Cyclic digene system as a control element of a bacterial biosensor

The Escherichia coli JC158(pCIA12/pGFK5) strain carrying a cyclic digene system with a negative feedback on the pCIA12 plasmid reacting to the DNA damage by changing the synthesis level of reporter genes—GFP and β-galactosidase—was tested. The acquired phenotype was inherited by the next generations after the removal of the genotoxic action when the concentration of the DNA-damaging compounds was above the threshold level. A potential has been shown for the application of bacterial biosensors to monitor the presence of genotoxicants in the environment and to test the consequences of short-term exposures to toxic compounds.     

Control of lytic development in the Streptomyces temperate phage φC31

The repressor gene. c, is required for maintenance of lysogeny in the Streptomyces phage φC31. The c gene expresses three in-frame N-terminally different protein isoforms at least one of which is thought to bind to a 17bp highly conserved inverted repeat (CIR) sequence found at 18 (or more) loci throughout the φC31 genome. Here we present evidence that one of these loci, CIR6, and its interaction with the products of the repressor gene are critical in the control of the lytic pathway in φC31. To the right of CIR6, according to the standard map of φC31, an ‘immediate-early’ promoter. ap1, was discovered after insertion of a fragment containing CIR6 upstream of a promoterless kanamycin-resistance gene. aphll, to form pCIA2. pCIA2 conferred kanamycin resistance upon Streptomyces coelicolor A3(2) but not upon a φC31 lysogen of S. coelicolor. Operator-constitutive (O^c) mutants of pCIA2 were isolated and the mutations lay in CIR6, i.e. CIR6:G14T and CIR6:C2A. Primer extension analysis of RNA prepared from an induced, temperature-sensitive lysogen of S. coelicolor localized a mRNA 5′ endpoint 21 bp to the right of CIR6. The importance of the ap1/CIR6 region in the regulation of lytic growth was demonstrated by the analysis of a virulent mutant, φC31 vir1, capable of forming plaques on an S. coelicolorφC31 lysogen, φC31 vir1 contained a DNA inversion with the breakpoints lying within the integrase gene (which lies approximately 7kbp to the right of CIR6) and in the essential early region between CIR6 and the -10 sequence for ap1. The separation of ap1 from its operator was thought to be the basis for the virulent phenotype in φC31 vir1. Band-shift assays and DNase I footprinting experiments using purified 42kDa repressor isoform confirmed that CIRs 5 and 6 were indeed the targets for binding of this protein. The 42 kDa repressor bound to CIR6 with higher affinity than to CIR5 in spite of their identical core sequences. Repressor bound at CIR6 facilitated binding at CIR5. The high-affinity binding to CIR6 was abolished with the O^c mutant, CIR6:G14T. Hydroxyl radical footprinting and dimethyl sulphate methylation protection of the 42 kDa repressor–CIR6 interaction suggested that the protein bound in the major groove and to one face of the DNA.     

Isolation and characterization of a new antifungal metabolite ofTrichoderma reesei

A mycelial extract ofTrichoderma reesei (P-12) was separated into four fractions by high performance liquid chromatography (HPLC). Out of these, two were found to exhibit antifungal activity when tested against plant pathogenic fungi. The level of antifungal compounds was higher in media containing glucose as the carbon source as compared to one containing cellulose. The synthesis of these antifungal compounds started after 3 days of inoculation at 30°C and continued upto 8 days. No further increase was recorded beyond this period.     

Form of Vitamin B(12) and Its Role in a Methanol-Utilizing Bacterium Protaminobacter ruber

A methanol-utilizing bacterium, Protaminobacter ruber, produced a large amount of vitamin B(12). The compounds were isolated from the cells and identified as methylcobalamin (methyl-B(12)) and adenosylcobalamin (adenosyl-B(12)) by various tests. The variation in the form of B(12) during cultivation was examined by bioautography with cellulose acetate membrane electrophoresis. Methyl-B(12) and adenosyl-B(12) were the two main B(12) compounds produced in the various phases of bacterial growth. The ratio of the amount of methyl-B(12) to total B(12) compounds was higher during the earlier phases of growth. After the logarithmic phase, adenosyl-B(12) was the predominant form. The existence of N-methyltetrahydrofolate:homocysteine transmethylase and methyl-B(12):homocysteine transmethylase was demonstrated in cell-free extracts of Protaminobacter ruber. Methyl-B(12) in P. ruber seems to function mainly in the B(12)-dependent methionine synthetase system.     

Evidence for a novel pathway of leukotriene formation in human platelets

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.     

Form of Vitamin B12 and Its Role in a Methanol-Utilizing Bacterium Protaminobacter ruber

A methanol-utilizing bacterium, Protaminobacter ruber, produced a large amount of vitamin B₁₂. The compounds were isolated from the cells and identified as methylcobalamin (methyl-B₁₂) and adenosylcobalamin (adenosyl-B₁₂) by various tests. The variation in the form of B₁₂ during cultivation was examined by bioautography with cellulose acetate membrane electrophoresis. Methyl-B₁₂ and adenosyl-B₁₂ were the two main B₁₂ compounds produced in the various phases of bacterial growth. The ratio of the amount of methyl-B₁₂ to total B₁₂ compounds was higher during the earlier phases of growth. After the logarithmic phase, adenosyl-B₁₂ was the predominant form. The existence of N⁵-methyltetrahydrofolate:homocysteine transmethylase and methyl-B₁₂:homocysteine transmethylase was demonstrated in cell-free extracts of Protaminobacter ruber. Methyl-B₁₂ in P. ruber seems to function mainly in the B₁₂-dependent methionine synthetase system.     

Calcium stimulation of a novel lipoxygenase

Homogenates of rat basophilic leukemia (RBL-1) cells have a novel lipoxygenase which was stimulated by calcium in a concentration dependent fashion and inhibited by epinephrine. The major compounds formed from [¹⁴C]-arachidonic acid were identified by gas chromatography — mass spectrometry to be 5-hydroxyeicosatetraenoic acid and 5,12-dihydroxyeicosatetraenoic acid. Other compounds present in small amounts were 12-hydroxy- and 5,6-dihydroxyeicosatetraenoic acid. The stimulation by calcium of this pathway in basophils links it closely to the release reaction which is calcium dependent.     

Ab initio parameterization of YFF1, a universal force field for drug-design applications

The YFF1 is a new universal molecular mechanic force field designed for drug discovery purposes. The electrostatic part of YFF1 has already been parameterized to reproduce ab initio calculated dipole and quadrupole moments. Now we report a parameterization of the van der Waals interactions (vdW) for the same atom types that were previously defined. The 6–12 Lennard-Jones potential terms were parameterized against homodimerization energies calculated at the MP2/6-31 G* level of theory. The Boys-Bernardi counterpoise correction was employed to account for the basis-set superposition error. As a source of structural information we used about 2,400 neutral compounds from the ZINC2007 database. About 6,600 homodimeric configurations were generated from this dataset. A special “closure” procedure was designed to accelerate the parameters fitting. As a result, dimerization energies of small organic compounds are reproduced with an average unsigned error of 1.1 kcal mol^-1. Although the primary goal of this work was to parameterize nonbonded interactions, bonded parameters were also derived, by fitting to PM6 semiempirically optimized geometries of approximately 20,000 compounds.     

Deoxycholic acid degradation by a Pseudomonas sp. Acidic intermediates with A-ring unsaturation.

The microbial catabolism of deoxycholic acid by a Pseudomonas sp. was studied, and six further acidic intermediates were isolated, as their methyl esters. Evidence is presented that the compounds are methyl 12 alpha-hydroxy-3-oxochol-4-en-24-oate, methyl 12 alpha-hydroxy-3-oxo-23,24-dinorchol-4-en-22-oate, methyl 12 alpha-hydroxy-3-oxochola-1,4-dien-24-oate, methyl 12 alpha-hydroxy-3-oxo-23,24-dinorchola-1,4-dien-22-oate, methyl 12 alpha-hydroxy-3-oxochola-1,4,22E-trien-24-oate and methyl 12 alpha-hydroxy-3-oxo-23,24-dinorchola-1,4,17(20)-trien-22-oate. On the basis of these compounds, together with the seven intermediates previously reported, a catabolic pathway is proposed.     

Seeds latency of Stylosanthes hamata (Leguminosae) and its relation with morphology of the seed-coat]

The seed of Stylosanthes hamata is characterized by it's a high level of dormancy, which is imposed by seed-coat impermeability to water entry (harseededness or hard-seed condition). The level of hardseededness between upper and lower articulations of seven diploid and five tetraploid accessions was studied under tropical conditions. Germination tests on naked seeds (relative humidity: 55-65%, constant temperature: 27-30 degrees C, photoperiod: 12/12 hr) showed that hard-seed percentage was, in most cases, over 85% independent of ploidy level and seed position within the fruit. Histochemical analysis indicated that hardseededness in this species was associated with a completely sealed microphile and hilum. This feature was also associated to: a) an epidermis consisting of a palisade-like layer of macrosclereids, b) a like-line at the outermost region of these cells and c) a high concentration of hydrophobic compounds located all across the seed-coat.     

Production of Antibacterial Compounds Analogous to Chloramphenicol by a n-Paraffin-grown Bacterium

Corynebacterium sp. KY 4339, when grown on n-paraffin (a mixture of C–12 to C–14 fractions) as the sole carbon source, produced three kinds of antibacterial compounds which were tentatively named Corynecins. These compounds were isolated by the extraction from the culture broth with ethyl acetate and by the chromatographies on silicic acid and alumina columns. Each component demonstrated some similarity to chloramphenicol on thin-layer chromatogram. Although their biological activities were not so remarkably as that of chloramphenicol, the patterns of antibacterial spectra against gram-positive and gram-negative bacteria resembled to it.

For the production of corynecins, n-paraffin was a preferable carbon source. By controlling the pH of the medium in the neutral range and keeping the aeration at a high level during the fermentation, approximately 3 g of corynecins per liter of the medium were produced after 72-hr incubation.     

Design, synthesis and cardioprotective effect of a new class of dual-acting agents: phenolic tetrahydro-beta-carboline RGD peptidomimetic conjugates

In this study, a new class of phenolic tetrahydro-beta-carboline RGD peptidomimetic conjugates was designed and synthesized. The radical scavenging activities of these newly synthesized compounds 12a-c were evaluated in PC12 cell survival assays. The NO scavenging activities of these compounds were confirmed in the acetylcholine-induced vasorelaxation assay. Compounds 12a-c were efficacious in a rat arterial thrombosis model, and were active in ADP- or PAF-induced in vitro platelet aggregation assays, which suggests these compounds also possess anti-thrombotic activity. The beneficial effects of dual-acting agent 12c were demonstrated on the ischemia-reperfusion induced cardiac infarct size and oxidative change in an in vivo rat model.     

Vitamin B12, a chlorophyll-related analog to pheophytin a from marine brown algae, promotes neurite outgrowth and stimulates differentiation in PC12 cells

We previously isolated an analog to chlorophyll-related compounds, pheophytin a, from the marine brown alga Sargassum fulvellum and demonstrated that it is a neurodifferentiation compound. In the current study, we investigated the effects of the pheophytin a analog vitamin B₁₂ on PC12 cell differentiation. In the presence of a low level of nerve growth factor (10 ng ml⁻¹), vitamin B₁₂ demonstrated neurite outgrowth-promoting activity in PC12 cells. The effect was dose-dependent in the range of 6–100 μM. In the absence of nerve growth factor, vitamin B₁₂ did not promote differentiation. To investigate the mechanism for this effect, we conducted differentiation assays and western blot analysis with signal transduction inhibitors and found that vitamin B₁₂ did not promote PC12 cell differentiation in the presence of K252a or U0126 inhibitors. These results suggest that vitamin B₁₂ stimulates PC12 cell differentiation through enhancement of the mitogen-activated protein kinase signal transduction pathway, which is also induced by nerve growth factor. Thus, vitamin B₁₂ may be a good candidate for treatment of neurodegenerative diseases such as Alzheimer’s disease.     

New N-pyridinyl(methyl)-indole-2- and 3-(alkyl)carboxamides and derivatives acting as systemic and topical inflammation inhibitors

A series of novel N-substituted-(indol-2-yl)carboxamides (12-18) and (indol-3-alkyl)carboxamides (25-31) were synthesized and evaluated as inhibitors of the inflammation process. Pharmacomodulation at the level of the amidic nitrogen by incorporation of the previously described pharmacophoric moieties 6-aminolutidine, beta-picolylamine, 4-aminopyridine and piperazine was investigated; only two compounds (12) and (31) exhibited significant (approximately 40%) inhibitory effect in the carrageenan-induced rat paw edema after oral administration of a dose of 0.1 mM kg(-1). Replacement of the indole core by indazole failed to increase activity. Incorporation of an alkyl chain spacer led to more efficient compounds (46-52) especially in the indolepropanamide sub-series. Determination of the efficiency of the most active compounds on topical inflammation, by measuring reduction of ear thickness in the acute tetradecanoyl phorbol acetate (TPA)-induced mouse ear swelling assay, confirmed the high potency of propanamides (49) and (51) after oral administration: ID50 = 0.041 +/- 0.013 and 0.042 +/- 0.016 mM kg(-1) respectively. The less toxic propanamide (51) exerted a high level of inhibitory activity after topical application of 2 x 100 microg/ear: 78 +/- 2%.     

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.     

Biosynthesis of corriphyrins - a new branch of metabolism of tetrapyrrole compounds

Experiments were carried out to study biosynthesis of tetrapyrrole pigments--corriphyrins which are methylated reduced derivatives of uroporphyrin III. It was shown that the pattern of corriphyrin synthesis was close to that of vitamin B12. The mutant strain of propionic bacteria which was active in the vitamin B12 formation synthesized significantly more corriphyrins than the original strain. The corriphyrin synthesis was stimulated by delta-aminolevulinic acid and methionine and was inhibited by hydroxylamine and aeration. The formation of corriphyrins was repressed by vitamin B12. It is concluded that corriphyrins are precursors of vitamin B12 at the stage between uroporphyrinogene III and cobyrinic acid. The paper discusses further investigations of metabolism of methylated derivatives of uroporphyrinogene III as a method of elucidating the evolution of tetrapyrrole compounds and clarifying the processes involved in porphyrin metabolism in higher plants and animals.     

Resistance to the CCR5 inhibitor 5P12-RANTES requires a difficult evolution from CCR5 to CXCR4 coreceptor use

Viral resistance to small molecule allosteric inhibitors of CCR5 is well documented, and involves either selection of preexisting CXCR4-using HIV-1 variants or envelope sequence evolution to use inhibitor-bound CCR5 for entry. Resistance to macromolecular CCR5 inhibitors has been more difficult to demonstrate, although selection of CXCR4-using variants might be expected. We have compared the in vitro selection of HIV-1 CC1/85 variants resistant to either the small molecule inhibitor maraviroc (MVC) or the macromolecular inhibitor 5P12-RANTES. High level resistance to MVC was conferred by the same envelope mutations as previously reported after 16-18 weeks of selection by increasing levels of MVC. The MVC-resistant mutants were fully sensitive to inhibition by 5P12-RANTES. By contrast, only transient and low level resistance to 5P12-RANTES was achieved in three sequential selection experiments, and each resulted in a subsequent collapse of virus replication. A fourth round of selection by 5P12-RANTES led, after 36 weeks, to a "resistant" variant that had switched from CCR5 to CXCR4 as a coreceptor. Envelope sequences diverged by 3.8% during selection of the 5P12-RANTES resistant, CXCR4-using variants, with unique and critical substitutions in the V3 region. A subset of viruses recovered from control cultures after 44 weeks of passage in the absence of inhibitors also evolved to use CXCR4, although with fewer and different envelope mutations. Control cultures contained both viruses that evolved to use CXCR4 by deleting four amino acids in V3, and others that maintained entry via CCR5. These results suggest that coreceptor switching may be the only route to resistance for compounds like 5P12-RANTES. This pathway requires more mutations and encounters more fitness obstacles than development of resistance to MVC, confirming the clinical observations that resistance to small molecule CCR5 inhibitors very rarely involves coreceptor switching.