Acute lymphoblastic leukemia (ALL) cells depend on the microenvironment of the host in vivo and do not survive in in vitro culture. Conversely, the suppression of non-malignant tissues is one of the leading characteristics of the course of ALL. Both the non-malignant suppression and malignant cell survival may be partly affected by soluble factors within the bone marrow (BM) environment. Here, we aimed to identify proteins in BM plasma of children with ALL that may contribute to ALL aggressiveness and/or the microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the time of ALL diagnosis was compared to control plasma of bone marrow (CBMp) or peripheral blood (CPBp) using a cytokine antibody array. The cytokine antibody array enabled simultaneous detection of 79 proteins per sample. Candidate proteins exhibiting significantly different profiles were further analyzed and confirmed by ELISA. mRNA expression of one of the candidate proteins (TIMP1) was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The cytokine antibody array experiments identified 23 proteins that differed significantly (p<0.05); of these, two proteins (TIMP1 and LIF) withstood the Bonferroni correction. In contrast, little difference was observed between CBMp and CPBp. At the diagnosis of ALL, changes in the soluble microenvironment are detectable in BM plasma. These changes probably participate in the pathogenesis and/or result from the changes in the cell composition.
- MeSH
- akutní lymfatická leukemie krev patologie MeSH
- čipová analýza proteinů MeSH
- cytokiny krev MeSH
- dítě MeSH
- kostní dřeň metabolismus MeSH
- leukemický inhibiční faktor biosyntéza MeSH
- lidé MeSH
- messenger RNA biosyntéza MeSH
- nádorové biomarkery krev MeSH
- tkáňový inhibitor metaloproteinasy 1 biosyntéza krev MeSH
- viabilita buněk MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied the early stages of pellicle formation by Mycobacterium smegmatis on the surface of a liquid medium [air-liquid interface (A-L)]. Using optical and scanning electron microscopy, we showed the formation of a compact biofilm pellicle from micro-colonies over a period of 8-30 h. The cells in the pellicle changed size and cell division pattern during this period. Based on our findings, we created a model of M. smegmatis A-L early pellicle formation showing the coordinate growth of cells in the micro-colonies and in the homogeneous film between them, where the accessibility to oxygen and nutrients is different. A proteomic approach utilizing high-resolution two-dimensional gel electrophoresis, in combination with mass spectrometry-based protein identification, was used to analyse the protein expression profiles of the different morphological stages of the pellicle. The proteins identified formed four expression groups; the most interesting of these groups contained the proteins with highest expression in the biofilm development phase, when the floating micro-colonies containing long and more robust cells associate into flocs and start to form a compact pellicle. The majority of these proteins, including GroEL1, are involved in cell wall synthesis or modification, mostly through the involvement of mycolic acid biosynthesis, and their expression maxima correlated with the changes in cell size and the rigidity of the bacterial cell wall observed by scanning electron microscopy.
- MeSH
- 2D gelová elektroforéza MeSH
- bakteriální proteiny genetika metabolismus MeSH
- biofilmy růst a vývoj MeSH
- biologické modely MeSH
- buněčná stěna metabolismus MeSH
- kultivační média MeSH
- mikroskopie elektronová rastrovací MeSH
- Mycobacterium smegmatis genetika růst a vývoj fyziologie ultrastruktura MeSH
- proteomika * MeSH
- regulace genové exprese u bakterií * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vzduch MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We studied the impact of a sublethal concentration of erythromycin on the fitness and proteome of a continuously cultivated population of Escherichia coli. The development of resistance to erythromycin in the population was followed over time by the gradient plate method and minimum inhibitory concentration (MIC) measurements. We measured the growth rate, standardized efficiency of synthesis of radiolabeled proteins, and translation accuracy of the system. The proteome changes were followed over time in two parallel experiments that differed in the presence or absence of erythromycin. A comparison of the proteomes at each time point (43, 68, and 103 h) revealed a group of unique proteins differing in expression. From all 35 proteins differing throughout the cultivation, only three were common to more than one time point. In the final population, a significant proportion of upregulated proteins was localized to the outer or inner cytoplasmic membranes or to the periplasmic space. In a population growing for more than 100 generations in the presence of antibiotic, erythromycin-resistant bacterial clones with improved fitness in comparison to early resistant culture predominated. This phenomenon was accompanied by distinct changes in protein expression during a stepwise, population-based development of erythromycin resistance.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- časové faktory MeSH
- erythromycin farmakologie MeSH
- Escherichia coli účinky léků genetika metabolismus MeSH
- genetická zdatnost účinky léků MeSH
- kultivační média MeSH
- mikrobiální testy citlivosti MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- proteom genetika metabolismus MeSH
- regulace genové exprese u bakterií * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Streptomycetes, soil-dwelling mycelial bacteria, can colonise surface of organic soil debris and soil particles. We analysed the effects of two different inert surfaces, glass and zirconia/silica, on the growth and antibiotic production in Streptomyces granaticolor. The surfaces used were in the form of microbeads and were surrounded by liquid growth media. Following the production of the antibiotic granaticin, more biomass was formed as well as a greater amount of antibiotic per milligram of protein on the glass beads than on the zirconia/silica beads. Comparison of young mycelium (6 h) proteomes, obtained from the cultures attached to the glass and zirconia/silica beads, revealed three proteins with altered expression levels (dihydrolipoamide dehydrogenase, amidophosphoribosyltransferase and cystathionine beta-synthase) and one unique protein (glyceraldehyde-3-phosphate dehydrogenase) that was present only in cells grown on glass beads. All of the identified proteins function primarily as cytoplasmic enzymes involved in different parts of metabolism; however, in several microorganisms, they are exposed on the cell surface and have been shown to be involved in adhesion or biofilm formation.
- MeSH
- antibakteriální látky metabolismus MeSH
- bakteriální adheze * MeSH
- bakteriální proteiny biosyntéza MeSH
- biomasa MeSH
- exprese genu MeSH
- hydrofobní a hydrofilní interakce * MeSH
- imobilizované buňky chemie metabolismus fyziologie MeSH
- naftochinony metabolismus MeSH
- povrchové vlastnosti * MeSH
- Streptomyces chemie růst a vývoj fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
135 l. : il., tab. ; 30 cm
ALL is the most frequent malignant disease of childhood. Surviving of leukemic cells is dependent on the bone marrow microenvironment, therefore we will focus on the analysis of soluble proteins pattern in the bone marrow plasma of children with ALL. Soluble proteins pattern will be analyzed by proteomic methods. Proteins differently expressed in ALL and control bone marrow plasma will be verified by flow cytometry and enzyme-linked immunoanalysis investigation. Identified proteins can be used as diagnostic markers of leukemias with worse prognosis or as the potential therapeutic targets, if these proteins significantly support the surviving of leukemic cells.
ALL je nejčastějším maligním onemocněním dětí. Přežívání leukemických buněk je závislé na mikroprostředí kostní dřeně, proto se v našem projektu zaměříme na analýzu solibilních faktorů přítomných v plazmě kostní dřeně dětí s ALL. Složení kostní plazmy dětí s ALL a kontrolní skupiny budeme analyzovat pomocí proteomických technik jako je "protein array" a dvoudimenzionální elektroforéza. Rozdíly v zastoupení proteinů ověříme pomocí průtokové cytometrie a enzymové imunoanalýzy. Zjištěné proteiny z kostní plazmy mohou sloužit jako diagnostické znaky leukémií s horší prognózou, nebo jako potenciální terapeutické cíle, bude-li se jednat o molekuly významně podporující přežívání leukemických buněk.
- MeSH
- akutní lymfatická leukemie diagnóza MeSH
- akutní nemoc MeSH
- dítě MeSH
- krevní plazma MeSH
- prognóza MeSH
- proteomika MeSH
- výsledek terapie MeSH
- Check Tag
- dítě MeSH
- Konspekt
- Pediatrie
- NLK Obory
- pediatrie
- hematologie a transfuzní lékařství
- onkologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
We present the results of analysis of membrane phosphoproteomes from individual morphological stages of Streptomyces coelicolor that reflect developmentally dependent heterogeneity and phosphorylation of intrinsic and externally added purified Strepomyces aureofaciens EF-Tu. Fast growing nonpathogenic Mycobacterium smegmatis was used as a non-differentiating actinomycetes comparative model. Streptomycetes membrane fraction was found to contain protein kinase(s) catalyzing phosphorylation of both its own and an externally added EF-Tu, whereas Mycobacterium membrane fraction contains protein kinase phosphorylating only its own EF-Tu.
- MeSH
- buněčná membrána enzymologie chemie metabolismus MeSH
- elongační faktor Tu izolace a purifikace metabolismus MeSH
- financování organizované MeSH
- fosforylace MeSH
- Mycobacterium smegmatis enzymologie chemie metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteinkinasy izolace a purifikace metabolismus MeSH
- Streptomyces enzymologie chemie metabolismus MeSH
- Publikační typ
- srovnávací studie MeSH
Monitoring the external environment and responding to its changes are essential for the survival of all living organisms. The transmission of extracellular signals in prokaryotes is mediated mainly by two-component systems. In addition, genomic analyses have revealed that many bacteria contain eukaryotic-type Ser/Thr protein kinases. The human pathogen Streptococcus pneumoniae encodes 13 two-component systems and has a single copy of a eukaryotic-like Ser/Thr protein kinase gene designated stkP. Previous studies demonstrated the pleiotropic role of the transmembrane protein kinase StkP in pneumococcal physiology. StkP regulates virulence, competence, and stress resistance and plays a role in the regulation of gene expression. To determine the intracellular signaling pathways controlled by StkP, we used a proteomic approach for identification of its substrates. We detected six proteins phosphorylated on threonine by StkP continuously during growth. We identified three new substrates of StkP: the Mn-dependent inorganic pyrophosphatase PpaC, the hypothetical protein spr0334, and the cell division protein DivIVA. Contrary to the results of a previous study, we did not confirm that the alpha-subunit of RNA polymerase is a target of StkP. We showed that StkP activation and substrate recognition depend on the presence of a peptidoglycan-binding domain comprising four extracellular penicillin-binding protein- and Ser/Thr kinase-associated domain (PASTA domain) repeats. We found that StkP is regulated in a growth-dependent manner and likely senses intracellular peptidoglycan subunits present in the cell division septa. In addition, stkP inactivation results in cell division defects. Thus, the data presented here suggest that StkP plays an important role in the regulation of cell division in pneumococcus.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčné dělení fyziologie MeSH
- klonování DNA MeSH
- protein-serin-threoninkinasy genetika metabolismus MeSH
- regulace genové exprese enzymů fyziologie MeSH
- regulace genové exprese u bakterií fyziologie MeSH
- Streptococcus pneumoniae enzymologie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Některé klinické symptomy u akutní leukemie (AL) mohou vznikat v důsledku působení solubilních faktorů secernovaných leukemickými buňkami do mikroprostředí kostní dřeně. Na druhé straně jsou leukemické buňky často závislé na mikroprostředí hostitele, takže většina leukemických buněk podlehne apoptóze během prvních dnů v in vitro podmínkách. I tento podpůrný vliv na leukemické buňky může být zprostředkován solubilními faktory. Většina pozornosti se logicky soustřeďuje na maligní buňky, a tak složení solubilních faktorů může být neprávem opomíjeno. Vyšetřovali jsme proteiny v kostní plazmě dětí s akutní lymfoblastickou leukemií (ALL), které mohou být zodpovědné za agresivitu ALL blastů nebo za mikroprostředím zprostředkované přežívání ALL buněk. Plazma z kostní dřeně a periferní krve byla analyzovaná pomocí proteinové array a dvojrozměrné elektroforézy (2-DE). Metodou proteinové array jsme detekovali 23 proteinů, jejichž koncentrace byla signifikantně odlišná u pacientů a kontrolních vzorků. Z kostní plazmy jsme před provedením 2-DE depletovali 12 abundatních proteinů pomocí afinitní chromatografie. Tím se nám podařilo zvýšit počet spotů, které jsme analyzovali pomocí softwaru PDQuest.
In acute leukemia (AL), several clinical symptoms may be caused by soluble factors secreted by AL cells into the bone marrow (BM) microenvironment. On the other hand, AL cells are often dependent on the microenvironment of the host, with most AL cells dying during first days after transferred to in vitro. This support of leukemic cells may be mediated by soluble factors as well. Attention is logically focused on malignant cells and the composition of soluble factors may be unjustly omitted. We aimed at identifying proteins in BM plasma of children with lymphoblastic AL (ALL), which may be responsible for ALL aggressiveness or for microenvironment-mediated survival of ALL cells. BM plasma and blood samples were analyzed by protein microarray and/or by two-dimensional electrophoresis (2D PAGE). We detected 23 proteins with a significantly different concentration in patients by Protein microarray. Before 2D PAGE, BM plasma was immuno-depleted from 12 abundant proteins by affinity chromatography. With this approach we succeeded to increase the number of spots that were analyzed by the PDQuest software.
- Klíčová slova
- akutní lymfoblastická leukemie, proteinová array, dvojrozměrná elektroforéza 2-DE, protein,
- MeSH
- 2D gelová elektroforéza metody využití MeSH
- akutní lymfatická leukemie genetika metabolismus patologie MeSH
- akutní nemoc MeSH
- dítě MeSH
- financování organizované MeSH
- leukemie genetika patologie MeSH
- lidé MeSH
- proteiny analýza MeSH
- vyšetřování kostní dřeně přístrojové vybavení MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
Chytridiopsis typographi Weiser, 1954, the microsporidian pathogen of the spruce bark beetle, Ips typographus L. (Coleoptera: Scolytidae), has an early developmental period with plurinucleate mother cells, each of which produces a single bud. The globular bud is connected with the mother cell by a collar and the cellular constituents are pushed to the distant end of the bud. Both the mother cell and the bud continue to develop; the bud then separates from the mother cell and grows to produce a cell of the same type. Both cells then continue sporogonial development and produce sporophorous vesicles with 16-32 spores. The process of a single mother cell producing a single bud that grows to an identical stage is new in the development of C. typographi and has no analogy in other Microsporidia.
