The purpose of the present study was to compare the myosin heavy chain (MHC) isoform composition of the deltoid and vastus lateralis muscles of the dominant and non-dominant limbs in handball players. Eleven male Greek elite handball players (age 22.6 ± 1.9 yrs, training experience 10.6 ± 2.1 yrs, height 184.1 ± 4.1 cm, and weight 81.0 ± 12.5 kg) participated in the study. Four muscle biopsies were obtained from the dominant and non-dominant deltoid and vastus lateralis muscles during the in-season period. The MHC composition was determined using SDS-PAGE. No significant difference was found between the dominant and non-dominant muscles; Deltoid muscle: MHC I [(95%CI = -1.22, 0.33), P = 0.228], MHC ΙΙa [(95%CI = -0.32, 1.59), P = 0.168] and MHC IIx [(95%CI = -1.49, 1.10), P = 0.749]; Vastus lateralis muscle: MHC I [(95%CI = -0.38, 0.63), P = 0.586], MHC ΙΙa [(95%CI = -0.50, 0.65), P = 0.783] and MHC IIx [(95%CI = -1.08, 0.42), P = 0.355]. The findings of the present study indicate that the greater use of the dominant limbs for throwing actions and body movements in handball do not lead to altered MHC isoform composition compared to the non-dominant limbs.

• Klíčová slova
• Deltoid, handball, muscle biopsies, myosin, vastus lateralis,
• MeSH
• čtyřhlavý sval stehenní chemie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• lidé MeSH
• mladý dospělý MeSH
• musculus deltoideus chemie   MeSH
• protein - isoformy analýza   MeSH
• sporty fyziologie   MeSH
• těžké řetězce myosinu analýza   chemie   MeSH
• Check Tag
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protein - isoformy MeSH
• těžké řetězce myosinu MeSH

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post-translational turnover, we devised a strategy combining pulse stable isotope-labeled amino acids in cells (pSILAC), data-independent acquisition mass spectrometry (DIA-MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome-wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation.

• Klíčová slova
• DIA mass spectrometry, alternative splicing, protein turnover, proteomics, pulsed SILAC,
• MeSH
• alternativní sestřih MeSH
• HeLa buňky MeSH
• hmotnostní spektrometrie MeSH
• izoformy RNA genetika   metabolismus   MeSH
• izotopové značení metody   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• protein - isoformy analýza   metabolismus   MeSH
• proteiny analýza   metabolismus   MeSH
• proteolýza MeSH
• proteomika metody   MeSH
• průběh práce MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izoformy RNA MeSH
• messenger RNA MeSH
• protein - isoformy MeSH
• proteiny MeSH

Microtubules formed by αβ-tubulin dimers represent cellular structures that are indispensable for the maintenance of cell morphology and for cell motility generation. Microtubules in intact cells are in highly regulated equilibrium with cellular pools of soluble tubulin dimers. Sensitive, reproducible and rapid assays are necessary to monitor tubulin changes in cytosolic pools after treatment with anti-mitotic drugs, during the cell cycle or activation and differentiation events. Here we describe new assays for α-tubulin quantification. The assays are based on sandwich ELISA, and the signal is amplified with biotinyl-tyramide or immuno-PCR. Matching monoclonal antibody pair recognizes phylogenetically highly conserved epitopes localized outside the C-terminal isotype-defining region. This makes it possible to detect α-tubulin isotypes in different cell types of various species. Biotinyl-tyramide amplification and immuno-PCR amplification enable detection of tubulin at concentrations 2.5ng/ml and 0.086ng/ml, respectively. Immuno-PCR detection shows enhanced sensitivity and wider dynamic range when compared to ELISA with biotinyl-tyramide detection. Our results on taxol-treated and activated bone marrow-derived mast cells demonstrate, that the assays allow sensitive quantification of tubulin in complex biological fluids.

• Klíčová slova
• 3,3′,5,5′-tetramethylbenzidine., BMMC, BSA, BSS, Biotinyl-tyramide, C(q), ELISA, Immuno-PCR, Mast cells, PBS, PCR, TBS, TBS containing 0.05% Tween 20, TBST, TMB, Tris buffer solution, bone marrow-derived mast cell, bovine serum albumin, buffered saline solution, enzyme-linked immunosorbent assay, phosphate buffer solution, polymerase chain reaction, quantification cycle, α-Tubulin isotypes,
• MeSH
• biotin analogy a deriváty   MeSH
• buněčné linie MeSH
• ELISA metody   statistika a číselné údaje   MeSH
• mapování epitopu MeSH
• mastocyty účinky léků   metabolismus   MeSH
• monoklonální protilátky MeSH
• myši MeSH
• paclitaxel farmakologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• protein - isoformy analýza   genetika   imunologie   MeSH
• thapsigargin farmakologie   MeSH
• tubulin analýza   genetika   imunologie   MeSH
• tyramin analogy a deriváty   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biotin MeSH
• biotinyltyramide MeSH Prohlížeč
• monoklonální protilátky MeSH
• paclitaxel MeSH
• protein - isoformy MeSH
• thapsigargin MeSH
• tubulin MeSH
• tyramin MeSH

Magnesium-protoporphyrin IX monomethylester cyclase is one of the key enzymes of the bacteriochlorophyll biosynthesis pathway. There exist two fundamentally different forms of this enzyme. The oxygen-dependent form, encoded by the gene acsF, catalyzes the formation of the bacteriochlorophyll fifth ring using oxygen, whereas the oxygen-independent form encoded by the gene bchE utilizes an oxygen atom extracted from water. The presence of acsF and bchE genes was surveyed in various phototrophic Proteobacteria using the available genomic data and newly designed degenerated primers. It was found that while the majority of purple nonsulfur bacteria contained both forms of the cyclase, the purple sulfur bacteria contained only the oxygen-independent form. All tested species of aerobic anoxygenic phototrophs contained acsF genes, but some of them also retained the bchE gene. In contrast to bchE phylogeny, the acsF phylogeny was in good agreement with 16S inferred phylogeny. Moreover, the survey of the genome data documented that the acsF gene occupies a conserved position inside the photosynthesis gene cluster, whereas the bchE location in the genome varied largely between the species. This suggests that the oxygen-dependent cyclase was recruited by purple phototrophic bacteria very early during their evolution. The primary sequence and immunochemical similarity with its cyanobacterial counterparts suggests that acsF may have been acquired by Proteobacteria via horizontal gene transfer from cyanobacteria. The acquisition of the gene allowed purple nonsulfur phototrophic bacteria to proliferate in the mildly oxygenated conditions of the Proterozoic era.

• MeSH
• bakteriální proteiny analýza   chemie   genetika   MeSH
• fotosyntéza genetika   MeSH
• fylogeneze MeSH
• genom bakteriální MeSH
• kyslík metabolismus   MeSH
• oxygenasy analýza   chemie   genetika   MeSH
• protein - isoformy analýza   chemie   genetika   MeSH
• Proteobacteria enzymologie   genetika   metabolismus   MeSH
• sinice enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• kyslík MeSH
• magnesium protoporphyrin monomethyl ester oxidative cyclase MeSH Prohlížeč
• oxygenasy MeSH
• protein - isoformy MeSH

Glutamate carboxypeptidase II (GCPII) and its splice variants, paralogs and human homologs represent a family of proteins with diverse tissue distribution, cellular localization and largely unknown function which have been explored only recently. While GCPII itself has been thoroughly studied from different perspectives, as clearly documented in this series of reviews, very little is known about other members of its family, even though they might be biologically relevant. Differential expression of individual GCPII splice variants is associated with tumor progression and prognosis of prostate cancer. The best studied GCPII homolog, GCPIII or NAALADase II, may be a valid pharmaceutical target for itself since it may compensate for a lack of normal GCPII enzymatic activity. Detailed molecular characterization of this family of proteins is thus very important not only with respect to the potential therapeutic use of GCPII inhibitors, but also for better understanding of the biological role of GCPII within as well as outside the nervous system.

• MeSH
• glutamátkarboxypeptidasa II analýza   antagonisté a inhibitory   genetika   metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• protein - isoformy analýza   antagonisté a inhibitory   genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• glutamátkarboxypeptidasa II MeSH
• inhibitory enzymů MeSH
• protein - isoformy MeSH

We developed a new method for the quantitative determination of myosin heavy chain (MyHC) isoforms taking advantage of immunochemical differences and based on the ELISA principle. In the present paper we compare analysis of MyHC isoforms using the SDS-PAGE and the ELISA methods in the same samples of adult female inbred Lewis strain euthyroid, hyperthyroid and hypothyroid rats. In all thyroid states, the same composition and corresponding changes of MyHC isoforms were determined using both methodological approaches in the slow soleus and the fast extensor digitorum longus muscles. Our results showed that ELISA can be used for a "semi-quantitative" or "comparative" measurement of MyHC isoforms in multiple muscle samples, but that it is neither more exact nor faster compared to SDS-PAGE.

• MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• ELISA MeSH
• inbrední kmeny potkanů MeSH
• krysa rodu Rattus MeSH
• potkani inbrední LEW MeSH
• protein - isoformy analýza   MeSH
• svalová vlákna typu I chemie   fyziologie   MeSH
• svalová vlákna typu II chemie   fyziologie   MeSH
• těžké řetězce myosinu analýza   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• protein - isoformy MeSH
• těžké řetězce myosinu MeSH

BACKGROUND: Identification of the content of asialotransferrin in the cerebrospinal fluid is a diagnostic method for childhood-onset ataxia and central nervous system hypomyelination (CACH), also known as vanishing white matter disease (VWM), and also for other types of CNS disorders. METHODS: In our work, we have determined the value of the ratio of the asialo form of transferrin to the total transferrin in the CSF using the commercially used Variant(TM) Bio-Rad system for the determination of carbohydrate-deficient transferrin (CDT) in serum. The peak corresponding to the asialo form of transferrin was identified with electrophoresis with subsequent immunofixation and mass spectrometry (MALDI-TOF/TOF). RESULTS: The intra-assay and inter-assay variations of the asialotransferrin value in CSF were 6.8% and 10.2%, respectively. Analysing CSF samples of 60 subjects (23 men aged 22-68 years and 37 women aged 18-77 years) with normal transferrin values and normal cytology as well as biochemistry parameters in the cerebrospinal fluid, and without apparent signs of neurological disorders, we have found the presence of 25.2 +/- 8.2% asialotransferrin. CONCLUSION: Except for the need to obtain approximately 1.5 mL of cerebrospinal fluid and a tenfold concentrating of the sample, there is no need to conduct any modifications of the preparation procedure for the analytic sample and chromatographic separation normally used for serum samples. The HPLC method of asialotransferrin determination in CSF provides clinically useful results.

• MeSH
• asialoglykoproteiny analýza   mozkomíšní mok   MeSH
• dospělí MeSH
• imunoelektroforéza MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• protein - isoformy analýza   krev   mozkomíšní mok   MeSH
• senioři MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• tandemová hmotnostní spektrometrie MeSH
• transferin analogy a deriváty   analýza   mozkomíšní mok   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asialoglykoproteiny MeSH
• asialotransferrins MeSH Prohlížeč
• protein - isoformy MeSH
• transferin MeSH

Changes in the levels of gamma-aminobutyric acid (GABA) are known to occur in different parts of the brain during aging. In our study we attempted to define the effect that aging has on glutamate decarboxylase (GAD), the key enzyme in the synthesis of GABA, in the central parts of the auditory system. Age-related changes in GAD65 and GAD67 levels were investigated using immunohistochemistry and Western blotting in the inferior colliculus (IC), the auditory cortex (AC) and the visual cortex in Long-Evans rats. The results show that aging is associated with a decrease in the numbers of GAD65- and 67-immunoreactive neurons and the optical density of their somas in both the IC and AC. Western blot analysis revealed a pronounced age-related decline in the levels of GAD65 and 67 proteins in both the IC and AC. For comparison, in the visual cortex the decrease in both proteins was less pronounced than in the IC and AC. A similar pattern of age-related changes was found in Fischer 344 rats, a strain that manifests a rapid loss of hearing function with aging. The observed age-related decline in the levels of GAD65 and 67 may contribute significantly to the deterioration of hearing function that accompanies aging in mammals, including man.

• MeSH
• analýza rozptylu MeSH
• colliculus inferior metabolismus   MeSH
• druhová specificita MeSH
• GABA metabolismus   fyziologie   MeSH
• glutamát dekarboxyláza metabolismus   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• potkani inbrední F344 MeSH
• potkani Long-Evans MeSH
• presbyakuze metabolismus   MeSH
• protein - isoformy analýza   metabolismus   MeSH
• sluchové korové centrum metabolismus   MeSH
• stárnutí metabolismus   MeSH
• western blotting metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GABA MeSH
• glutamát dekarboxyláza MeSH
• protein - isoformy MeSH

A method for the determination and quantification of collagen types I-V in rat tissues has been developed. This method is based on collagen fragmentation by cyanogen bromide followed by trypsin digestion. After that, HPLC-MS/MS (HPLC coupled to an IT mass spectrometer) analyses of the resulting peptide mixtures (peptide maps) were performed. Specific peptides for each collagen type were selected. According to online databases, these peptides are present in human, bovine, and rat collagens. As a result, this method can be potentially applied to other species' tissues as well, such as human tissues, and provides a universal and simple method of quantifying collagen types. The applicability of this method for analyzing collagen types was demonstrated on rat tissues (skin, tendon, and aorta).

• MeSH
• kalibrace MeSH
• kolagen analýza   genetika   MeSH
• krysa rodu Rattus MeSH
• lidé MeSH
• Lod skóre MeSH
• protein - isoformy analýza   genetika   MeSH
• sekvence aminokyselin MeSH
• skot MeSH
• tandemová hmotnostní spektrometrie MeSH
• tkáňové extrakty chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kolagen MeSH
• protein - isoformy MeSH
• tkáňové extrakty MeSH

Interactions of boar, bull, and human seminal plasma proteins with heparin and phosphorylcholine were studied by affinity LC using heparin immobilized to a Toyopearl support. A step gradient elution from 0.15 to 1.50 M NaCl was employed to elute the seminal plasma proteins. Relative amounts of the heparin-binding fraction of seminal plasma proteins (H+) in seminal plasma of three species were determined. Further on, the fraction of seminal plasma proteins interacting with phosphorylcholine-binding proteins (P+) was evaluated. P+ proteins were not found in human seminal plasma and their highest amount was present in bull seminal plasma. A CE method was developed for separation of seminal plasma proteins. Various capillaries and separation conditions were tested; the best resolution was obtained in a bare-silica capillary, with a micellar system consisting of a 0.02 M borate buffer and 0.05 M SDS pH 10.0. The optimized conditions were applied to the identification of the components in boar plasma.

• MeSH
• chromatografie afinitní metody   MeSH
• chromatografie kapalinová metody   MeSH
• elektroforéza kapilární metody   MeSH
• fosforylcholin chemie   MeSH
• heparin chemie   MeSH
• lidé MeSH
• protein - isoformy analýza   MeSH
• proteiny semenné plazmy analýza   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosforylcholin MeSH
• heparin MeSH
• protein - isoformy MeSH
• proteiny semenné plazmy MeSH