Acetonitrile-assisted enzymatic digestion can facilitate the bottom-up identification of proteins of cancer origin.

The main objective of this study was to develop an effective in-gel trypsin digestion protocol using aqueous-acetonitrile solvent system to facilitate MS analysis and maximize the number of identified proteins from biological samples. The procedure, where 80% acetonitrile was present in the trypsin reaction mixture, increased the number of matched peptides, and allowed the identification of more proteins with higher coverage than the common digestion protocol. Vimentin, annexins, tubulin, actin, peptidyl-prolyl cis-trans isomerase or alpha-enolase are examples of important proteins that change during the progress cancer. These were isolated from human breast cancer cells and were used for this study.

Consequences of the natural retinoid/retinoid X receptor ligands action in human breast cancer MDA-MB-231 cell line: Focus on functional proteomics.

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all-trans and 9-cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e.g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial-mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1.

Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination.

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.

Effects of retinoic acid isomers on proteomic pattern in human breast cancer MCF-7 cell line.

Retinoids, acting through their cognate nuclear receptors, are crucial transcriptional regulators of many cellular processes such as differentiation, development, apoptosis, carbohydrate and lipid metabolism, homeostasis, etc. The aim of this study was the exploration of molecular mechanisms in relation to therapy of human breast cancer. One of the efficient strategies is identification of biomarkers as important tools in early cancer diagnosis and advisable treatment. Retinoids have been regarded as important therapeutic agents for many types of cancers, including human breast cancer. The effects of all-trans retinoic acid and 9-cis retinoic acid or their combination on proteomic pattern in human MCF-7 breast cancer line were investigated. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and identified by matrix assisted laser desorption ionization technique with time of flight mass analyzer (MALDI-TOF/TOF). Our data offer novel information on the proteomic pattern of proteins evaluated after treatment of MCF-7 cells with retinoic acid isomers.

An efficient proteomic approach to analyze agriculture crop biomass.

While a plant cell wall is formed by a complex of various components, including polysaccharides and structural proteins, its composition and representation may vary during cell growth. Currently, plant research targets the proteins participating in wall loosening. Multiple classes of enzymes, including various hemicellulases and cellulases, are required for plant material degradation to achieve the maximum decomposition. Identifying the set of proteins involved in the breakdown of cell-wall polymers is important to understand plant material conversion into suitable products. The objective of this study was to describe a method which can be used to carry out proteomics analysis of complex plant samples and identify enzymes degrading biomass. For this purpose we used proteomic techniques including gel electrophoresis, high pressure liquid chromatography combinated with mass spectrometry followed by data evaluation using databases searching. Results show that more than 50 % of these activities correspond to enzymes with proteolytic function. This study was focused primarily on enzymes able to breakdown the lignocellulosic and hemicellulosic parts that are very important for the material conversion into required products of degradation.

Thyroid hormone receptors and type I iodothyronine 5'-deiodinase activity of human thyroid toxic adenomas and benign cold nodules.

The majority of thyroid adenomas are of clonal origin. In a subset of toxic adenomas (TAs) and cold nodules (CNs) activating mutations in the thyrotropin (TSH) receptor or G s -alpha gene may explain the altered functions in these benign tumours. The present study was undertaken to investigate the status of functional thyroid hormone receptors, major thyroid hormone signal mediators, in both the human TAs and CNs in comparison with a normal thyroid tissue from the same patient. Electrophoretic mobility shift assays using a DR4 ("direct repeats" 4), a thyroid hormone responsive element (TRE) of human type I iodothyronine 5'-deiodinase demonstrated the DNA-binding of thyroid hormone receptors (TRs) in thyroid tissue nuclear extracts. A significant increase (p < 0.05) in the functional binding properties of TRs to the DR4 thyroid hormone responsive element was found in TAs when compared to normal thyroid tissue. Contrary, a marked diminution in the TR-TRE complex formation was found in CNs in comparison with normal thyroid tissue. In addition, functional activity of the iodothyronine 5'-deiodinase (5'DI) was analyzed in benign tumours, thyroid TAs and CNs in comparison with that of normal thyroid tissue. A significantly increased (p < 0.01) activity of 5'DI was demonstrated in TAs, and in contrast, decreased values of the enzyme activity were found in CNs when compared to a normal tissue. From the data it is suggested that both the status of TR-TRE complex formation and the activity of the 5'DI may be altered in benign tumours of human thyroid gland.

Presynaptic alpha2-adrenoceptor-mediated modulation of adenosine 5' triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein.

1. The modulatory effects of agonists and antagonists of prejunctional alpha2-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5' triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2. The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 microM) and guanethidine (10 microM) in the artery and vein. Desipramine (10 microM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS-evoked overflow of ATP was equal in the artery and vein. 3. Stimulation of alpha2-adrenoceptors with clonidine (0.1 microM) and oxymethazoline (0.3 microM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the alpha2-adrenoceptor agonists in the artery and vein. 4. In artery, blockade of alpha2-adrenoceptors with yohimbine at a concentration of 0.1 microM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 microM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 microM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 microM) increased the NA overflow in artery only at 4 Hz, whereas in vein idazoxan increased the NA overflow at both 4 and 16 Hz. No changes of EFS-evoked ATP overflow were observed in the presence of 0.1 microM yohimbine in both artery and vein. Greater concentration of yohimbine (i.e. 1 microM) increased the overflow of ATP in both the artery and vein only at 4 Hz EFS. Idazoxan (1 microM) enhanced the ATP overflow only at 16 Hz in vein. The overflow of ADP was affected by both yohimbine and idazoxan in a similar manner to the ATP overflow so that the ATP:ADP ratios were not changed. 5. In conclusion, sympathetic nerves in both mesenteric arteries and veins appear to release ATP along with NA. Release of NA in veins exceeds release of NA in arteries, whereas both the canine artery and vein release equal amount of ATP. At long-duration nerve stimulation (as might occur during stress) the alpha2-adrenoceptors appear to rather modulate release of NA than release of the cotransmitter ATP. The prejunctional autoinhibition of NA release is more effective at lower frequencies of nerve stimulation. The alpha2-adrenoceptor-mediated neuromodulation plays a greater role in veins than arteries. Quantitative differences in alpha2-adrenoceptor-mediated neuromodulation in the arteries and veins may participate to differing contributions of mesenteric blood vessels to the control of blood flow and volume distribution in splanchnic circulation.

Co-release of endogenous ATP and noradrenaline from guinea-pig mesenteric veins exceeds co-release from mesenteric arteries.

1. The present study was designed to compare the overflow of sympathetic neurotransmitters of guinea-pig inferior mesenteric artery and mesenteric vein evoked by electrical field stimulation (EFS) with special emphasis on the simultaneous release of ATP and noradrenaline (NA). The stimulation-evoked overflow of ADP, AMP and adenosine was also evaluated. 2. Endothelium-denuded segments of inferior mesenteric arteries or veins were superfused in a small volume (200 microL)-chamber for EFS and subsequent detection of NA (HPLC- electrochemical detection) and adenine nucleotides and adenosine (HPLC-fluorescence detection) in samples of the superfusate. 3. Both arteries and veins responded to EFS (15 V, 4-16 Hz, 0.3 msec for 60 s) with overflow of ATP and NA in a tetrodotoxin (1 micromol/L)- and guanethidine (10 micromol/L)-sensitive manner. The EFS-evoked overflow of NA in veins exceeded the overflow of NA in arteries at all frequencies of stimulation, whereas the EFS-evoked overflow of ATP, ADP and AMP in veins exceeded the overflow of adenine nucleotides in arteries at 4 and 8 Hz but not at 16 Hz stimulation. The EFS-evoked overflow of adenosine was similar in arteries and veins. 4. Activation of alpha1-adrenoceptors with methoxamine (10 micromol/L) did not produce overflow of ATP. 5. Blockade of alpha1/alpha2-adrenoceptors with phentolamine (1 micromol/L) did not affect EFS-evoked overflow of ATP, ADP, AMP and adenosine. 6. It is concluded that overflow of ATP and NA from sympathetic nerves may constitute an effective mechanism in the complex balance between capacitance and resistance in splanchnic circulation.

Inhibition studies of porphobilinogen synthase from Escherichia coli differentiating between the two recognition sites.

Porphobilinogen synthase condenses two molecules of 5-aminolevulinate in an asymmetric way. This unusual transformation requires a selective recognition and differentiation between the substrates ending up in the A site or in the P site of porphobilinogen synthase. Studies of inhibitors based on the key intermediate first postulated by Jordan allowed differentiation of the two recognition sites. The P site, whose structure is known from X-ray crystallographic studies, tolerates ester functions well. The A site interacts very strongly with nitro groups, but is not very tolerant to ester functions. This differentiation is a central factor in the asymmetric handling of the two identical substrates. Finally, it could be shown that the keto group of the substrate bound at the A site is not only essential for the recognition, but that an increase in electrophilicity of the carbon atom also increases the inhibition potency considerably. This has important consequences for the recognition process at the A site, whose exact structure is not yet known.

Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig inferior mesenteric vein.

Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2-64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance.

Inhibition of Escherichia coli porphobilinogen synthase using analogs of postulated intermediates.

BACKGROUND: Porphobilinogen synthase is the second enzyme involved in the biosynthesis of natural tetrapyrrolic compounds, and condenses two molecules of 5-aminolevulinic acid (ALA) through a nonsymmetrical pathway to form porphobilinogen. Each substrate is recognized individually at two different active site positions to be regioselectively introduced into the product. According to pulse-labeling experiments, the substrate forming the propionic acid sidechain of porphobilinogen is recognized first. Two different mechanisms for the first bond-forming step between the two substrates have been proposed. The first involves carbon-carbon bond formation (an aldol-type reaction) and the second carbon-nitrogen bond formation, leading to an iminium ion. RESULTS: With the help of kinetic studies, we determined the Michaelis constants for each substrate recognition site. These results explain the Michaelis-Menten behavior of substrate analog inhibitors - they act as competitive inhibitors. Under standard conditions, however, another set of inhibitors demonstrates uncompetitive, mixed, pure irreversible, slow-binding or even quasi-irreversible inhibition behavior. CONCLUSIONS: Analysis of the different classes of inhibition behavior allowed us to make a correlation between the type of inhibition and a specific site of interaction. Analyzing the inhibition behavior of analogs of postulated intermediates strongly suggests that carbon-nitrogen bond formation occurs first.

Cotransmission from sympathetic vasoconstrictor neurons: differences in guinea-pig mesenteric artery and vein.

Vasoconstrictor responses to electrical field stimulation (EFS, 0.2-32 Hz, 0.1 ms, 12 V, for 1 min) were measured in endothelium-denuded segments of guinea-pig mesenteric vein and compared to responses in mesenteric artery. The distribution of both tyrosine-hydroxylase-like immunoreactivity (TH-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) was also studied using anti-TH and anti-NPY antibodies. The effect of exogenous NPY (10 nM) on EFS (8 Hz, 0.3 ms, 12 V, for 1 min)-evoked overflow of noradrenaline (NA) was also studied using an HPLC technique with electrochemical detection. Veins responded with contractions at lower frequencies of stimulation than arteries. Prazosin (0.1 microM) abolished the EFS-evoked contractions in artery at 0.5-32 Hz and in vein at 0.2-1 Hz of stimulation. However, in vein, the contractile responses to EFS at 2-32 Hz of stimulation were only reduced by prazosin. Phentolamine (1 microM) abolished the responses to 0.5-4 Hz and reduced the responses to 8-32 Hz of EFS in artery. In vein, phentolamine (1 microM) abolished the responses to 0.2-1 Hz and facilitated the contractions elicited by 16-32 Hz. The NPY-receptor antagonist BIBP3226 (1 microM), in combination with phentolamine, abolished contractions in vein. Yohimbine (0.1 microM) abolished the responses to lower frequencies of stimulation in both artery (0.5-2 Hz) and vein (0.2-1 Hz). The responses to greater frequency stimulation were not affected by yohimbine in artery, and were facilitated in vein. Pre-treatment of animals for 24 h with reserpine abolished contractile responses to EFS in artery, whereas in vein, responses to 0.2-2 Hz were abolished while responses to 4-32 Hz were unchanged. Suramin (100 microM) or alpha,beta-methylene ATP (alpha,beta MeATP; 10-100 microM) treatment did not affect the contractile responses to EFS in either artery or vein. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 30 microM), even potentiated the responses to 2-16 Hz in vein. However, following resperine-treatment, both PPADS and suramin reduced the nerve-evoked contractions of vein. Either BIBP3226 (1 microM) alone or BIBP3226 in combination with PPADS or suramin abolished the contractile response to EFS in reserpine-treated veins. NPY (100 nM) produced significantly more contraction in vein than in artery (i.e., 93 +/- 2.5 versus 7 +/- 4% of the response to 70 mM KCl, respectively). NPY (10 nM) significantly reduced the NA overflow evoked by EFS at 8 Hz. Flat mount preparations and cryostat sections of both mesenteric artery and vein revealed that TH-LI and NPY-LI were co-localized in a dense network of fibers within the adventitial layer. In conclusion, NA exclusively mediates the contractile response to sympathetic nerve stimulation in guinea-pig mesenteric artery, whereas at least three neurotransmitters [i.e., NA, adenosine 5'-triphosphate (ATP) and NPY] are involved in the neural response of mesenteric vein.