Chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), is a major structural component in chitin-containing organism including crustaceans, insects and fungi. Mammals express two chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Here, we report that pig AMCase is stable in the presence of other digestive proteases and functions as chitinolytic enzyme under the gastrointestinal conditions. Quantification of chitinases expression in pig tissues using quantitative real-time PCR showed that Chit1 mRNA was highly expressed in eyes, whereas the AMCase mRNA was predominantly expressed in stomach at even higher levels than the housekeeping genes. AMCase purified from pig stomach has highest activity at pH of around 2-4 and remains active at up to pH 7.0. It was resistant to robust proteolytic activities of pepsin at pH 2.0 and trypsin and chymotrypsin at pH 7.6. AMCase degraded polymeric chitin substrates including mealworm shells to GlcNAc dimers. Furthermore, we visualized chitin digestion of fly wings by endogenous AMCase and pepsin in stomach extract. Thus, pig AMCase can function as a protease resistant chitin digestive enzyme at broad pH range present in stomach as well as in the intestine. These results indicate that chitin-containing organisms may be a sustainable feed ingredient in pig diet.
- MeSH
- chitin metabolismus MeSH
- chitinasy genetika izolace a purifikace metabolismus MeSH
- chymotrypsin metabolismus MeSH
- dieta * MeSH
- Drosophila chemie MeSH
- endopeptidasy metabolismus MeSH
- gastrointestinální trakt metabolismus MeSH
- křídla zvířecí chemie MeSH
- messenger RNA genetika metabolismus MeSH
- orgánová specificita MeSH
- pepsinogen A metabolismus MeSH
- prasata genetika MeSH
- rozpustnost MeSH
- substrátová specifita MeSH
- Tenebrio MeSH
- tkáňové extrakty MeSH
- trypsin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Systémová enzymoterapie je definována jako léčebná metoda, která zahrnuje perorální aplikaci kombinovaných enzymových směsí ve formě acidorezistentních tablet. Hlavní účinné látky těchto přípravků tvoří proteolytické enzymy původu živočišného (trypsin a chymotrypsin) a rostlinného (bromelain a papain). Základní podmínkou systémového účinku takto aplikovaných proteáz je jejich podávání nalačno a následné vstřebávání střevní sliznicí, při kterém zůstává zachována jejich enzymatická aktivita. Léčivé přípravky určené pro systémovou enzymoterapii jsou známy svými účinky protizánětlivými, protiotokovými, analgetickými a působí též imunomodulačně. Díky svému fibrinolytickému a antiagregačnímu účinku příznivě ovlivňují reologické vlastnosti krve. Současné podávání přípravků určených pro systémovou enzymoterapii společně s antibiotiky (ale i s dalšími léčivy) zlepšuje jejich tkáňovou dostupnost (efekt vehikula). Léčivé přípravky pro systémovou enzymoterapii Wobenzym® a Phlogenzym® jsou v České republice registrovány jako volně prodejné léky. Oborem, kde má využití systémové enzymoterapie v ČR nejdelší tradici, je patrně sportovní medicína.
Systemic enzyme therapy is defined as a therapeutic method using oral application of combined enzyme mixtures in the form of acid resistant tablets. The key active substances in these tablets are proteolytic enzymes of both animal (trypsin and chymotrypsin) and plant (bromelain and papain) origin. The systemic effect of thus applied proteases fully depends on their administration before meals and on their absorption through the intestinal mucosa, after which their enzymatic activity is preserved. Therapeutic agents for systemic enzyme therapy are known for their anti inflammatory, anti edematous, analgesic, and immunomodulatory properties. Thanks to their fibrinolytic and anti aggregation effects, they also improve the rheologic properties of the blood. Parallel application of systemic enzyme therapy and antibiotics (but also other drugs) improves their tissue availability (effect of the vehiculum). Therapeutic agents for systemic enzyme therapy Wobenzym® and Phlogenzym® are registered in the Czech Republic as over the counter drugs. The longest tradition of systemic enzyme therapy use in the Czech Republic can probably be seen in the sport medicine.
- MeSH
- alfa-makroglobuliny MeSH
- aplikace orální MeSH
- bromelainy farmakokinetika metabolismus MeSH
- chymotrypsin farmakokinetika metabolismus MeSH
- cytokiny účinky léků MeSH
- enzymoterapie * MeSH
- imunitní systém enzymologie MeSH
- klinické zkoušky kontrolované jako téma MeSH
- lidé MeSH
- makrofágy MeSH
- papain farmakokinetika metabolismus MeSH
- proteasy * farmakokinetika metabolismus MeSH
- střevní sliznice účinky léků MeSH
- trypsin farmakokinetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
Oil bodies, lipid-storage organelles, are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by "classical" proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a modified method comprising standard three-step procedure (SDS-PAGE, in-gel digestion, and LC-MS/MS analysis). In this method, chymotrypsin, single or in combination with trypsin, was used, which enabled to obtain proteolytic peptides from the hydrophobic regions and to identify new oil bodies' proteins. Our method can be easily applied to identification of other hydrophobic proteins.
- MeSH
- Arabidopsis metabolismus MeSH
- chromatografie kapalinová MeSH
- chymotrypsin metabolismus MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hmotnostní spektrometrie MeSH
- hydrofobní a hydrofilní interakce * MeSH
- lipidy * MeSH
- organely metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- proteomika metody MeSH
- trypsin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The effect of long-term exposure to tributyltin (TBT) on the intestine-related biochemical biomarkers in common carp was investigated in this study. Fish were exposed at sub-lethal concentrations of TBT (75 ng/L, 0.75 and 7.5 μg/L) for 60 days. Multiple biomarkers were measured, including digestive enzymes (trypsin, lipase and amylase), antioxidant responses (malondialdehyde (MDA) and total antioxidative capacity (T-AOC)), RNA/DNA ratio and the expression of digestive-related genes (try, lipc and amy). TBT exposure at 0.75 and 7.5 μg/L led to significantly inhibited activities of all digestive enzymes. At higher concentration of TBT, oxidative stress was apparent as reflected by the significant higher MDA content in the fish intestine, associated with an inhibition of T-AOC activities. After 60 days, the RNA/DNA ratio in fish intestine was significantly lower in groups exposed to TBT at higher concentrations (0.75 and 7.5 μg/L). In addition, the expression levels of try, lipc and amy in intestine of all treated fish were inhibited, even at the environmental concentration (75 ng/L). Our results suggest that long-term exposure to TBT could result in different responses of intestine-related biochemical biomarkers in fish, which could be used as new potential indicators for monitoring residual TBT present in aquatic environment.
- MeSH
- antioxidancia metabolismus MeSH
- biologické markery metabolismus MeSH
- chymotrypsin metabolismus MeSH
- DNA metabolismus MeSH
- kapři metabolismus MeSH
- malondialdehyd metabolismus MeSH
- oxidační stres účinky léků MeSH
- pankreatická elastasa metabolismus MeSH
- pesticidy chemie toxicita MeSH
- RNA metabolismus MeSH
- střeva účinky léků metabolismus MeSH
- trialkylcínové sloučeniny chemie toxicita MeSH
- trypsin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol-gel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of α-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. α-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.
- MeSH
- celulosa chemie MeSH
- chymotrypsin chemie metabolismus MeSH
- enzymy imobilizované chemie metabolismus MeSH
- lidé MeSH
- magnety chemie MeSH
- mikrosféry * MeSH
- molekulární sekvence - údaje MeSH
- pepsin A chemie metabolismus MeSH
- proteolýza MeSH
- rozpustnost MeSH
- sekvence aminokyselin MeSH
- skot MeSH
- sulfony chemie MeSH
- žaludeční šťáva enzymologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The proteasome inhibitors are used as research tools to study of the ATP-dependent ubiquitin-proteasome system. Some of them are at present undergoing clinical trials to be used as therapeutic agents for cancer or inflammation. These diseases are often accompanied by muscle wasting. We herein demonstrate findings about new proteasome inhibitors, belactosin A and C, and their direct effect on protein metabolism in rat skeletal muscle. M. soleus (SOL) and m. extensor digitorum longus (EDL) were dissected from both legs of male rats (40-60 g) and incubated in a buffer containing belactosin A or C (30 microM) or no inhibitor. The release of amino acids into the medium was estimated using high performance liquid chromatography to calculate total and myofibrillar proteolysis. Chymotrypsin-like activity (CTLA) of proteasome and cathepsin B, L activity were determined by fluorometric assay. Protein synthesis and leucine oxidation were detected using specific activity of L-[1-14C] leucine added to medium. Inhibited and control muscles from the same rat were compared using paired t-test. The results indicate that after incubation with both belactosin A and C total proteolysis and CTLA of proteasome decreased while cathepsin B, L activity did not change in both SOL and EDL. Leucine oxidation was significantly enhanced in SOL, protein synthesis decreased in EDL. Myofibrillar proteolysis was reduced in both muscles in the presence of belactosin A only. In summary, belactosin A and C affected basic parameters of protein metabolism in rat skeletal muscle. The response was both muscle- and belactosin-type-dependent.
- MeSH
- aminokyseliny metabolismus MeSH
- chymotrypsin antagonisté a inhibitory metabolismus MeSH
- inhibitory proteasomu MeSH
- kathepsin B metabolismus MeSH
- kosterní svaly metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- peptidy farmakologie MeSH
- potkani Wistar MeSH
- proteasomový endopeptidasový komplex MeSH
- svalové proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
BACKGROUND: Rb(a), Tr(a), and Wd(a) are three low-incidence blood group antigens that have not been assigned to a particular structure of the red cell membrane. Recent genetic and serologic data suggested erythroid band 3 as a possible carrier of these three antigens. STUDY DESIGN AND METHODS: Ten band 3 gene exons that encode the membrane domain of band 3 were screened for single strand conformation polymorphism (SSCP). Exons displaying SSCP were cloned and sequenced, and the presence of the mutations was verified by restriction digestion. RESULTS: Substitutions 548 Pro-->Leu, 551 Lys-->Asn, and 557 Val-->Met, all located in the third ectoplasmic loop of band 3, were detected in the subjects with Rb(a+), Tr(a+), and Wd(a+) red cells, respectively. The presence of the Rb(a) and Wd(a) mutations was confirmed in additional carriers of these blood group antigens. Chymotryptic cleavage at Tyr 553 and Tyr 555 abolished the agglutinability of Tr(a+) and Wd(a+) cells with the corresponding antisera, further demonstrating that the epitopes are located in the third ectoplasmic loop of band 3. Similar quantities of mRNA corresponding of the two band 3 alleles, a normal pattern of red cell membrane proteins, and normal DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt)-inhibitable sulfate flux were detected, which suggests that the mutations do not affect band 3 mRNA stability or band 3 protein expression and transport function. CONCLUSION: Wd(a) and Rb(a), and tentatively Tr(a), can be assigned to the Diego blood group system.
- MeSH
- antigeny krevních skupin * imunologie MeSH
- chymotrypsin metabolismus MeSH
- DNA krev MeSH
- epitopy analýza MeSH
- erytrocyty - protein 1 vyměňující anionty * genetika imunologie imunologie MeSH
- erytrocyty chemie metabolismus ultrastruktura MeSH
- isoantigeny * analýza MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- mutace MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus konformace jednovláknové DNA MeSH
- retikulocyty chemie MeSH
- RNA krev MeSH
- sekvenční analýza DNA MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
