Leishmaniasis is an infectious disease caused by protozoan parasites of the genusLeishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin.L. majorpromastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50= 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubatedL. majorpromastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin toLeishmaniapromastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect onL. majorpromastigotes. Also, we showed thatLeishmaniaconstitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation ofLeishmanianitric oxide synthase as a new therapeutic approach.
Acquisition of genes by plastid genomes (plastomes) via horizontal gene transfer (HGT) seems to be a rare phenomenon. Here, we report an interesting case of HGT revealed by sequencing the plastomes of the eustigmatophyte algae Monodopsis sp. MarTras21 and Vischeria sp. CAUP Q 202. These plastomes proved to harbour a unique cluster of six genes, most probably acquired from a bacterium of the phylum Bacteroidetes, with homologues in various bacteria, typically organized in a conserved uncharacterized putative operon. Sequence analyses of the six proteins encoded by the operon yielded the following annotation for them: (i) a novel family without discernible homologues; (ii) a new family within the superfamily of metallo-dependent hydrolases; (iii) a novel subgroup of the UbiA superfamily of prenyl transferases; (iv) a new clade within the sugar phosphate cyclase superfamily; (v) a new family within the xylose isomerase-like superfamily; and (vi) a hydrolase for a phosphate moiety-containing substrate. We suggest that the operon encodes enzymes of a pathway synthesizing an isoprenoid-cyclitol-derived compound, possibly an antimicrobial or other protective substance. To the best of our knowledge, this is the first report of an expansion of the metabolic capacity of a plastid mediated by HGT into the plastid genome.
- MeSH
- anotace sekvence MeSH
- bakteriální geny MeSH
- DNA řas genetika MeSH
- genom plastidový * MeSH
- Heterokontophyta genetika MeSH
- molekulární evoluce MeSH
- multigenová rodina MeSH
- přenos genů horizontální * MeSH
- sekvenční analýza DNA metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine.
- MeSH
- biosenzitivní techniky * MeSH
- konfokální mikroskopie MeSH
- kontrolní body buněčného cyklu účinky léků účinky záření MeSH
- lasery škodlivé účinky MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- oprava DNA MeSH
- poškození DNA * účinky léků účinky záření MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In my article I tried to present the results of early experiments suggesting a significant role for cell association in Rous sarcoma virus transformation of non-permissive cells and revealing that infectious virus can be efficiently rescued from such cells by their fusion with permissive chicken fibroblasts.
- MeSH
- krysa rodu rattus MeSH
- kur domácí virologie MeSH
- proviry patogenita fyziologie MeSH
- ptačí sarkom virologie MeSH
- replikace viru MeSH
- virová transformace buněk MeSH
- virus Rousova sarkomu patogenita fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
OBJECTIVES: Cytochromes P450 (CYP) are monooxygenases, which metabolize mostly hydrophobic endogenous and exogenous compounds. CYPs without any clear connection to metabolism are called "orphans". Interestingly, these "orphan" CYPs are over-expressed in tumor tissues. Thus, the main aim of the paper is the development of antibodies for immunodetection of these CYPs as potential malignancy markers. METHODS: Unique sequences of CYP2S1 and 2W1 were selected and peptides synthesized. Chickens were immunized with peptides bound to hemocyanin (KLH). The antibodies were isolated from egg yolks and their reactivity was tested by ELISA. Antibodies were further affinity purified on immobilized peptides. Western blots containing CYP2S1 and 2W1 standards were developed with purified antibodies. RESULTS: Using unique peptide immunogens of CYP2S1 and 2W1 the antibodies were developed. As judged from ELISA all chickens produced specific antibodies against the respective peptides. Both affinity purified antibodies against CYP2S1 peptide recognized the CYP2S1 standard on Western blots, but only one of four anti-peptide antibodies against CYP2W1 reacted with CYP2W1 standard. The antibodies were used for the detection of CYPs in cancer cell lines and human tissues samples. Although both CYPs were frequently co-expressed in cancer cells, CYP2S1 was solely induced in the cell line BxPC3, while CYP2W1 was predominantly present in cell lines MCF7 and HeLa. Our data show that anti-peptide antibodies are an indispensable tool for detection of homologous CYPs. CONCLUSIONS: The anti-peptide antibodies successfully recognized CYP2S1 and 2W1 in the cancer cell lines and tissue samples.
- MeSH
- 2D gelová elektroforéza MeSH
- ELISA MeSH
- imunoglobuliny imunologie MeSH
- imunologické techniky metody MeSH
- kur domácí MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory enzymologie MeSH
- peptidy MeSH
- protilátky imunologie MeSH
- systém (enzymů) cytochromů P-450 imunologie metabolismus MeSH
- tvorba protilátek * MeSH
- vejce MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.
- MeSH
- buňky 3T3 MeSH
- dvouvláknová RNA metabolismus MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- imunoprecipitace MeSH
- lidé MeSH
- malá interferující RNA genetika MeSH
- myši MeSH
- plazmidy genetika MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- průtoková cytometrie MeSH
- regulace genové exprese genetika MeSH
- reportérové geny genetika MeSH
- transfekce metody MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
Sitagliptin is a dipeptidyl peptidase IV (DPP-IV) inhibitor that exerts an anti-hyperglycaemic effect by preventing degradation of glucagon-like peptide 1 with subsequent β-cell stimulation and potential regeneration. We tested whether sitagliptin therapy in symptomatic non-obese diabetic (NOD) mice would lead to changes in the immune cell profile, improve β-cell survival and induce diabetes remission. Flow cytometry analysis of immune cells in the spleen and peripheral lymph nodes, immunohistology of the pancreas and DPP-IV activity were investigated in diabetic NOD mice, either treated or non-treated with sitagliptin, at 0, 7, 14 and 28 days after hyperglycaemia onset, and in non-diabetic NOD controls. While compared to diabetic controls sitagliptin prevented increase of the CD8+/CD4+ ratio in pancreatic nodes after four weeks (0.443 ± 0.067 vs. 0.544 ± 0.131; P < 0.05), the population of Tregs in lymph nodes increased from day 0 to 28 in both treated and non-treated diabetic groups (8 ± 1.76 vs. 13.45 ± 5.07 % and 8 ± 1.76 vs. 13.19 ± 5.58 %, respectively). The severity of islet infiltration was similar in both diabetic groups and decreased in parallel with β-cell loss. Surprisingly, sitagliptin blocked the DPP-IV activity only temporarily (on day 7, 277.68 ± 89.2 vs. 547.40 ± 94.04 ng/ml in the diabetic control group) with no apparent effect later on. In conclusion, sitagliptin administered after the onset of overt hyperglycaemia in NOD mice had only a marginal immunological effect and did not lead to diabetes remission. Failure to block DPP-IV over time represents an important finding that requires further explanation.
- MeSH
- časové faktory MeSH
- diabetes mellitus 1. typu farmakoterapie imunologie patologie MeSH
- dipeptidylpeptidasa 4 účinky léků MeSH
- inhibitory dipeptidylpeptidasy 4 krev farmakologie terapeutické užití MeSH
- Langerhansovy ostrůvky imunologie patologie MeSH
- lymfatické uzliny účinky léků imunologie patologie MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední NOD MeSH
- myši MeSH
- pyraziny farmakologie terapeutické užití MeSH
- slezina imunologie patologie MeSH
- T-lymfocyty - podskupiny účinky léků imunologie patologie MeSH
- triazoly farmakologie terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
