Transcribed enhancer sequences are required for maize p1 paramutation.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr' state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr'. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr' epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr' sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.

Polymorphic modifications of a 1H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide possessing strong diuretic properties.

6-Hydroxy-N-(4-methoxyphenyl)-4-oxo-2,4-dihydro-1H-pyrrolo[3,2,1-ij]quinoline-5-carboxamide, C19H16N2O4, possesses strong diuretic properties and can be used as a new hypertension remedy. Two polymorphic modifications of this compound have been found, namely the triclinic polymorph (space group P-1), with one molecule in the asymmetric unit, and the monoclinic polymorph (space group P21/n), with two molecules in asymmetric unit. An analysis of the pairwise interaction energies between the molecules in the crystal phase revealed differences in the crystal packing. The triclinic form has only one level of organization, namely a chain/column, while the monoclinic form has two levels of organization, with a chain/column as the primary basic structural motif and a layer as the secondary basic structural motif.

The Study of the Structure-Diuretic Activity Relationship in a Series of New N-(Arylalkyl)-6-hydroxy-2-methyl-4-oxo-2,4-dihydro-1H-pyrrolo-[3,2,1-ij]quinoline-5-carboxamides.

In accordance with the principles of "me-too" technique, the preparative method for obtaining has been proposed, and the synthesis of a large series of new N-(arylalkyl)-6-hydroxy-2-methyl-4-oxo-2,4-dihydro-1H-pyrrolo[3,2,1-ij]quinoline-5-carboxamides as structurally close analogs of tricyclic pyrrolo- and pyridoquinoline diuretics has been carried out. All target compounds were obtained with high yields and purity by amidation of ethyl ester of the corresponding 2-methyl-pyrroloquinoline-5-carboxylic acid with arylalkylamines in boiling ethanol. Their structure was confirmed by the data of elemental analysis, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and polarimetry. Moreover, interpretations of their ¹H and 13C-NMR spectra, their mass spectrometric behavior, as well as peculiarities of the polarimetric studies were discussed. The effect of N-(arylalkyl)-6-hydroxy-2-methyl-4-oxo-2,4-dihydro-1H-pyrrolo[3,2,1-ij]quinoline-5-carboxamides on the urinary function of the kidneys was studied in white rats by the standard method of oral administration in the dose of 10 mg/kg compared to hydrochlorothiazide. According to the results of the primary pharmacological screening, the structural and biological regularities that were unexpected, but interesting for further studies were revealed. Among the substances studied, the samples, which by their diuretic effect are not inferior and even superior to both the known hydrochlorothiazide and the lead structure of the pyrroloquinoline group, have been found. On this basis, it can be argued that the introduction of the methyl group made by us in position 2 of pyrrolo[3,2,1-ij]quinoline nucleus can be considered as a successful and promising implementation of the "me-too" cloning of tricyclic 4-hydroxyquinoline-2-one diuretics.

Synthesis, Crystal Structure, and Biological Activity of Ethyl 4-Methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate Polymorphic Forms.

Continuing the search for new potential analgesics among the derivatives of 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid, the possibility of obtaining its esters by the alkylation of the corresponding sodium salt with iodoethane in dimethyl sulfoxide (DMSO) at room temperature was studied. It was found that under such conditions, together with the oxygen atom of the carboxyl group, a heteroatom of nitrogen is also alkylated. Therefore, the product of the reaction studied is a mixture of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate (major) and its 1-ethyl-substituted analog (minor). A simple but very effective method of preparative separation of these compounds was proposed. Moreover, the heterogeneous crystallization from ethanol was revealed to result in a monoclinic polymorphic form of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate, while the homogeneous crystallization results in its orthorhombic form. The molecular and crystal structures of both forms were confirmed by X-ray diffraction analysis, and the phase purity by powder diffraction study. The pharmacological tests carried out on the model of a carrageenan edema showed that the screening dose of 20 mg/kg of 1-ethyl-substituted ester and the orthorhombic form of its analog unsubstituted in position 1 exhibited weak anti-inflammatory and moderate analgesic effects. At the same time, the monoclinic form of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate appeared to be both a powerful analgesic and an anti-inflammatory agent that exceeded Piroxicam and Meloxicam in the same doses by these indicators. A detailed comparative analysis of the molecular and crystal structures of two polymorphic forms of ethyl 4-methyl-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate was carried out using quantum chemical calculations of the energies of pairwise interactions between molecules. An explanation of the essential differences of their biological properties based on this was offered.

N-Aryl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido-[3,2,1-ij]quinoline-6-carboxamides. The Synthesis and Effects on Urinary Output.

Continuing a targeted search for new leading structures with diuretic action among tricyclic derivatives of hydroxyquinolines, which are of interest as potential inhibitors of aldosterone synthase, the synthesis of a series of the corresponding pyrido[3,2,1-ij]quinoline-6-carboxanilides was carried out by amidation of ethyl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline-6-carboxylate with aniline, aminophenols and O-alkylsubstituted analogs with high yields and purity. The optimal conditions of this reaction are proposed; they make it possible to prevent partial destruction of the original heterocyclic ester and thereby avoid formation of specific impurities of 7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one. To confirm the structure of all substances obtained, elemental analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry were used. Moreover, the peculiarities of their ¹H and 13C-NMR spectra, as well as their mass spectrometric behavior under conditions of electron impact ionization, were discussed. The effect of pyrido[3,2,1-ij]quinoline-6-carboxanilides on the urinary function of the kidneys was studied in white rats of both genders by the standard method of oral administration at a dose of 10 mg/kg. Testing was conducted in comparison with hydrochlorothiazide, as well as with structurally close pyrrolo[3,2,1-ij] quinoline-5-carboxanilides studied earlier with the same substituents in the anilide fragments. It was found that addition of one methylene unit to the heterocycle partially hydrogenated and annelated with the quinolone core has a positive impact on biological properties-most of the substances studied exhibit a statistically significant diuretic effect exceeding the activity of not only hydrochlorothiazide, in some cases, but also the action of the structural analogs. The important structural and biological regularities, which are common with pyrroloquinolines and introduced by a chemical modification, were revealed. The importance of the presence in the structure of terminal amide fragments of tricyclic quinoline-3-carboxamides of a 4-methoxy-substituted aromatic ring was particularly marked. The expediency of further study of pyridoquinolines as promising diuretic agents has been shown.

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid.

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

The Study of Structure-Analgesic Activity Relationships in a Series of 4-Hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic Acid Toluidides and Xylidides.

In continuing the search for new analgesics among derivatives of 2,1-benzothiazines, a series of corresponding toluidides and xylidides of 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylic acid has been synthesized by the reaction of ethyl 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate with equimolar amounts of mono- and dimethyl-substituted anilides in boiling dry xylene. Their structure has been confirmed by the data of elemental analysis, nuclear magnetic resonance (NMR) spectroscopy (¹Ð and 13С), as well as mass spectrometry. All compounds obtained were subjected to pharmacological screening to identify their analgesic properties. Testing was carried out in male rats using the standard model of the thermal tail-flick (tail immersion test) in parallel and in comparison with the structurally related drugs meloxicam and piroxicam. Among the substances studied, highly active oral painkillers have been found; they exceed the analgesic effect of the reference drugs using the same dose. Interesting structural and biological regularities have been described; they will be useful in further research on creating promising new analgesics based on 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxamides.

Synthesis, Structure, and Analgesic Properties of Halogen-Substituted 4-Hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxanilides.

As potential new analgesics, the corresponding 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxanilides have been obtained by amidation of ethyl 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxylate with aniline and its halogenated analogsin boiling dry xylene. The peculiarities of the mass and nuclear magnetic resonance (¹Ð and 13С) spectra of the synthesized compounds are discussed. Using X-ray diffraction analysis, the ability of the compounds to form stable solvates with N,N-dimethylformamide has been shown on the example of 4-bromo-substituted derivative. It should be further studied to be considered in their crystallization. According to the results of the pharmacological testing conducted on the model of the thermal tail-flick (tail immersion test) among halogen-substituted 4-hydroxy-2,2-dioxo-1H-2λ6,1-benzothiazine-3-carboxanilides, substances which are considerably superior to meloxicam and piroxicam by their analgesic activity have been found. They are of interest for further profound studies.