The reported method allows for a simple and rapid monitoring of DNA replication and cell cycle progression in eukaryotic cells in vitro. The DNA of replicating cells is labeled by incorporation of a metabolically-active fluorescent (Cy3) deoxyuridine triphosphate derivative, which is delivered into the cells by a synthetic transporter (SNTT1). The cells are then fixed, stained with DAPI and analyzed by flow cytometry. Thus, this protocol obviates post-labeling steps, which are indispensable in currently used incorporation assays (BrdU, EdU). The applicability of the protocol is demonstrated in analyses of cell cycles of adherent (U-2 OS, HeLa S3, RAW 264.7, J774 A.1, Chem-1, U-87 MG) and suspension (CCRF-CEM, MOLT-4, THP-1, HL-60, JURKAT) cell cultures, including those affected by a DNA polymerase inhibitor (aphidicolin). Owing to a short incorporation time (5-60 min) and reduced number of steps, the protocol can be completed within 1-2 h with a minimal cell loss and with excellent reproducibility.

• MeSH
• barvení a značení metody   MeSH
• bromodeoxyuridin aplikace a dávkování   MeSH
• buněčný cyklus * MeSH
• DNA analýza   MeSH
• fluorescenční barviva aplikace a dávkování   MeSH
• HeLa buňky MeSH
• HL-60 buňky MeSH
• Jurkat buňky MeSH
• karbocyaniny aplikace a dávkování   MeSH
• lidé MeSH
• průtoková cytometrie metody   MeSH
• replikace DNA * MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• formuláře a záznamy - kontrola a vedení * MeSH
• léčivé přípravky zubní * zásobování a distribuce   MeSH
• léčivé přípravky * zásobování a distribuce   MeSH
• řízení stomatologické praxe * MeSH
• skladování léků MeSH
• záznamy jako téma MeSH

• MeSH
• analýza nákladů a výnosů MeSH
• efektivita organizační MeSH
• lidé MeSH
• organizace a řízení MeSH
• stomatologie * ekonomika   organizace a řízení   zákonodárství a právo   MeSH
• Check Tag
• lidé MeSH

• MeSH
• inzerce jako téma MeSH
• nebezpečný odpad * klasifikace   zákonodárství a právo   MeSH
• zdravotnický odpad MeSH
• zubní zařízení * MeSH

Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches. Our data unequivocally show that even a slight disruption of the Ca2+ -binding site destabilizes the three-dimensional fold of GCPII and is associated with impaired secretion, a high propensity to form nonphysiological oligomers, and an inability to bind active site-targeted ligands. Additionally, the Ca2+ -binding site is critical for maintenance of the native homodimeric quaternary arrangement of GCPII, which is indispensable for its enzymatic activity. Overall, our results offer a clear picture of the importance of Ca2+ for the structural integrity and hydrolytic activity of human GCPII and by extension homologous members of the M28 zinc-dependent metallopeptidase family.

• MeSH
• dimerizace MeSH
• glutamátkarboxypeptidasa II chemie   genetika   metabolismus   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• stabilita proteinů MeSH
• teplota * MeSH
• vápník chemie   metabolismus   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

UNLABELLED: Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body. DATABASE: The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.

• MeSH
• antigeny povrchové chemie   metabolismus   MeSH
• glutamátkarboxypeptidasa II chemie   metabolismus   MeSH
• glutamáty chemie   metabolismus   MeSH
• karboxypeptidasy chemie   metabolismus   MeSH
• katalytická doména MeSH
• lidé MeSH
• molekulární struktura MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

Předmět a metodika řešení projektu jsou následující: 1) Vytvoření souborů pacientů s hypodoncií detekovanou na základě RTG resp. CT vyšetření; 2) screening vzorků na mutace v genech PAX9, MSX1, AXIN2 a dalších relevantních genů metodou kapilárního sekvenování – analýza prevalence mutací; 3) analýza tkání po autotransplantacích (molekulární a buněčná analýza komplexu zub-kost-periodont).; The project is focused on: 1) Creation of a hypodontic patients collection diagnosed by RTG, CT and clinical examination; 2) Screening of samples for known mutations in PAX9, MSX1 and AXIN2 by capillary sequencing – prevalence analysis; 3) Analysis of cells and tissues after tooth autotransplantations (molecular and cellular analysis of the tissue complex tooth - bone - periodontal apparatus).

• MeSH
• anodoncie MeSH
• autologní transplantace MeSH
• imunohistochemie MeSH
• mutace MeSH
• polymorfismus genetický MeSH
• sekvenční analýza DNA MeSH

• Konspekt
• Stomatologie
• NLK Obory
• zubní lékařství
• molekulární biologie, molekulární medicína
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Transplantace zubů je ve stomatologii dlouho známou technikou, která byla zdokonalena v posledních desetiletích. Některé parametry úspěšného přenosu zubu můžeme hodnotit přímo u lidí. V soudobém výzkumu však nadále hrají důležitou roli zvířecí modely, které umožňují pozorovat změny ve tkáních na molekulární i buněčné úrovni. Zvířecí modely navíc přispívají k optimalizaci postupů u alotransplantací a kryoprezervacízubů. Tento článek je zaměřen na hodnocení transplantovaných zubů mikroskopickými metodami, zejména buněčných pochodů a molekulárních markerů souvisejících s regenerací měkkých tkání a tvorbou tvrdých tkání u myšís cílem shrnout a doplnit nejaktuálnější poznatky a možnosti výzkumu.

Transplantations of teeth are long known techniques used in human dentistry, which have been mastered within the last few decades. Several parameters of successful transplantation can be evaluated directly in the human; however, there is still an important role for research in animal models, enabling changes at molecular and cellular levels to be uncovered. Additionally, animals help with the optimization of allotransplantations and cryopreservat/on procedures. This paper is focused on microscopical evaluations of transplanted teeth, particularly cellular processes and molecular markers related to soft tissue regeneration and hard tissue formation in the mouse, with the aim of summarizing and supplementating the latest knowledge and recent research.

• MeSH
• apoptóza MeSH
• autologní transplantace * MeSH
• fyziologie buňky MeSH
• histologické techniky * MeSH
• homologní transplantace MeSH
• kryoprezervace MeSH
• krysa rodu rattus MeSH
• modely nemocí na zvířatech * MeSH
• morfologické a mikroskopické nálezy MeSH
• myši MeSH
• regenerace MeSH
• zuby * transplantace   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

In addition to its well-characterized role in the central nervous system, human glutamate carboxypeptidase II (GCPII; Uniprot ID Q04609) acts as a folate hydrolase in the small intestine, participating in the absorption of dietary polyglutamylated folates (folyl-n-γ-l-glutamic acid), which are the provitamin form of folic acid (also known as vitamin B9 ). Despite the role of GCPII as a folate hydrolase, nothing is known about the processing of polyglutamylated folates by GCPII at the structural or enzymological level. Moreover, many epidemiologic studies on the relationship of the naturally occurring His475Tyr polymorphism to folic acid status suggest that this polymorphism may be associated with several pathologies linked to impaired folate metabolism. In the present study, we report: (a) a series X-ray structures of complexes between a catalytically inactive GCPII mutant (Glu424Ala) and a panel of naturally occurring polyglutamylated folates; (b) the X-ray structure of the His475Tyr variant at a resolution of 1.83 Å; (c) the study of the recently identified arene-binding site of GCPII through mutagenesis (Arg463Leu, Arg511Leu and Trp541Ala), inhibitor binding and enzyme kinetics with polyglutamylated folates as substrates; and (d) a comparison of the thermal stabilities and folate-hydrolyzing activities of GCPII wild-type and His475Tyr variants. As a result, the crystallographic data reveal considerable details about the binding mode of polyglutamylated folates to GCPII, especially the engagement of the arene binding site in recognizing the folic acid moiety. Additionally, the combined structural and kinetic data suggest that GCPII wild-type and His475Tyr variant are functionally identical.

• MeSH
• antigeny povrchové chemie   genetika   MeSH
• glutamátkarboxypeptidasa II chemie   genetika   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kyselina polyglutamová chemie   metabolismus   MeSH
• lidé MeSH
• molekulární modely MeSH
• polymorfismus genetický MeSH
• stabilita enzymů MeSH
• vazebná místa genetika   MeSH
• vysoká teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring nonpolar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of nonpolar interactions governing GCPII affinity toward novel substrates as well as formerly unnoticed plasticity of the S1' specificity pocket. On the basis of those data, we designed, synthesized, and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.

• MeSH
• antigeny povrchové genetika   MeSH
• dipeptidy chemická syntéza   chemie   farmakologie   MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   genetika   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely MeSH
• mutageneze cílená MeSH
• substrátová specifita MeSH
• termodynamika MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH