosphoserine is shown to yield a 3-membered aziridine ring and 5-membered oxazoline ring, respectively, and not the dehydroalanine moiety. This is in contrast to results presented by DeGnore and Qin (J. Am. Soc. Mass Spectrom. 1998, 9, 1175-1188) for the loss of H3PO4 from larger peptides, where dehydroalanine was observed. Alternate mechanisms to cis 1,2 elimination, for the formation of dehydroalanine in larger phosphoserine or phosphothreonine containing peptides, are proposed.

Mass Spectrometry↗
PubMed1999

Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564.

BACKGROUND: The mitomycins are natural products that contain a variety of functional groups, including aminobenzoquinone- and aziridine-ring systems. Mitomycin C (MC) was the first recognized bioreductive alkylating agent, and has been widely used clinically for antitumor therapy. Precursor-feeding studies showed that MC is derived from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, L-methionine and carbamoyl phosphate. A genetically linked AHBA biosynthetic gene and MC resistance genes were identified previously in the MC producer Streptomyces lavendulae NRRL 2564. We set out to identify other genes involved in MC biosynthesis. RESULTS: A cluster of 47 genes spanning 55 kilobases of S. lavendulae DNA governs MC biosynthesis. Fourteen of 22 disruption mutants did not express or overexpressed MC. Seven gene products probably assemble the AHBA intermediate through a variant of the shikimate pathway. The gene encoding the first presumed enzyme in AHBA biosynthesis is not, however, linked within the MC cluster. Candidate genes for mitosane nucleus formation and functionalization were identified. A putative MC translocase was identified that comprises a novel drug-binding and export system, which confers cellular self-protection on S. lavendulae. Two regulatory genes were also identified. CONCLUSIONS: The overall architecture of the MC biosynthetic gene cluster in S. lavendulae has been determined. Targeted manipulation of a putative MC pathway regulator led to a substantial increase in drug production. The cloned genes should help elucidate the molecular basis for creation of the mitosane ring system, as well efforts to engineer the biosynthesis of novel natural products.

Amino Acid Sequence↗