Effect of the kanamycin resistance marker on stability of 2μ-based expression plasmids

U ovom radu opisan je uticaj gena za kanamicinsku rezistenciju (Kmr) na održavanje 2μm plazmida u Saccharomyces cerevisiae. Prisustvo ovog marker gena dovodi do gubitka stabilnog model-vektora konstantnom stopom nezavisnom od izvora ugljenika i stope rasta kulture. Kod sojeva za sintezu heterolognih proteina sa galaktoznog promotora (GALUAS) uvođenje Kmr rezultira "čišćenjem" od kvaščevih epizomalnih plazmida (YEp) u svega nekoliko generacija. Primena selektivnog pritiska na sojeve koji proizvode rekombinantnu penicilin G amidazu (rPGA) nije dovela do očekivanog povećanja prinosa proteina. Ispitivanjem uticaja samih genetičkih elemenata za proizvodnju heterolognih proteina na stabilnost vektora pokazano je da najjače destabilišuće dejstvo ima prisustvo i eksprecija stranog gena.In this paper we describe the effect of the kanamycin resistance gene (Kmr) on 2μm-based plasmid maintenance in Saccharomyces cerevisiae. The influence of this marker gene on the loss of the stable model-vectors proved to be constant, as well as independent of carbon source and culture growth rates. In strains for GALUAS - driven heterologous protein production introduction of Kmr resulted in curing of the yeast episomal plasmid (YEp) from the population in a small number of generations. Application of selective pressure on the strain producing recombinant penicillin G amidase (rPGA) did not provide the expected increase of protein yield. The influence of genetic elements for heterologous protein production on vector stability was examined, and the most destabilizing factors prove to be the presence and expression of the foreign gene

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view

Opšta psihopatologija i impulsivnost kao pokazatelji potencijalnog ishoda medicinske nutritivne terapije kod predgojaznih i gojaznih žena

Background/Aim. Obesity is the chronic disease and health threatening condition. The number of obese people in the world has taken epidemic conditions. Medical nutritional therapy is the first choice in the treatment of obesity, but it is also accompanied with a great percentage of attrition and a significant weight regain. The aim of our study was to evaluate if psychiatric and specific psychological factors (impulsivity) could be the predictors of successful weight loss. Methods. A study sample consisted of 84 consecutive overweight/obesity women, 20 to 40 years old, who were willing to adhere to the medical nutrition therapy after the initial anthropometric measurements were performed. All participants received a personalized nutritional counseling and a daily diet treatment with 20% caloric restriction from estimated daily energy requirement. At the beginning of the study, the 90-item Symptom Check-list (SCL-90) and the 30-item Barratt Impulsiveness scale (BIS11) were administrated. At the end of six months of caloric restriction, the conventional diet therapy and control weight measurement, the patients were divided into two groups: the group I - 40 participants (48%) who lost ≥ 10% of their initial body weight (successful group) and the group II - 44 participants (52%) who lost lt 10% of their initial weight (unsuccessful). Results. There were no significant differences between groups in the demographic data (age, education level, employment, marital status), baseline anthropometric measurements, and in the general psychopathology total score. Groups I and II were significantly different in the total BIS-11 score (p lt 0.001) and in Factor II (motor impulsivity) (p lt 0.05). Conclusion. Even though the successful and unsuccessful diet responded participants did not differ in the general psychopathology and symptom dimensions, our results pointed out impulsivity as a discriminative factor between them. The total impulsivity and Factor II (motor impulsivity) as an indicator of binge eating were higher in the unsuccessfully responding overweight/obese women on the conventional diet treatment. A focus on impulsivity in a psychotherapeutic work might lead to better outcomes in the medical nutrition therapy.Uvod/Cilj. Gojaznost je hronično oboljenje sa značajnim nepovoljnim efektima na zdravlje, a broj gojaznih osoba u svetu dostigao je epidemijske razmere. Medicinska nutritivna terapija je terapija izbora, ali je njena uspešnost ograničena zbog značajnog procenta odustajanja, kao i ponovnog dobijanja na težini koje je prate. Cilj ove studije je bio da se proceni da li psihijatrijski i specifični psihološki faktori (impulsivnost) mogu biti pokazatelji pozitivnog ishoda lečenja. Metode. Studijom je bilo obuhvaćeno ukupno 84 predgojaznih i gojaznih žena životnog doba 20-40 godina, koje su bile motivisane za medicinsku nutritivnu terapiju. Sve ispitanice su prošle individualno savetovanje i dobile dnevni plan ishrane koji je podrazumevao kalorijsku restrikciju od 20% u odnosu na procenjene dnevne energetske potrebe. Na početku studije sprovedeno je antropometrijsko merenje i primenjene su Lista simptoma od 90 pitanja (90item Symptom Check-list) i Baratova skala impulsivnosti (Barratt Impulsiveness scale (BIS-11)). Nakon šest meseci primene kalorijski restriktivnog načina ishrane ponovljena su antropometrijska merenja. Ispitanice su podeljene u dve grupe: 40 ispitanica (48%) koje su izgubile ≥ 10% od početne telesne mase (uspešne) i 44 ispitanice (52%) koje su izgubile lt 10% od početne telesne mase (neuspešne). Rezultati. Nije bilo značajne razlike između grupa ispitanica u odnosu na demografske karakteristike (godine života, stepen obrazovanja, zaposlenost, bračni status) i antropometrijske parametre na početku studije, kao i na skor opšte psihopatologije i dimenzije simptoma. Statistički značajna razlika između uspešnih i neuspešnih predgojaznih/gojaznih ispitanica dobijena je za ukupni BIS-11 skor (p lt 0.001) i za Faktor II (motorna impulsivnost) (p lt 0.05). Zaključak. Ispitanice sa ponovljenim lošim odgovorom nisu se razlikovale po opštoj psihopatologiji od onih sa uspešnim odgovorom na nutritivnu terapiju. Naši rezultati ukazuju na impulsivnost kao mogući pokazatelj ishoda lečenja. Ukupna impulsivnost i motorna impulsivnost kao indikator povremenog impulsivnog prejedanja bili su viši u grupi predgojazih/gojaznih ispitanica koje nisu postigle terapijski cilj. Psihoterapijski rad na impulsivnosti možda bi mogao doprineti boljem ishodu medicinske nutritivne terapije

Didehydroroflamycoin pentaene macrolide family from Streptomyces durmitorensis MS405(T): production optimization and antimicrobial activity

AimsThe aim of this study was to improve production of pentaene 32,33-didehydroroflamycoin (DDHR) in Streptomyces durmitorensis MS405 strain to obtain quantities sufficient for in depth analysis of antimicrobial properties. Methods and ResultsThrough classical medium optimization conditions for stable growth, DDHR production within 7days of incubation was established. Yields of 215mgl(-1) were achieved in shake flask experiments in complex medium with mannitol as the primary carbon source. DDHR had poor antibacterial activity with minimal inhibitory concentrations (MIC) of 400gml(-1) for Staphylococcus aureus and Bacillus subtilis, while MIC of 70gml(-1) was determined for Candida albicans. Using flow cytometry and fluorescent microscopy, it was demonstrated that DDHR induced membrane damage in C.albicans followed by cell death. Combination studies with known antifungal nystatin showed that DDHR is a promising agent for the development of novel antimycotic treatments potentially less toxic for human cells. ConclusionsPentaene didehydroroflamycoin has no antibacterial activity but can be further developed for the application in antifungal therapy. Significance and Impact of the StudyThis study is the first report on the stable and production in high yields of a novel pentaene family that acts on Candida cell membranes and can be used in combination with known antifungals. Polyenes are still antifungal antibiotics of choice, and therefore, isolation and production of new lead structures are highly significant

A comparative antimicrobial and toxicological study of gold(III) and silver(I) complexes with aromatic nitrogen-containing heterocycles: synergistic activity and improved selectivity index of Au(III)/Ag(I) complexes mixture

Five aromatic nitrogen-containing heterocycles, pyridazine (pydz, 1), pyrimidine (pm, 2), pyrazine (pz, 3), quinoxaline (qx, 4) and phenazine (phz, 5) have been used for the synthesis of gold(III) and silver(I) complexes. In contrast to the mononuclear Au1-5 complexes all having square-planar geometry, the corresponding Ag1-5 complexes have been found to be polynuclear and of different geometries. Complexes Au1-5 and Ag1-5, along with K[AuCl4], AgNO3 and N-heterocyclic ligands used for their synthesis, were evaluated by in vitro antimicrobial studies against a panel of microbial strains that lead to many skin and soft tissue, respiratory, wound and nosocomial infections. All tested complexes exhibited excellent to good antibacterial activity with minimal inhibitory (MIC) values in the range of 2.5 to 100 mu g mL(-1) against the investigated strains. The complexes were particularly efficient against pathogenic Pseudomonas aeruginosa (MIC = 2.5-30 mu g mL(-1)) and had a marked ability to disrupt clinically relevant biofilms of strains with high inherent resistance to antibiotics. Moreover, the Au1-4 and Ag1-5 complexes exhibited pronounced ability to competitively intercalate double stranded genomic DNA of P. aeruginosa, which was demonstrated by gel electrophoresis techniques and supported by molecular docking into the DNA major groove. Antiproliferative effect on the normal human lung fibroblast cell line MRC5 has also been evaluated in order to determine therapeutic potential of Au1-5 and Ag1-5 complexes. Since the investigated gold(III) complexes showed much lower negative effects on the viability of the MRC5 cell line than their silver(I) analogues and slightly lower antimicrobial activity against the investigated strains, the combination approach to improve their pharmacological profiles was applied. Synergistic antimicrobial effect and the selectivity index of 10 were achieved for the selected gold(III)/silver(I) complexes mixtures, as well as higher P. aeruginosa PAO1 biofilm disruption activity, and improved toxicity profile towards zebrafish embryos, in comparison to the single complexes. To the best of our knowledge, this is the first report on synergistic activity of gold(III)/silver(I) complexes mixtures and it could have an impact on development of new combination therapy methods for the treatment of multi-resistant bacterial infections

A comparative antimicrobial and toxicological study of gold(III) and silver(I) complexes with aromatic nitrogen-containing heterocycles: synergistic activity and improved selectivity index of Au(III)/Ag(I) complexes mixture

Five aromatic nitrogen-containing heterocycles, pyridazine (pydz, 1), pyrimidine (pm, 2), pyrazine (pz, 3), quinoxaline (qx, 4) and phenazine (phz, 5) have been used for the synthesis of gold(III) and silver(I) complexes. In contrast to the mononuclear Au1-5 complexes all having square-planar geometry, the corresponding Ag1-5 complexes have been found to be polynuclear and of different geometries. Complexes Au1-5 and Ag1-5, along with K[AuCl4], AgNO3 and N-heterocyclic ligands used for their synthesis, were evaluated by in vitro antimicrobial studies against a panel of microbial strains that lead to many skin and soft tissue, respiratory, wound and nosocomial infections. All tested complexes exhibited excellent to good antibacterial activity with minimal inhibitory (MIC) values in the range of 2.5 to 100 mu g mL(-1) against the investigated strains. The complexes were particularly efficient against pathogenic Pseudomonas aeruginosa (MIC = 2.5-30 mu g mL(-1)) and had a marked ability to disrupt clinically relevant biofilms of strains with high inherent resistance to antibiotics. Moreover, the Au1-4 and Ag1-5 complexes exhibited pronounced ability to competitively intercalate double stranded genomic DNA of P. aeruginosa, which was demonstrated by gel electrophoresis techniques and supported by molecular docking into the DNA major groove. Antiproliferative effect on the normal human lung fibroblast cell line MRC5 has also been evaluated in order to determine therapeutic potential of Au1-5 and Ag1-5 complexes. Since the investigated gold(III) complexes showed much lower negative effects on the viability of the MRC5 cell line than their silver(I) analogues and slightly lower antimicrobial activity against the investigated strains, the combination approach to improve their pharmacological profiles was applied. Synergistic antimicrobial effect and the selectivity index of 10 were achieved for the selected gold(III)/silver(I) complexes mixtures, as well as higher P. aeruginosa PAO1 biofilm disruption activity, and improved toxicity profile towards zebrafish embryos, in comparison to the single complexes. To the best of our knowledge, this is the first report on synergistic activity of gold(III)/silver(I) complexes mixtures and it could have an impact on development of new combination therapy methods for the treatment of multi-resistant bacterial infections.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3334

Accumulation of nickel in red clover

Red clover (Trifoilum pratense L.) is considered as one of the most important legumes, and today is the second most important, after alfalfa. Production of high-quality feed from red clover may be affected, among other things, the increased concentration of heavy metals in some agricultural areas, primarily due to anthropogenic influences. The aim of this study was to determine the level of nickel in red clover grown on soils with different content of nickel, in order to obtain information on safety of these nutrients. The examination was conducted on four types of soil: chernozem, vertisol, eutric cambisol and humofluvisol. Sampling of soil and plant material was carried out during May 2011, in the second year of red clover production. The total content of nickel in soil samples, at a depth 0.30 cm, was in the range 3.36-129.67 mg/kg. Maximum permitted level of nickel in soil is 50 mg/kg. The content of nickel in red clover was in the range 0,41- 6,87 mg/kg, which is below the critical and toxic concentrations to plants. It was concluded that the accumulation of heavy metals in plants did not depend only on the total content in soil, but also the affinity of the plant, and individual and interactive effects of various soil properties. It is necessary to further control of nickel in contaminated area, in order to prevent his entry into the food chain and provide safety food

Supplementary data for the article: Savić, N. D.; Milivojevic, D. R.; Glišić, B. D.; Ilic-Tomic, T.; Veselinovic, J.; Pavic, A.; Vasiljevic, B.; Nikodinovic-Runic, J.; Djuran, M. I. A Comparative Antimicrobial and Toxicological Study of Gold(III) and Silver(i) Complexes with Aromatic Nitrogen-Containing Heterocycles: Synergistic Activity and Improved Selectivity Index of Au(III)/Ag(i) Complexes Mixture. RSC Advances 2016, 6 (16), 13193–13206. https://doi.org/10.1039/c5ra26002g

Supplementary material for: [https://doi.org/10.1039/c5ra26002g]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2041

Akumulacija teških metala u Medicago sativa L. i Trifolium pratense L. na kontaminiranom fluvisolu

Recently, heavy metals concentrations increased in some agricultural areas due to the consequences of anthropogenic impacts. The aim of this study was to determine the level of heavy metals (As, Cr, Ni and Pb) in Medicago sativa L. and Trifolium pratense L. grown on fluvisol, in order to obtain information on safety of these nutrients. The total content of Pb, As, Cr and Ni in the samples of fluvisol was above the maximum allowable amount. The content of heavy metals in Medicago sativa L. and Trifolium pratense L. was below the critical and toxic concentrations in all samples originating from contaminated soil. It was concluded that the accumulation of heavy metals in plants did not depend only on the total content in soil, but also the affinity of the plant, and individual and interactive effects of various soil properties. No statistically significant differences in the accumulation of heavy metals between Medicago sativa L. and Trifolium pratense L were observed. It is necessary to further control of heavy metals in the investigated area, in order to prevent their entry into the food chain and provide healthy food.U poslednje vreme povećana je koncentracija teških metala na nekim poljoprivrednim površinama usled antropogenog uticaja. Cilj rada je bio da se utvrdi nivo teških metala (As, Cr, Ni i Pb) u nekarbonatnom aluvijalnom (fluvijanom) zemljištu, te njihova akumulacija u Medicago sativa L. i Trifolium pratense L., radi dobijanja informacije o zdravstvenoj ispravnostinosti ovih hraniva. Ukupan sadržaj ispitivanih teških metala u uzorcima zemljišta bio je iznad maksimalno dozvoljene količine, što znači da svi uzorci analiziranih lokaliteta pripadaju kategoriji kontaminiranog zemljišta. Sadržaj teških metala u Medicago sativa L. i Trifolium pratense L. bio je ispod kritičnih i toksičnih koncentracija, kao i maksimalno dozvoljenih količina u hrani za životinje u svim uzorcima poreklom sa kontaminiranog zemljišta. Konstatovano je da akumulacija teških metala u biljkama nije zavisila samo od ukupnog sadržaja u zemljištu, nego i afiniteta biljke, te individualnog ili interaktivnog dejstva raznih zemljišnih svojstava. Nije bilo značajnih razlika u akumulaciji teških metala izmeću Medicago sativa L. i Trifolium pratense L

Postmarketinška studija efikasnosti i bezbednosti primene losartana u lečenju bolesnika s umerenom i blagom arterijskom hipertenzijom - studija Lotnar

Introduction. Losartan, the angiotensin type 1 receptor blocker (ARB) exercises its main antihypertensive effect by vasodilatation of peripheral arteries. Objective. The aim of this study was to evaluate the antihypertensive effect and safety of losartan in patients with mild and moderate arterial hypertension (AH). Methods. This was an open post-marketing study with losartan as monotherapy in previously treated or untreated patients with AH. Primary efficacy parameter was the percentage of patients that achieved target blood pressure after 8-week treatment with a single daily dose of losartan of 50-100 mg. Safety parameters were assessed according to the percentage of adverse events and metabolic effects of therapy. Results. The study included 550 patients with AH (59% female and 41% male), mean age 56.8±11.4 years, BMI=27±4 kg/m2. Losartan was applied in 31% of untreated and 69% of previously treatment-resistant patients After 8 weeks target blood pressure was achieved in 67.8% (SBP) and in 81.1% (DBP) of patients, respectively. The mean decrease was 21.8% for SBP and 21.1% for DBP (p lt 0.001). Out of all, 65% of patients achieved both target SBP and DBP values. Hydrochlorothiazide was added to the therapy in 11.6% of patients. There were no significant differences in drug efficacy between the entire group and subgroups of patients with diabetes mellitus and impaired renal function (p=ns). Adverse events were rare and metabolic effect was favorable. Conclusion. Monotherapy with losartan in a dosage of 50-100 mg applied during 8 weeks resulted in achieving target values of blood pressure in 65% of patient with mild and moderate hypertension, also including the patients with diabetes mellitus and impaired renal function. Losartan is a safe and metabolically neutral medication.Uvod. Losartan, blokator angiotenzinskog receptora tip 1 (ARB), antihipertenzivni efekat ostvaruje vazodilatacijom perifernih arterija. Cilj rada. Cilj studije je bio da se procene efikasnost i bezbednost primene losartana kod bolesnika s arterijskom hipertenzijom blagog i umerenog stepena. Metode rada. U otvorenoj postmarketinškoj studiji losartan je primenjen kao monoterapija kod bolesnika s prethodno lečenom ili nelečenom umerenom i blagom hipertenzijom tokom osam nedelja u dozi od 50 mg i 100 mg jednom dnevno, uz posmatranje promene procenta bolesnika kod kojih su dostignute ciljne vrednosti sistolnog (SAP) i dijastolnog arterijskog krvnog pritiska (DAP). Bezbednost primene leka procenjena je prema procentu neželjenih dejstava i metaboličkim efektima terapije. Rezultati. U studiju je uključeno 550 bolesnika (59% žena i 41% muškaraca) prosečne starosti od 56,8±11,4 godine i prosečnog indeksa telesne mase (BMI) od 27±4 kg/m2. Losartan je primenjen kod 31% nelečenih i 69% prethodno lečenih bolesnika ali s neregulisanom hipertenzijom. Posle osam nedelja ciljne vrednosti SAP su postignute kod 67,8% bolesnika, a DAP kod 81,1% (i SAP i DAP kod 65% bolesnika), s prosečnim smanjenjem SAP od 21,8% i DAP od 21,1% (p lt 0,001). Hidrohlorotijazid je dodat terapiji kod samo 11,6% bolesnika. Terapijski efekti se nisu statistički značajno razlikovali između svih bolesnika i podgrupa ispitanika sa dijabetes melitusom (DM) i smanjenom funkcijom bubrega. Neželjena dejstva leka su bila retka, a metabolički efekat bio je povoljan. Zaključak. Monoterapija losartanom dovodi do postizanja ciljnih vrednosti krvnog pritiska kod 65% bolesnika sa blagom i umerenom hipertenzijom tokom osam nedelja primene, uključujući i bolesnike sa DM i sa smanjenom funkcijom bubrega. Losartan je siguran i metabolički neutralan lek