Biohemijske promene u krvnom serumu pasa tretiranih fenobarbitonom

Despite being described as the safest antiepileptic drug of first choice the presented literature data are much varied as far as dog blood serum biochemical parameters are considered. The aim of this study was to investigate the effect of phenobarbitone at different per os doses on the values of selected blood serum biochemical parameters in dogs during both short and long term application. The study was conducted on 30 dogs of different races, both sexes, ranging from 2 to 8 years of age. A total of 15 healthy and 15 dogs suffering from idiophatic epilepsy were observed. During the short term per os application of phenobarbitone (given at 3 week intervals) to the healthy population in varied doses a statistically significant increase in ALT and AP was recorded. Application of 16 mg/kg/day of phenobarbitone to the healthy population during 14 days resulted in a significant increase of ALT and AP. This increase was dependant on the duration of the treatment. During chronic application of phenobarbitone to dogs suffering from idiopathic epilepsy a significant increase in values of AP and ALT depending on the given dose was recorded. In two of the studied epileptic dogs treated with high therapeutic doses of phenobarbitone clinical signs of hepatotoxicity were recorded. Hepatotoxicity resulted in decreased albumin and total protein concentrations, as well as increased blood serum total bilirubin, AST, ALP and AP. The obtained results indicate that a long term application of phenobarbitone at high therapeutic doses can cause hepatotoxicity. However, there was no relationship between phenobarbitone dosage and duration of therapy and blood glucose, urea, creatinine, total proteins, albumins, total bilirubin, triglycerides and cholesterol.Iako označen kao antiepileptik prvog izbora i veoma bezbedan lek, u literaturi su prezentovani različiti podaci o uticaju fenobarbitona na vrednosti biohemijskih parametara krvnog seruma kod pasa. S obzirom na to, cilj ovog istraživanja bio je da se ispita uticaj fenobarbitona pri različitim per os dozama na vrednosti biohemijskih parametara krvnog seruma kod pasa, tokom kratkotrajne i dugotrajne aplikacije. Istraživanje je sprovedeno na 30 pasa različitih rasa, oba pola, starosne kategorije od dve do osam godina, od kojih su 15 bili zdravi psi a 15 psi oboleli od idiopatske epilepsije. Tokom kratkotrajne per os aplikacije fenobarbitona (intervali od po tri nedelje) zdravoj populaciji pasa pri različitim per os dozama, u krvnom serumu je registrovano značajno povećanje aktivnosti ALT i vrlo značajno povećanje aktivnosti AP u zavisnosti od doze leka. Aplikacija fenobarbitona u dozi od 16 mg/kg/dnevno tokom 14 nedelja zdravoj populaciji pasa ukazala je, na vrlo značajno povećanje aktivnosti ALT i AP u zavisnosti od dužine trajanja aplikacije. Tokom hronične aplikacije fenobarbitona psima obolelim od idiopatske epilepsije, ustanovljeno je statistički vrlo značajno povećanje aktivnosti AP i ALT u krvnom serumu u zavisnosti od doze leka. Kod dve jedinke u bolesnoj grupi pasa pri visokim terapeutskim dozama fenobarbitona registrovana je hepatotoksičnost, što je dovelo do statistički značajnog smanjenja koncentracije albumina i ukupnih proteina, statistički značajnog povećanja koncentracije ukupnog bilirubina, statistički značajnog povećanja aktivnosti AST i statistički vrlo značajnog povećanja aktivnosti ALT i AP u krvnom serumu Dobijeni rezultati ukazuju da dugotrajna aplikacija fenobarbitona, pri visokim terapeutskim dozama može izazvati hepatotoksičnost. Nije ustanovljeno postojanje veze između koncentracije glukoze, uree, kreatinina, ukupnih proteina, albumina, ukupnog bilirubina, triglicerida i holesterola u krvnom serumu pasa sa per os dozom fenobarbitona, niti sa dužinom trajanja terapije

Proliferation and differentiation potential of canine synovial fluid cells

The aim of this study was to determine whether synovial fluid (SF) of dogs contains cells that have characteristics of MSCs and to describe their differentiation potential. SF adherent cells from 5 young German shepherd dogs (average 3.8 +/- 0.9 years) were expanded (37 degrees C, 5% CO2, humidified atmosphere) three weeks before their phenotype was characterized by flow-cytometry for the presence of CD90 and CD34. Population doubling time (PDT), number of CFU-F and adipogenic, osteogenic and chondrogenic potentials have been determined in vitro. In early passages PTD was 31 +/- 10 hours and expansion fold after 3 sub cultivations (9 days) theoretically could be 372 +/- 134. At P1, 0.55 +/- 0.05% of SF cells had the ability to form CFU-F. Sixty-six percent of cells expressed CD90 and none of the cells expressed markers of hematopoietic cells. Oil Red O staining has shown accumulation of fat droplets in cells grown in adipogenic medium, while deposits of calcium in the osteogenic medium were evidenced with Alizarin red staining. SF cultured in hondrogenic and control medium in three-dimensional conditions formed a cartilage-like tissue. Alcian blue staining of pellets' slides have shown a significant amount of glycosaminoglycans (GAGs) and immunohistochemistry analysis documented collagen type II expression. The amount of GAGs in pellets grown in both conditions showed no difference. SF cells in vitro exhibited osteogenic, adipogenic and chondrogenic differentiation potentials, suggesting the presence of different mesenchymal progenitors. These results also demonstrated that SF cells have a spontaneous chondrogenic potential that should be further explored for possible tissue engineering protocols

Karakterizacija matičnih ćelija izolovanih iz zubne pulpe mlečnih zuba dece

Background/Aim. The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption. Methods. The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics. Results. According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 ± 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation. Conclusion. Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and tri- lineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.Uvod/Cilj. Prošla dekada je bila posebno obeležena naporima na polju korišćenja ex vivo razvijenih i usmeravanih mezenhimskih matičnih ćelija (MSCs), kao sredstva za različite tipove regenerativne terapije. Cilj ove studije bio je da se utvrdi imunofenotip i potencijal za proliferaciju i diferencijaciju ćelija izolovanih iz zubne pulpe mlečnih zuba dece eksfoliranih u periodu kada je koren zuba bio u poslednjoj fazi resorpcije. Metode. Primarna adherentna populacija ćelija poreklom od pet donora dobijena je metodom eksplanta. Prisustvo progenitorskih ćelija koje obrazuju kolonije fibroblasta (CFU-F) pokazano je u prvoj pasaži. Do treće pasaže ćelije su ekspandirane, a potom korišćene za analiziranje. Imunofenotip je određen korišćenjem protočne citometrije. Proliferativni potencijal i vreme udvajanja ćelija (PDT) u kulturi je definisano na osnovu apsolutnog broja ćelija na početku i na kraju svake pasaže. Posle tronedeljne kultivacije ćelija u komercijalnim medijumima za stimulaciju osteogeneze, hondrogeneze i adipogeneze, citološkim i histološkim metodama je određena morfologija ćelija i karakteristike vanćelijskog matriksa. Rezultati. Antigeni koji karakterišu mezenhimske matične ćelije CD29 i CD73 su bili eksprimirani na svim nediferenciranim ćelijama, dok su antigeni CD146 i CD106 bili eksprimirani na ograničenom broju ćelija. Antigen CD34 (karakterističan za ćelije hematopoetske loze) nije bio eksprimiran. Incidencija CFU-F bila je 4,7 ± 0,5/100 ćelija. PDT se nije menjao tokom osam pasaža i u proseku je iznosio 25 h. Posle tronedeljne stimulacije diferencijacije u kulturama sa adipogenim medijumom došlo je do stvaranja ćelija sa masnim kapljicama, a u kulturama sa osteogenim medijumom došlo je do formiranja vanćelijskog matriksa sa deponovanim kalcijumovim solima. U kulturama sa hondrogenim medijumom došlo je do stvaranja tkiva sličnog hrskavici i vanćelijskog matriksa sa glikozaminoglikanima i kolagenom II. Zaključak. Zubna pulpa mlečnih zuba dece sadrži ćelijsku populaciju koja odgovara mezenhimskim matičnim ćelijama prema svojim karakteristikama, ima visok proliferativni potencijal i potencijal da se diferencira u tri ćelijske linije što je čini pogodnom za dalje in vitro analize i evaluaciju ćelijske terapije

Crystal structure of dicholoro-(3,5-dimethyl-1H-pyrazole-1-carboxamidine-N,N )copper(II), Cu(C6H10N4)Cl-2

C6H10Cl2CuN4, monoclinic, P2(1)/n (no. 14), a = 7.316(2) angstrom b = 16.002(2) angstrom, c = 9.202(6) angstrom, beta = 113.15 (2)degrees, V = 990.6 angstrom(3), Z = 4, R-gt(F) = 0.054, wR(ref)(F-2) = 0.125, T = 293 K

The relBE2Spn Toxin-Antitoxin System of Streptococcus pneumoniae: Role in Antibiotic Tolerance and Functional Conservation in Clinical Isolates

Type II (proteic) chromosomal toxin-antitoxin systems (TAS) are widespread in Bacteria and Archaea but their precise function is known only for a limited number of them. Out of the many TAS described, the relBE family is one of the most abundant, being present in the three first sequenced strains of Streptococcus pneumoniae (D39, TIGR4 and R6). To address the function of the pneumococcal relBE2Spn TAS in the bacterial physiology, we have compared the response of the R6-relBE2Spn wild type strain with that of an isogenic derivative, ΔrelB2Spn under different stress conditions such as carbon and amino acid starvation and antibiotic exposure. Differences on viability between the wild type and mutant strains were found only when treatment directly impaired protein synthesis. As a criterion for the permanence of this locus in a variety of clinical strains, we checked whether the relBE2Spn locus was conserved in around 100 pneumococcal strains, including clinical isolates and strains with known genomes. All strains, although having various types of polymorphisms at the vicinity of the TA region, contained a functional relBE2Spn locus and the type of its structure correlated with the multilocus sequence type. Functionality of this TAS was maintained even in cases where severe rearrangements around the relBE2Spn region were found. We conclude that even though the relBE2Spn TAS is not essential for pneumococcus, it may provide additional advantages to the bacteria for colonization and/or infection

Charge Density Analysis of 2-Pyridineformamide N(4)-Methylthiosemicarbazone (Z =4): Role of an Enhanced N-H center dot center dot center dot S Thioureido Dimer

Charge density distribution in 2-pyridineformamide N(4)-methylthiosemicarbazone (TSC4) has been determined using high-resolution X-ray diffraction data (100 K) and Hansen-Coppens multipole formalism. The results are interpreted in terms of Baders quantum theory of atoms in molecules (QTAIM), electrostatic potential analysis, and theoretical calculations (CRYSTAL09). The study highlights the molecular diSsimilarity at the electronic level. The N-H center dot center dot center dot S interactions have a dominant role in Stabilization of the TSC4 crystal Structure. As each of the four S acceptors simultaneously engages in several interactions, the focus of study is on the lone pairs electron density of these acceptors which is particularly able to adjust to the various spatial distributions of the interacting donor groups. The main structural feature of TSC4 is the formation of specific N-H center dot center dot center dot S bonded dimers between two pairs of independent molecules. These dimers are the main building block in the orkstal structure, and with the two additional N-H center dot center dot center dot S contacts they represent a significant enhancement of a typical R-2(2)(8) dirtier synthon which dominates among the thioureido-based crystal structures. Two TSC4 dimers are structurally very similar and exhibit considerable cohesive energy (-20.8 and 21.4 kcal/mol). This: type of dimer is the only structural motif that is common for S acceptors of all four independent molecules

Crystal structure of the antitoxin toxin protein complex RelB RelE from Methanococcus jannaschii

Here we present the crystal structure of the Methanococcus jannaschii RelE RelB RelBE toxin antitoxin TA protein complex determined by the MIRAS multiple isomorphous replacement with anomalous signal method. The genes encoding this TA system are located in the chromosome of this archaeon and involved in stress response. RelE acts as an endoribonuclease that cleaves mRNA on the ribosome, and we compare the RelBE complex to the known structures of other TA systems belonging to this group and to endoribonucleases. M. jannaschii RelBE forms a heterotetramer with the antitoxin in the centre of the complex, a configuration that differs vastly from the heterotetramer structure of the previously published RelBE from another archaeon, Pyrococcus horikoshii. The long N terminal amp; 945; helix of the tightly bound M. jannaschii antitoxin RelB covers the presumed active site of the toxin RelE that is formed by a central amp; 946; sheet, a loop on one side and a C terminal amp; 945; helix on the other side. The active site of the M. jannaschii toxin RelE harbours positive charges that are thought to neutralize the negative charges of the substrate mRNA, including Arg62 that was changed to Ser62 by the Escherichia coli expression system, thereby leading to inactive toxin RelE. Comparative studies suggest that Asp43 and His79 are also involved in the activity of the toxi

Electronic features and hydrogen bonding capacity of the sulfur acceptor in thioureido-based compounds. Part 2. Further insight by theoretical charge density study

Theoretical charge density analysis of the thioureido based compound 4-methyl-3-thiosemicarbazide (MeTSC) is used in this study with the aim to provide additional insight into electronic features of the thioureido sulfur acceptor and the corresponding D-H...S hydrogen bonding (D=N, C). The present work is motivated by our earlier experimental charge density study on the same compound that pointed out to a great flexibility of the thioureido S acceptor and its ability to adjust the lone pair electron density to the donor groups in simultaneous hydrogen bonding. Through the additional theoretical approach we were able to single out different fragments of MeTSC crystal and to carefully follow the changes in electron density properties of the S acceptors belonging to: isolated MeTSC monomers (i.e. two crystallographically independent molecules), eight D-H...S bonded dimers and MeTSC molecules placed in full crystal environment, where each S atom engages in four hydrogen bonds. Deformation density of the sulfurs lone electron pair, topological properties of D-H...S interactions and electrostatic potential are here examined in order to comprehend the mutual influence and potential cooperative effects of the four simultaneously formed interactions to each of the S acceptors. The hydrogen bonding involving thioureido S acceptor is also investigated in terms of the energetic properties of the eight real MeTSC dimers existing in the crystal structure, and additional systems MeTSC/MeOH and acetone/MeOH. The latter systems are designed with the purpose of direct comparison and ranking of interactions involving thioureido S to those involving more conventional, carbonyl O acceptor. Energetic features were thoroughly studied through electrostatic interactions energies (XD2006), cohesive energies (CRYSTAL09) and ab initio approach employing the coupled-cluster single S and doubles augmented by a perturbational correction for connected triple excitations (CCSD(T)) method. (C) 2015 Elsevier B.V. All rights reserved