For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.
- MeSH
- bakteriální nálož MeSH
- Bifidobacterium klasifikace růst a vývoj izolace a purifikace metabolismus MeSH
- Clostridium butyricum růst a vývoj izolace a purifikace metabolismus MeSH
- feces mikrobiologie MeSH
- kultivační média MeSH
- laktosa metabolismus MeSH
- lidé MeSH
- mateřské mléko chemie MeSH
- mléko chemie MeSH
- oligosacharidy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pseudallescheria boydii sensu lato is an emerging fungal pathogen causing fatal infections in both immunocompromised and immunocompetent hosts. In this work, two P. boydii isolates (human and animal origin) have been identified as being producers of cyclic peptides. Five putative nonribosomal peptides with a unique structure, which have been named pseudacyclins, were characterized by nuclear magnetic resonance spectroscopy and mass spectrometry. The most abundant representative of the pseudacyclins was quantified also on fungal spores. The presence of these peptides on inhaled fungal spores creates the possibility for exploitation of pseudacyclins as early indicators of fungal infections caused by Pseudallescheria species.
- MeSH
- cyklické peptidy chemie izolace a purifikace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- molekulární struktura MeSH
- myši MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- Pseudallescheria chemie patogenita MeSH
- sekvence aminokyselin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.
- MeSH
- amidy chemie MeSH
- antibiotika antitumorózní farmakologie MeSH
- antigeny CD43 metabolismus MeSH
- apoptóza MeSH
- doxorubicin analogy a deriváty farmakologie MeSH
- endoplazmatické retikulum metabolismus MeSH
- galektin 1 metabolismus MeSH
- glykosylace MeSH
- Golgiho aparát metabolismus MeSH
- kyseliny polymethakrylové farmakologie MeSH
- lymfom T-buněčný farmakoterapie metabolismus patologie MeSH
- myši MeSH
- nádorové buněčné linie účinky léků MeSH
- nosiče léků MeSH
- proliferace buněk MeSH
- průtoková cytometrie MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Penicillin G acylase of Escherichia coli (PGAEc) is a commercially valuable enzyme for which efficient bacterial expression systems have been developed. The enzyme is used as a catalyst for the hydrolytic production of beta-lactam nuclei or for the synthesis of semi-synthetic penicillins such as ampicillin, amoxicillin and cephalexin. To become a mature, periplasmic enzyme, the inactive prepropeptide of PGA has to undergo complex processing that begins in the cytoplasm (autocatalytic cleavage), continues at crossing the cytoplasmic membrane (signal sequence removing), and it is completed in the periplasm. Since there are reports on impressive cytosolic expression of bacterial proteins in Pichia, we have cloned the leader-less gene encoding PGAEc in this host and studied yeast production capacity and enzyme authenticity. RESULTS: Leader-less pga gene encoding PGAEcunder the control of AOX1 promoter was cloned in Pichia pastoris X-33. The intracellular overproduction of heterologous PGAEc(hPGAEc) was evaluated in a stirred 10 litre bioreactor in high-cell density, fed batch cultures using different profiles of transient phases. Under optimal conditions, the average volumetric activity of 25900 U l-1 was reached. The hPGAEc was purified, characterized and compared with the wild-type PGAEc. The alpha-subunit of the hPGAEc formed in the cytosol was processed aberrantly resulting in two forms with C- terminuses extended to the spacer peptide. The enzyme exhibited modified traits: the activity of the purified enzyme was reduced to 49%, the ratios of hydrolytic activities with cephalexin, phenylacetamide or 6-nitro-3-phenylacetylamidobenzoic acid (NIPAB) to penicillin G increased and the enzyme showed a better synthesis/hydrolysis ratio for the synthesis of cephalexin. CONCLUSIONS: Presented results provide useful data regarding fermentation strategy, intracellular biosynthetic potential, and consequences of the heterologous expression of PGAEc in P. pastoris X-33. Aberrant processing of the precursor of PGAEc in the cytosol yielded the mature enzyme with modified traits.
- MeSH
- bioreaktory MeSH
- Escherichia coli enzymologie genetika MeSH
- fermentace MeSH
- klonování DNA MeSH
- penicilinamidasa biosyntéza genetika izolace a purifikace MeSH
- Pichia genetika metabolismus MeSH
- promotorové oblasti (genetika) MeSH
- proteiny z Escherichia coli biosyntéza genetika izolace a purifikace MeSH
- průmyslová mikrobiologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Fungal beta-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal beta-N-acetylhexosaminidase. The fungal beta-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined. RESULTS: The complete primary structure of the fungal beta-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate - chitobiose with a stable value of binding energy during the molecular dynamics simulation. CONCLUSION: Whereas the intracellular bacterial beta-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal beta-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys448 with Cys483 stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.
- MeSH
- Aspergillus oryzae enzymologie MeSH
- beta-N-acetylhexosaminidasy chemie izolace a purifikace metabolismus MeSH
- dimerizace MeSH
- financování organizované MeSH
- glykosylace MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- počítačová simulace MeSH
- Ramanova spektroskopie metody MeSH
- spektroskopie infračervená s Fourierovou transformací metody MeSH
- stabilita enzymů MeSH
Filamentous fungi produce and secrete beta-N-acetylhexosaminidases, Hex, as important components of the binary chitinolytic systems involved in the formation of septa and hyphenation. Enzyme reconstitution experiments published previously indicate that Hex can occur in the form of two molecular species containing either one or two molecules of the propeptide noncovalently associated with the enzyme dimer. Here, we describe a novel mechanism for the regulation of the activity of Hex based on the association of their catalytic subunits with the large N-terminal propeptides in vivo. We show that the enzyme precursor is processed early in the biosynthesis, shortly after the addition of N-glycans through the action of a dibasic peptidase, cleaving both before and after the dibasic sequence. The processing site for this unique dibasic peptidase, different from that of kexins, is conserved among the beta-N-acetylhexosaminidases from filamentous fungi, and inhibition of the dibasic peptidase abrogates enzyme folding and activation. Binding of the released propeptide to the catalytic subunit of Hex is essential for its activation. An examination of the kinetics of Hex activation and dimerization in vitro allowed us to understand the unusually high efficiency of the assembly of this enzyme. We also report that the fungus is able to actively regulate the concentration of the processed propeptide in endoplasmic reticulum and thus the specific activity of the produced Hex. This novel regulatory mechanism enables the control of the catalytic activity and architecture of the secreted enzyme according to the needs of the producing cell at various stages of its growth cycle.
- MeSH
- acetylglukosamin analogy a deriváty farmakologie MeSH
- aktivace enzymů MeSH
- beta-N-acetylhexosaminidasy metabolismus sekrece MeSH
- biologický transport MeSH
- dimerizace MeSH
- endoplazmatické retikulum metabolismus MeSH
- financování organizované MeSH
- furin metabolismus MeSH
- genetická transkripce imunologie MeSH
- houby enzymologie MeSH
- katalýza MeSH
- molekulární sekvence - údaje MeSH
- prekurzory enzymů metabolismus sekrece MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- stabilita enzymů MeSH
- thiazoly farmakologie MeSH
- MeSH
- autonomní látky MeSH
- buňky NK MeSH
- experimentální nádory terapie MeSH
- finanční podpora výzkumu jako téma MeSH
- hexosaminy imunologie MeSH
- interakce mezi receptory a ligandy imunologie MeSH
- lidé MeSH
- ligandy MeSH
- metabolismus sacharidů MeSH
- nádory terapie MeSH
- sacharidy terapeutické užití MeSH
- Check Tag
- lidé MeSH
