Physiologically Aggregated LacZ Applied in Trehalose Galactosylation in a Recycled Batch Mode.

Galactooligosaccharides obtained via ß-galactosidase transgalactosylation have health-promoting properties and are widely recognized as effective prebiotics. Trehalose-based galactooligosaccharides could be introduced into food and pharmaceutical industries similarly to trehalose. In light of this, new technological approaches are needed. Recently, in vivo enzyme immobilizations for recombinant proteins have been introduced, and physiological aggregation into active inclusion bodies (aIBs) has emerged as one such method of in vivo immobilization. To prepare LacZ ß-galactosidase in the form of aIBs, we used a short 10 amino acid aggregation-prone tag. These native protein particles were simply washed from the cell lysate and applied in trehalose galactosylation in a recycled batch mode. In this study, aIBs entrapped in alginate beads, encapsulated in alginate/cellulose sulfate/poly(methylene-co-guanidine) capsules and magnetized were compared with free aIBs. Alginate/cellulose sulfate/PMCG capsules showed more suitable properties and applicability for biotransformation of trehalose at its high concentration (25%, w/v) and elevated temperature (50 °C).

Pull-Down Into Active Inclusion Bodies and Their Application in the Detection of (Poly)-Phosphates and Metal-Ions.

Inclusion bodies are typically ignored as they are considered unwanted protein waste generated by prokaryotic host cells during recombinant protein production or harmful protein inclusions in human cell biology. However, these protein particles may have applications for in vivo immobilization in industrial biocatalysis or as cell-tolerable protein materials for the pharmaceuticals industry and clinical development. Thus, there is a need to in vivo "pull-down" (insolubilize) soluble enzymes and proteins into inclusion bodies. Accordingly, in this study, sequences from the short-chain polyphosphatase ygiF were used to design pull-down tags capable of detecting (poly)-phosphates and metal ions. These tags were compared with the entire CHAD domain from Escherichia coli ygiF and SACS2 CHAD from Saccharolobus solfataricus. The results demonstrated that highly soluble green fluorescent protein variants could be pulled down into the inclusion bodies and could have modified sensitivity to metals and di-/tri-inorganic phosphates.

Insoluble Protein Applications: The Use of Bacterial Inclusion Bodies as Biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase, UDP-glucose pyrophosphorylase) physiologically aggregated within inclusion bodies retaining their biological activity as immobilized biocatalysts.

Prebiotic Peptides Based on the Glycocodon Theory Analyzed with FRET.

In modern protein-carbohydrate interactions, carbohydrate-aromatic contact with CH-π interactions are used. Currently, they are considered driving forces of this complexation. In these contacts, tryptophan, tyrosine, and histidine are preferred. In this study, we focus on primary prebiotic chemistry when only glycine, alanine, aspartic acid, and valine are available in polypeptides. In this situation, when the aromatic acids are not available, hydrogen-bonding aspartic acid must be used for monosaccharide complexation. It is shown here that (DAA)n polypeptides play important roles in primary "protein"-glucose recognition, that (DGG)n plays an important role in "protein"-ribose recognition, and that (DGA)n plays an important role in "protein"-galactose recognition. Glucose oxidase from Aspergillus niger, which still has some ancient prebiotic sequences, is chosen here as an example for discussion.

ADMA, homocysteine and redox status improvement affected by 7-nitroindazole in spontaneously hypertensive rats.

Inhibition of nitric oxide (NO) production can influence blood pressure regulation and increase hypertension. Asymmetric dimethylarginine, ADMA, an analogue of L-arginine, can inhibit NO synthesis, impair endothelial function, and is a risk marker of cardiovascular diseases. Homocysteine (Hcy) level affects oxidative stress production of reactive oxygen species (ROS) in hypertension and also influences changes in signaling and cell damage. The present study was focused on experimental effects of exogenous NOS inhibitors and their effect on ADMA, an endogenous NOS inhibitor, homocysteine and ROS production measured as reactive oxidative metabolites (ROM). We compared effects of the two potential exogenous NO-inhibitors: NG-nitro L-arginine methyl ester (L-NAME) and 7-nitroindazole (7-NI). Levels of ADMA, Hcy, ROM and total thiols (TTL) were not changed in the L-NAME group. With 7-NI administration, we observed unchanged NOS activity in the left ventricle and a pronounced decrease of ADMA and Hcy levels, accompanied by ROM over-production in plasma. TTL/ROM ratio was more favorable than in the L-NAME group. We observed that 7-NI, an exogenous NOinhibitor, can decrease and improve the levels of ADMA, Hcy, and ROM, and increase TTL/ROM ratio in the plasma of spontaneously hypertensive rats.

A semi-multifunctional sialyltransferase from Bibersteinia trehalosi and its comparison to the Pasteurella multocida ST1 mutants.

Sialic acids are well known for their crucial roles in many physiological and pathological processes. Improvement in the efficacy of protein drugs, an increase in the anti-inflammatory activity of intravenous immunoglobulin, preparation of infant milk and the diagnosis of diseases are examples of why there is a need for efficient in vitro sialylation. Sialyltransferases are crucial enzymes for the synthesis of sialo-oligosaccharides. Here, we introduce a new α2,3-sialyltransferase from bacteria Bibersteinia trehalosi (BtST1), which is homological to sialyltransferase from Pasteurella multocida (PmST1), Pasteurella dagmatis (PdST1) and Haemophilus ducreyi (Hd0053). BtST1 is active in a wide pH range and shows considerable acceptor flexibility. Very good specific activities have been detected with lactose and LacNAc as acceptors, and these activities were comparable to those of efficient multifunctional PmST1 and higher than PdST1, Hd0053 and also PmST1 M144D which was constructed to decrease the high sialidase activity of PmST1. Testing of PmST1 mutant forms revealed that mutations that included S143 caused only the restriction of sialyltransferase activity, whereas mutations including G142 resulted in the loss of activity with lactose. BtST1 possesses only low sialidase and trans-sialidase activities that are comparable to mutant PmST1 M144D, which are detected only in the presence of CMP. The combination of large acceptor flexibility, high activity for lactose and LacNAc and naturally low sialidase activity make BtST1 an attractive enzyme for biotechnological applications.

Protein-RNA and protein-glycan recognitions in light of amino acid codes.

BACKGROUND: RNA-binding proteins, in cooperation with non-coding RNAs, play important roles in post-transcriptional regulation. Non-coding micro-RNAs control information flow from the genome to the glycome by interacting with glycan-synthesis enzymes. Glycan-binding proteins read the cell surface and cytoplasmic glycome and transfer signals back to the nucleus. The profiling of the protein-RNA and protein-glycan interactomes is of significant medicinal importance. SCOPE OF REVIEW: This review discusses the state-of-the-art research in the protein-RNA and protein-glycan recognition fields and proposes the application of amino acid codes in profiling and programming the interactomes. MAJOR CONCLUSIONS: The deciphered PUF-RNA and PPR-RNA amino acid recognition codes can be explained by the protein-RNA amino acid recognition hypothesis based on the genetic code. The tripartite amino acid code is also involved in protein-glycan interactions. At present, the results indicate that a system of four codons ("gnc", where n=g - guanine, c - cytosine, u - uracil or a - adenine) and four amino acids (G - glycine, A - alanine, V - valine, D - aspartic acid) could be the original genetic code that imprinted "rules" into both recognition processes. GENERAL SIGNIFICANCE: Amino acid recognition codes have provocative potential in the profiling and programming of the protein-RNA and protein-glycan interactomes. The profiling and even programming of the interactomes will play significant roles in diagnostics and the development of therapeutic procedures against cancer and neurodegenerative, developmental and other diseases.

Insoluble protein applications: the use of bacterial inclusion bodies as biocatalysts.

Biocatalysis and biotransformations have a broad application in industrial synthetic chemistry. In addition to the whole cell catalysis, purified recombinant enzymes are successfully used for biocatalysis of specific chemical reactions. In this contribution, we report characterization, immobilization, and application of several model target enzymes (D-amino acid oxidase, sialic acid aldolase, maltodextrin phosphorylase, polyphosphate kinase) physiologically aggregated within inclusion bodies (IBs) retaining their biological activity as immobilized biocatalysts.

Bacterial inclusion bodies as potential synthetic devices for pathogen recognition and a therapeutic substance release.

BACKGROUND: Adhesins of pathogens recognise the glycans on the host cell and mediate adherence. They are also crucial for determining the tissue preferences of pathogens. Currently, glyco-nanomaterials provide potential tool for antimicrobial therapy. We demonstrate that properly glyco-tailored inclusion bodies can specifically bind pathogen adhesins and release therapeutic substances. RESULTS: In this paper, we describe the preparation of tailored inclusion bodies via the conjugation of indicator protein aggregated to form inclusion bodies with soluble proteins. Whereas the indicator protein represents a remedy, the soluble proteins play a role in pathogen recognition. For conjugation, glutaraldehyde was used as linker. The treatment of conjugates with polar lysine, which was used to inactivate the residual glutaraldehyde, inhibited unwanted hydrophobic interactions between inclusion bodies. The tailored inclusion bodies specifically interacted with the SabA adhesin from Helicobacter pylori aggregated to form inclusion bodies that were bound to the sialic acids decorating the surface of human erythrocytes. We also tested the release of indicator proteins from the inclusion bodies using sortase A and Ssp DNAB intein self-cleaving modules, respectively. Sortase A released proteins in a relatively short period of time, whereas the intein cleavage took several weeks. CONCLUSIONS: The tailored inclusion bodies are promising "nanopills" for biomedical applications. They are able to specifically target the pathogen, while a self-cleaving module releases a soluble remedy. Various self-cleaving modules can be enabled to achieve the diverse pace of remedy release.

Free-radical degradation of high-molar-mass hyaluronan induced by ascorbate plus cupric ions: evaluation of antioxidative effect of cysteine-derived compounds.

Based on our previous findings, the present study has focused on free-radical-mediated degradation of the synovial biopolymer hyaluronan. The degradation was induced in vitro by the Weissberger's system comprising ascorbate plus cupric ions in the presence of oxygen, representing a model of the early phase of acute synovial joint inflammation. The study presents a novel strategy for hyaluronan protection against oxidative degradation with the use of cysteine-derived compounds. In particular, the work objectives were to evaluate potential protective effects of reduced form of L-glutathione, L-cysteine, N-acetyl-L-cysteine, and cysteamine, against free-oxygen-radical-mediated degradation of high-molar-mass hyaluronan in vitro. The hyaluronan degradation was influenced by variable activity of the tested thiol compounds, also in dependence of their concentration applied. It was found that L-glutathione exhibited the most significant protective and chain-breaking antioxidative effect against the hyaluronan degradation. Thiol antioxidative activity, in general, can be influenced by many factors such as various molecule geometry, type of functional groups, radical attack accessibility, redox potential, thiol concentration and pK(a), pH, ionic strength of solution, as well as different ability to interact with transition metals. Antioxidative activity was found to decrease in the following order: L-glutathione, cysteamine, N-acetyl-L-cysteine, and L-cysteine. These findings might be beneficial in future development of potential drugs in the treatment of synovial hyaluronan depletion-derived diseases.

Pro-oxidative effect of peroxynitrite regarding biological systems: a special focus on high-molar-mass hyaluronan degradation.

Current understanding on the role of peroxynitrite in etiology and pathogenesis of some human diseases, such as cardio-vascular diseases, stroke, cancer, inflammation, neurodegenerative disorders, diabetes mellitus and diabetic complications has recently led to intensive investigation of peroxynitrite involvement in physiology and pathophysiology. Mechanism of cytotoxic effects of peroxynitrite involve its reactions with lipids, DNA/RNA, proteins, and polysaccharides, thus triggering cellular responses ranging from subtle changes of cell functioning to severe oxidative damage of the affected macromolecules leading to necrosis or apoptosis. The present work is aimed at providing a brief overview of i) peroxynitrite biosynthesis and reaction pathways in vivo, ii) its synthetic preparation in vitro, and iii) to reveal its potential damaging role in vivo, on actions studied via monitoring in vitro hyaluronan degradation. The complex biochemical behavior of peroxynitrite is determined by a number of variables, such as chemistry of the reaction itself, depending mostly on the involvement of conformational structures of different energy states, concentration of the species involved, content of reactive intermediates and trace transition metal ions, contribution of carbon dioxide, presence of trace organics, and by the reaction kinetics. Recently, in vitro studies of oxidative cleavage of hyaluronan have, in fact, been the subject of growing interest. Here we also describe our experimental set-up for studying peroxynitrite-mediated degradation of hyaluronan, a system, which may be suitable for testing prospective pharmacological substances.

Aurothiomalate as preventive and chain-breaking antioxidant in radical degradation of high-molar-mass hyaluronan.

The potential anti- or pro-oxidative effects of a disease-modifying antirheumatic drug, aurothiomalate, to protect high-molar-mass hyaluronan against radical degradation were investigated along with L-glutathione - tested in similar functions. Hyaluronan degradation was induced by the oxidative system Cu(II) plus ascorbate known as the Weissberger's oxidative system. The time- and dose-dependent changes of the dynamic viscosity of the hyaluronan solutions were studied by the method of rotational viscometry. Additionally, the antioxidative activity of aurothiomalate expressed as a radical-scavenging capacity based on a decolorization 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay was inspected. At the higher concentrations tested, L-glutathione showed excellent scavenging of (.) OH and peroxyl-type radicals, however, at the lowest concentration applied, its pro-oxidative effect was revealed. The effects of aurothiomalate on hyaluronan degradation were similar to that of L-glutathione, however, at the lowest concentration tested, no significant pro-oxidant effect was observed.

Free-radical degradation of high-molecular-weight hyaluronan induced by ascorbate plus cupric ions. Testing of bucillamine and its SA981-metabolite as antioxidants.

High-molecular-weight hyaluronan (HA) samples were exposed to free-radical chain-degradation reactions induced by ascorbate in the presence of Cu(II) ions - the so-called Weissberger's oxidative system. The concentrations of both reactants [ascorbate, Cu(II)] were comparable to those that may occur during an early stage of the acute phase of joint inflammation. The time-dependent changes of the viscosity of the HA solution in the absence of the substance tested were monitored by rotational viscometry. However, when the anti- or pro-oxidative effects of the antioxidants/drugs were investigated, their dose-dependency was also examined. Additionally, the anti-oxidative activities of these substances were screened by the well-established ABTS and DPPH decolorization assays. The actions of the disease-modifying anti-rheumatic drugs, namely bucillamine and D-penicillamine, were compared to those of L-cysteine and of SA981, the oxidized metabolite of bucillamine. The results indicated that bucillamine was the most efficient scavenger of hydroxyl- and/or peroxyl-type radicals, even at the lowest drug concentration. In contrast, SA981 demonstrated no scavenging activity against the aforementioned free radicals. D-Penicillamine and L-cysteine showed a dual effect, i.e. a pronounced anti-oxidative effect was, after a given time period, followed by a significant pro-oxidative effect.

Carnosine inhibits degradation of hyaluronan induced by free radical processes in vitro and improves the redox imbalance in adjuvant arthritis in vivo.

OBJECTIVE: New ways of supplementary or combinatory therapy of rheumatoid arthritis (RA) are of great importance. The aim is to find an additive to classical RA therapy with natural molecules without side effects possessing anti-inflammatory and anti-oxidative properties. In this study we investigated the anti-oxidative and anti-inflammatory properties of the endogenous natural compound carnosine (CARN) in vitro and in vivo. Moreover, we tested also the inhibitory properties of the drug methotrexate (MTX) on dynamic viscosity of hyaluronan (HA) solutions in the same manner. METHODS: For in vitro testing of the inhibitory properties of CARN against degradation of HA solutions, we used the model of degradation of hyaluronan (HA) induced by free radicals. Both substances, CARN and MTX, were compared to glutathione (GSH). Rotational viscometry was used in evaluation of protective properties of compounds studied. The ability of CARN to restore the redox imbalance occurring in adjuvant arthritis (AA) of rats was also tested. We monitored the effect of CARN on hind paw volume (HPV) and on the levels of protein carbonyls, and thiobarbituric acid reacting substances (TBARS) in AA. RESULTS: In the reaction system with the prevalence of •OH and/or peroxy-type radicals, CARN in 200 µmol/L concentration tested was shown to exert a protective action on HA degradation. MTX was less effective than CARN in preventing HA degradation. Its ability to protect HA against radical degradation was evident only at the highest concentration of 400 µmol/L. In AA, carnosine significantly reduced TBARS and protein carbonyls in plasma, and also decreased the HPV of animals most effectively on the day 14. CONCLUSIONS: CARN proved its inhibitory properties against degradation of HA solutions at experimental conditions in vitro and showed its beneficial efficiency in vivo. Moreover, it reduced also HPV, the clinical marker of inflammation in AA.

Ascorbate and Cu(II)-induced oxidative degradation of high-molar-mass hyaluronan. Pro- and antioxidative effects of some thiols.

OBJECTIVE: This study presents the results of antioxidative and pro-oxidative efficacy of cysteamine and D-penicillamine (D-pen) in comparison to L-glutathione (L-GSH) on high-molar-mass hyaluronan (HA) degradation by cupric ions plus ascorbic acid. METHODS: The substance tested was applied in the degradative system cupric ions plus ascorbate: (i) before the reaction onset or also (ii) 1 h after the reaction started. The results obtained were compared with that one recorded by using the degradative system in the absence of the substance tested. To monitor HA degradation kinetics, rotational viscometry was applied. Moreover, the standard ABTS and DPPH assays were used. RESULTS: By using the method of rotational viscometry, D-pen showed dual effect: initial inhibitory effect on •OH radicals was changed to a pro-oxidative one in the dose and time dependent manner. Both L-GSH and cysteamine were recorded to be more effective scavengers of •OH radicals than D-pen. Cysteamine demonstrated to be an excellent scavenger also of alkoxyl- and peroxyl- type radicals. Based on IC50 values, gained by ABTS assay, it is evident that D-pen showed higher radical scavenging capacity compared to cysteamine. Similar results were observed also in DPPH assay, although in this assay less effective radical scavenging capacities of both substances tested were recorded. CONCLUSIONS: On the basis of the results obtained, it can be stated that D-pen can produce hydrogen peroxide or •OH radicals and can inhibit the production of these oxidants. Our results showed that both L-GSH and cysteamine are similarly effective in inhibiting of HA degradation. Moreover, cysteamine demonstrated to be a significant inhibitor of alkoxyl- and peroxyl- type radicals generated from C-type macroradical of HA.

An alternative standard for Trolox-equivalent antioxidant-capacity estimation based on thiol antioxidants. Comparative 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] decolorization and rotational viscometry study regarding hyaluronan degradation.

Comparison of the effectiveness of antioxidant activity of three thiol compounds, D-penicillamine, reduced L-glutathione, and 1,4-dithioerythritol, expressed as a radical-scavenging capacity based on the two independent methods, namely a decolorization 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay and a rotational viscometry, is reported. Particular concern was focused on the testing of potential free-radical scavenging effects of thiols against hyaluronan degradation, induced by hydroxyl radicals. A promising, solvent-independent, antioxidative function of 1,4-dithioerythritol, comparable to that of a standard compound, Trolox(®), was confirmed by the 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay. The new potential antioxidant 1,4-dithioerythritol exhibited very good solubility in a variety of solvents (e.g., H(2)O, EtOH, and DMSO) and could be widely accepted and used as an effective antioxidant standard instead of a routinely used Trolox(®) on 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid] assay.

Degradation of high-molar-mass hyaluronan by ascorbate plus cupric ions: effects of D-penicillamine addition.

Pro- and anti-oxidative effects of an anti-rheumatoid drug, D-penicillamine (D-PN), on the kinetics of high-molar-mass hyaluronan (HA) degradation were monitored using the method of rotational viscometry. The degradation of the dissolved HA macromolecules was attained by applying the Weissberger's system comprising ascorbic acid plus cupric ions. Electron paramagnetic resonance (EPR) spectroscopy was used to identify the generated free radicals. The results obtained indicate that the initial anti-oxidative action of D-PN is followed by induction of pro-oxidative conditions due to the generation of reactive free radicals. It is speculated, however, that the latter situation may be considered as an advantageous property of D-PN. Hydroxyl radicals formed in this way may participate in decomposition of proteinases, which are believed to be responsible for the destruction of joint cartilage under rheumatoid arthritic conditions.

Lectinomics II. A highway to biomedical/clinical diagnostics.

The review assesses current status and attempts to forecast trends in the development of lectin biorecognition technology. The progressive trend is characterized scientometrically and reflects the current transient situation, when standard low-throughput lectin-based techniques are being replaced by a novel microarray-based techniques offering high-throughput of detection. The technology is still in its infancy (validation phase), but already shows promise as an efficient tool to decipher the enormous complexity of the glycocode that influences physiological status of the cell. Further enhancement in robustness and flexibility of lectin microarrays is predicted by using recombinant and artificial lectins that will render production of lectin microarrays cost-effective and more affordable. Mass spectrometry is expected to play an important role to characterize the binding profile of new lectins. Differences in glycan recognition by lectins and anti-carbohydrate antibodies are given on a molecular basis, and strong and weak points of both biorecognition molecules in diagnosis are briefly discussed.

Study of pro- and anti-oxidative properties of D-penicillamine in a system comprising high-molar-mass hyaluronan, ascorbate, and cupric ions.

OBJECTIVES: The study presents results of pro- and anti-oxidative effects of D-penicillamine on hyaluronan degradation by ascorbate plus cupric ions. METHODS: The well established degradative system comprising high-molar-mass hyaluronan and ascorbate plus Cu(II) ions was used. Primarily, the effects of replacement of ascorbic acid in this system by D-penicillamine were investigated. Then, D-penicillamine was added into the above degradative system before reaction onset or 1h after the reaction had started. To monitor hyaluronan degradation kinetics, rotational viscometry was applied. RESULTS: No hyaluronan degradation occurred when ascorbate was replaced by D-penicillamine. The drug addition into the complete degradative system at the reaction onset caused a marked inhibition of hyaluronan degradation. However, the inhibitory effect turned to a pro-oxidative one within appr. 1 h. CONCLUSION: The dual behavior of D-penicillamine on hyaluronan degradation can relate to: (i) the drug completely traps *OH radicals generated from ascorbate plus Cu(II) ions under aerobic conditions; (ii) thiyl radicals generated from D-penicillamine react with D-penicillamine anions resulting in novel radical reactive species, which e.g. by reducing dioxygen molecules can generate further *OH radicals.

A crosslinked inclusion body process for sialic acid synthesis.

The propensity of a recombinant protein produced in bacteria to aggregate has been assumed to be unpredictable, and inclusion bodies have been thought of as wasted cell material. However, a target protein can be purposely driven to inclusion bodies, which demonstrate full cell tolerable activity. Sialic acid aldolase, N-terminally fused with the cellulose-binding module of Clostridium cellulovorans, was almost quantitatively physiologically aggregated into active inclusion bodies. These inclusion bodies were entrapped in alginate beads and crosslinked by glutaraldehyde. The immobilized biocatalyst generated by this crosslinked inclusion bodies (CLIB) technology was used in a repetitive batch protocol for sialic acid production that was monitored on-line by flow calorimetry. The required substrate, N-acetyl-D-mannosamine, was obtained by partially improved alkaline epimerization.