Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358

The regulation of the p-hydroxybenzoate hydroxylase gene (pobA) of Pseudomonas putida WC5358 involved in the catabolism of p-hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the beta -ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria pobA has been found genetically linked to a regulator called pobR which activates pobA expression in response to PHB, In this study the identification and characterization of the pobC-pobA locus of P, putida WCS358 is presented. The p-hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated PobC, Using the pobA promoter transcriptionally fused to a promoterless lad gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA, This PobC-PCA response was genetically demonstrated by making use of pobC::Tn5 and pcaH::Tn5 mutants of strain WC5358 constructed in this study. In pobC mutants both the p-hydroxybenzoic and PCA response were not observed, whereas in the pcaH mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent pobA activation was still observed. Finally, the activation of pobA by PHB varied according to the concentration and it was observed that in the pcaR::Tn5 regulatory mutant of strain WCS358 the pobA promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the beta -ketoadipate pathway, one of which is pcaK, It was postulated that the reduction of pobA activation in pcaR::Tn5 mutants was because there was no expression of the pcaK gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell

Theoretical study of structure and relaxation mechanisms of photoexcited avobenzone using ab initio methods

Avobenzon (4-terc-butil-4’metoksidibenzoilmetan) je jedan od najpoznatijih najˇsire koriˇs´cenih filtera za zaˇstitu od UVA zraˇcenja. Avobenzon spada u grupu β-dikarbonilnih jedinjenja i ˇcija fotohemija ukljuˇcuje keto-enol tautomerizaciju, cis-trans izomerizaciju, rotaciju oko jednostruke veze kao i raskidanje karbonilnih grupa. Kako bi istraˇzili fotohemiju avobenzona izuˇcavani su tautomeri avobenzona: helatni i ne-helatni enol, rotamer Z, rotamer E kao i keto oblik u osnovnom i pobudenim stanjima u gasnoj fazi i acetonitrilu pomo´cu DFT i metode spregnutih klastera. Istraˇzivanje je pokazalo da torzija oko dvostruke C2-C3 veze fotopobudenog enolnog oblika vodi do unutraˇsnje konverzije u osnovno elektronsko stanje i formiranje rotamera E. Takode, otvaranje helatnog prstena i raskidanje vodoniˇcne veze rotacijom hidroksilne grupe uzrokuje stvaranje ne-helatnog enolnog oblika. Izuˇcavani su i mogu´ci mehanizmi za formiranje rotamera Z. Studija je pokazala da je zavisnost fotostabilnosti avobenzona od rastvaraˇca u direktnoj vezi sa relativnim rasporedom najniˇzeg tripletnog ππ∗ i nπ∗ stanja. Opis apsorpcionih osobina samo na osnovu vertikalnog ekscitacionog spektra raˇcunatog u minimumu povrˇsi potencijalne energije osnovnog elektronskog stanja, dovodi do zakljuˇcka da ne dolazi do inicijalnog pobudivanja nπ∗ stanja. S obzirom da pod uticajem termalnih i kvantnih efekata dolazi do znaˇcajnih promena u strukturi molekula avobenzona, za potpunije opisivanje sistema neophodno je uraˇcunati ne samo jednu konfiguraciju ve´c ansambl konfiguracija molekula. Tako je u ovom radu izvrˇsena simulacija spektara primenom DFT/MRCI metode zajedno sa ansamblom konfiguracija dobijenih pomo´cu metoda molekulske dinamike. Takode, izraˇcunati su i Frank-Kondonovi faktori za prelazak na ππ∗ stanje. Istraˇzivanje je pokazalo da termalne i kvantne fluktuacije simetriˇcno ˇsire apsorpcioni spektar u okviru poluklasiˇcne aproksimacije zbog ˇcega je neophodno uraˇcunati i vibronske efekte kako bi eksperimentalni spektar u potpunosti mogao biti reprodukovan. Proces apsorpcije uglavnom ukljuˇcuje pobudivanje ππ∗ stanja, mada ima je i nπ∗ stanje takode pobudeno u manjoj meri. Prelaz izmedu ova dva pobudena stanja se javlja prilikom intramolekulskog transfera protona. Takode postulirana su dva termalna tautomerizaciona mehanizma i prodiskutovane su njihove intermedijerne strukture.Avobenzone (4-tert-butyl-4’-methoxydibenzoylmethane, AB) is one of the most popular and widely used organic filter for skin photoprotection in the UVA band. AB belongs to the class of a β-dicarbonyl compounds which undergoes keto-enol tautomerization, rotation around single bond, bond breaking of carbonyl groups and cis-trans isomerization. All of these structural changes of AB lead to formation of different forms of AB which we labeled as: rotamer E, rotamer Z, chelated enol, non-chelated enol and keto tautomer. In order to study the photochemistry of AB we examined AB tautomers in the ground and excited states in the gas phase and acetonitrile using the coupled cluster and DFT computational methods. Based on the results of this study, it can be concluded that the internal conversion to the ground state of photoexcited chelated enol and creation of rotamer E is a consequence of the rotation around C2-C3 double bond of the chelated enol form. In addition, non-chelated enol tautomer is formed as a result of torsion of the hydroxyl group and breaking of the hydrogen bond of the chelated ring. The possible mechanisms of rotamer Z creation are discussed. Examination of the keto tautomer triplet states revealed that the lowest triplet ππ∗ and nπ∗ states exchange their relative order depending on the solvent. Results acquired from the calculation of the vertical excitation spectrum at the minimum of the ground state potential energy surface indicate that nπ∗ state does not exhibit initial photoexcitation. Because thermal and quantum fluctuations cause major structural changes of AB molecule and in order to create overall picture of photoexcitation, it is necessary to consider an ensemble of configurations instead of one molecular configuration. In order to do so, we sampled different configurations using DFT-based classical and path integral molecular dynamics, which are later used to calculate UVA response at DFT/MRCI level of theory. Also, Franck-Condon factors are calculated for the ππ∗ state. Results obtained using the semi-classical method show that there is symmetrical broadening of the excited states absorption due to the thermal and nuclear fluctuation, which lead to the conclusion that vibronic effects need to be included in order to achieve better matching of simulated and experimental spectrum. During the intramolecular proton transfer, crossing between the ππ∗ and nπ∗ state occurs, which results in minor contribution of the nπ∗ state in absorption, although the ππ∗ state contributes considerably more. Also, two thermal tautomerization mechanisms are postulated and their transient structures are discussed

The acetyl xylan esterase of Bacillus pumilus belongs to a family of esterases with broad substrate specificity

The Bacillus pumilus gene encoding acetyl xylan esterase tare) was identified and characterized. The axe gene was expressed and the recombinant enzyme produced in Escherichia coli was purified and characterized. The recombinant enzyme displayed similar properties to the acetyl xylan esterase (AXE) from B. pumilus. The AXE primary structure was 76% identical to the cephalosporin C deacetylase of B. subtilis, and 40% to two recently identified AXEs from Thermoanaerobacterium and Thermotoga maritima. These four proteins are of similar sire and represent a new family of esterases having a broad substrate specificity. The recombinant AXE was demonstrated to have activity on several acetylated substrates, including on cephalosporin C

Novel target genes of PsrA transcriptional regulator of Pseudomonas aeruginosa

The PsrA transcriptional regulator is involved in stationary phase induced transcriptional regulation of rpoS and in negative auto-regulation in Pseudomonas aeruginosa. This study was designed to determine whether other loci were regulated by PsrA in P. aeruginosa. Computer search was performed of the PsrA binding motif (G/CAAAC N2-4 GTTTG/C) against the P. aeruginosa genome sequence. Four of 14 analysed promoters responded to and bound PsrA; (i) divergent promoters controlling PA2952/ PA2951 and PA2953, (ii) promoter of PA0506 and (iii) upstream region of PA3571. Promoters PA0506 and PA2952-PA2951 were regulated negatively whereas promoters of PA2953 and PA3571 were regulated positively by PsrA. Two dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (21) SDS-PAGE) analysis on total proteins from P. aeruginosa PAO1 and psrA knock-out derivative was also performed resulting in the identification of 11 protein spots which were differentially regulated. These studies have indicated PsrA as a global regulator

Stabilnost morfoloških osobina semena kukuruza u različitim proizvodnim uslovima

The analyses were performed with the hybrid seeds of four hybrid combinations derived at the Maize Research Institute, Zemun Polje and produced in three locations. Under laboratory conditions, the following seed traits were analysed in the working sample of 10x100 seeds: seed test weight and shelling percentage, which was determined as the seed weight to ear weight ratio. In the three-year study, the greatest (352.71 g), i.e. smallest (280.09 g) test weight was recorded in the hybrids ZP 434 and ZP 704, respectively. The highest shelling percentage of 60.53 % was determined in the hybrid ZP 704, while the lowest one (48.20 %) was recorded the hybrid ZP 434. Obtained results point out that both traits in all four hybrids are highly expressed under all observed agro-ecological conditions and that effects of factors on the seed weight and the shelling percentage are great.U ovim istraživanja posmatrane su masa 1000 semena i randman semena, u odnosu na genotipsku kombinaciju i agroekološke uslove proizvodnje semena. Analize su rađene na hibridnom semenu četiri komercijalne hibridne kombinacije Instituta za kukuruz 'Zemun Polje', proizvedene na tri lokaliteta. U laboratorijskim uslovima, na radnom uzorku od 10x100 semena, izvršena su ispitivanja apsolutne mase semena, randman semena utvrđen je kao procentualni odnos mase semena i klipa. U trogodišnjem istraživanju najveća izmerena masa 1000 semena je kod hibrida ZP 434 (352,71 g) dok je ZP 704 hibrid sa najmanjom postignutom masom(280,09 g). Randman semena najveći je kod ZP 704 (60,53%), najmanji 48,20% kod ZP 434. Lokalitet sa najvećim variranjem mase bio je Turija (41,58%), a druga osobina najviše varijabilnosti u toku istraživanja bila je na lokalitetu Vrbas (28,13%). Eksperimentalni podaci obrađeni su na srednju vrednost i ukupnu varijabilnost (x i C.V.) za obe osobine semena i svaku varijantu istraživanja. Dvofaktorijalnom analizom varijanse utvrđen je uticaj faktora (hibrid i lokacija), kao i njihova interakcija na ispitivane osobine semena. Dobijeni rezultati pokazali su da sva četiri hibrida imaju visok nivo ispoljavanja obe osobine u svim posmatranim agroekološkim uslovima, kao i visok uticaj faktora (hibrid i lokacija) na masu semena i randman semena

Cell-selective toxicity of hydroxyapatite-chitosan oligosaccharide lactate particles loaded with a steroid cancer inhibitor

The applicative potential of synthetic calcium phosphates, especially hydroxyapatite (HAp), has become intensely broadened in the past 10 years, from bone tissue engineering to multiple other fields of biomedicine. Hybrid systems based on nano hydroxyapatites (HAp) are the subject of numerous studies in preventive and regenerative medicine. HAp nanoparticles coated with bioresorbable polymers have been successfully used as fillers, carriers of antibiotics, vitamins and stem cells in bone tissue engineering, etc. In this study we utilize an emulsification process and freeze drying to load the hybrid system made of nano HAp particles coated with chitosan oligosaccharide lactate (ChOSL) with two different but similar steroid derivatives: 3β-hydroxy- 16-hydroxymino-androst-5-ene-17-one (A), C19H27NO3 and 3β, 17β-dihydroxy-16-hydroxyminoandrost- 5-ene (B), C19H29NO3. The cell-selective toxicity of HAp particles coated with of A- or B-loaded ChOSL was examined simultaneously on the following cell lines: human breast carcinoma (MCF-7, MDA-MB-231), human lung carcinoma (A549) and human lung fibroblasts (MRC-5), using dye exclusion (DET) and MTT assays. 1H NMR, 13C NMR and high-resolution time-of-flight mass spectrometry (MS) techniques confirmed the intact structure of the derivatives A or B. FT-IR, XRD, DTA, TGA and DSC techniques confirmed the drug loading process of steroide (A or B) in core–shell particles based on nano hydroxyapatite. Atomic force microscopy and particle size analyses were used to confirm that the particles were spherical with sizes between 80 and 240 nm. The measured values of electrokinetic parameters (zeta potential, electrophoretic mobility and conductivity) were significantly different for the steroid free carrier (HAp/ChOLS) and A- or B-loaded ChOSL. The value of the topological molecular polar surface area (TPSA, the sum of the surfaces of polar atoms and groups in the molecule), were also different for drug free carrier and A- or BHAp/ ChOLS. Highly selective anticancer activity was noted towards breast cancer cells (MDAMB- 231) by B-loaded HAp/ChOLS. DET testing after 48 hours (after incubation and recovery) of the treatment with A-HAp/ChOSL and B-HAp/ChOSL particles showed a high viability of healthy cells (over 80%). The lowest viability was found in MDA-MB-231 cancer cells treated with B-HAp/ChOSL (28%). The obtained results of the DET and MTT tests showed that the particles of A-HAp/ChOLS exhibited nearly four-fold greater cytotoxicity towards breast cancer cells (MDA-MB-231) than towards healthy cells (MRC-5). B-HAp/ChOSL particles exhibited nearly six times greater cytotoxicity to all breast cancer cells than to healthy ones

Highly selective anticancer activity of core shell particles based on hydroxyapatite, chitosan lactate and different androstane derivatives

Hybrid systems based on nano hydroxyapatites (HAp) are the subject of numerous studies in preventive and regenerative medicine. Special interests are directed towards the creation of a system based on HAp for use in a nano-oncology. The main objective of this research is directed towards the creation of a system with cytotoxic properties towards the cancer cells with the same time, minimum side effects. Carriers base on core shell of HAp/chitosan-poly(D,L)-lactide-coglycolide (PLGA) loaded with androstane-based cancer inhibitor could be seen as promising drug delivery platforms for selective cancer therapies. In this study we utilize an emulsification process and freeze drying to load the composite particles based on HAp nanocarrier, chitosane (Ch), PLGA and chitosan oligosaccharide lactate (ChOL) with 17β-hydroxy-17α-picolyl-androst-5-en-3β-acetate (A) and 3β,17β-dihydroxy-16-hydroxymino-androst-5-en (B), a chemotherapeutic derivatives of androstane. The picolyl androstane derivatives showed high potency in the cell inhibitors of hormone-dependent cancers (lung, prostate and colon cancer; adeno and cervix carcinoma; etc.). 1H NMR, 13C NMR and high-resolution time-of-flight mass spectrometry (MS) techniques confirmed the intact structure of the derivatives A and B. The thermogravimetric and differential thermal analysis (TGA, DTA) coupled with mass spectrometry was used to qualitatively confirm the drug loading process. FT-IR, XRD, AFM and DSC techniques have confirmed the success of androstane (A and B) loading process in core shell particles base on nano hydroxyapatite. All the synthesized particles were found to be spherical in shape with a uniform size distribution from d50=167 to d50=231 nm. Highly selective anticancer activity was noted towards the human lung carcinoma (A549) by A loaded HAp/Ch-PLGA and towards the human breast adenocarcinoma (MDA-MB-231) by B loaded HAp/ChOL. The obtained results of the DET and MTT tests were in agreement

Tumor-selective hybrid system based on hydroxyapatite nanocarrier, chitosan, poly(lactic-co-glycolic acid) and androstan derivate

The applicative potential of synthetic calcium phosphates, especially hydroxyapatite (HAp), has become intensely broadened in the past 10 years, from bone tissue engineering to multiple other fields of biomedicine. Previously we have shown that hydroxyapatite nanoparticles coated with chitosan-poly(D,L)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous administration into mice. For this purpose radioactive 125-Iodine (125I), a low energy gamma emitter, was used to develop a novel in situ method for radiolabeling of particles and investigation of their biodistribution. In this study we utilize an emulsification process and freeze drying to load the composite particles based on hydroxyapatite nanocarrier, chitosane and poly(lactic-co-glycolic acid) with 17β- hydroxy-17α-picolyl-androst-5-en-3β-acetate (A), a chemotherapeutic derivative of androstane. The picolyl androstane derivatives showed high potency in the cell inhibitors of hormonedependent cancers (adenocarcinoma, prostate cancer, cervix carcinoma, colon cancer, etc.). 1H NMR, 13C NMR and high-resolution time-of-flight mass spectrometry (MS) techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The synthesized particles of A-loaded HAp/Ch-PLGA were found to be spherical in shape with a uniform size distribution of d50=168 nm. The release of A from HAp/Ch-PLGA was sustained, with no burst release or plateauing after three weeks. The obtained results of the DET and MTT tests show that the particles of A-loaded HAp/Ch-PLGA exhibit almost three times higher cytotoxicity towards lung adenocarcinoma cells (A549) than towards healthy cells (MRC5), while at the same time allowing twice as fast recovery of healthy cells. We have also analyzed the period of recovery of healthy, as well as cancer cells, following the treatment with A-loaded HAp/Ch-PLGA. After treatment with A-loaded HAp/Ch-PLGA, healthy cells recover twice as fast as the malignant ones. Immunofluorescent staining of primary fibroblasts interacting with HAp/Ch-PLGA and A-HAp/Ch-PLGA particles demonstrates no negative morphological or proliferative effects on cells