Effect of advanced glycation end-products in a wide range of medical problems including COVID-19.

Glycation is a physiological process that determines the aging of the organism, while in states of metabolic disorders it is significantly intensified. High concentrations of compounds such as reducing sugars or reactive aldehydes derived from lipid oxidation, occurring for example in diabetes, atherosclerosis, dyslipidemia, obesity or metabolic syndrome, lead to increased glycation of proteins, lipids and nucleic acids. The level of advanced glycation end-products (AGEs) in the body depends on rapidity of their production and the rate of their removal by the urinary system. AGEs, accumulated in the extracellular matrix of the blood vessels and other organs, cause irreversible changes in the biochemical and biomechanical properties of tissues. As a consequence, micro- and macroangiopathies appear in the system, and may contribute to the organ failure, like kidneys and heart. Elevated levels of AGEs also increase the risk of Alzheimer's disease and various cancers. In this paper, we propose a new classification due to modified amino acid residues: arginyl-AGEs, monolysyl-AGEs and lysyl-arginyl-AGEs and dilysyl-AGEs. Furthermore, we describe in detail the effect of AGEs on the pathogenesis of metabolic and old age diseases, such as diabetic complications, atherosclerosis and neurodegenerative diseases. We summarize the currently available data on the diagnostic value of AGEs and present the AGEs as a therapeutic goal in a wide range of medical problems, including SARS-CoV-2 infection and so-called long COVID.

Matrix Metalloproteinase-2 Inhibition in Acute Ischemia-Reperfusion Heart Injury-Cardioprotective Properties of Carvedilol.

Matrix metalloproteinase 2 (MMP-2) is activated in hearts upon ischemia-reperfusion (IR) injury and cleaves sarcomeric proteins. It was shown that carvedilol and nebivolol reduced the activity of different MMPs. Hence, we hypothesized that they could reduce MMPs activation in myocytes, and therefore, protect against cardiac contractile dysfunction related with IR injury. Isolated rat hearts were subjected to either control aerobic perfusion or IR injury: 25 min of aerobic perfusion, followed by 20 min global, no-flow ischemia, and reperfusion for 30 min. The effects of carvedilol, nebivolol, or metoprolol were evaluated in hearts subjected to IR injury. Cardiac mechanical function and MMP-2 activity in the heart homogenates and coronary effluent were assessed along with troponin I content in the former. Only carvedilol improved the recovery of mechanical function at the end of reperfusion compared to IR injury hearts. IR injury induced the activation and release of MMP-2 into the coronary effluent during reperfusion. MMP-2 activity in the coronary effluent increased in the IR injury group and this was prevented by carvedilol. Troponin I levels decreased by 73% in IR hearts and this was abolished by carvedilol. Conclusions: These data suggest that the cardioprotective effect of carvedilol in myocardial IR injury may be mediated by inhibiting MMP-2 activation.

Association of Novel Advanced Glycation End-Product (AGE10) with Complications of Diabetes as Measured by Enzyme-Linked Immunosorbent Assay.

Advanced glycation end-products (AGEs) contribute to vascular complications and organ damage in diabetes. The unique AGE epitope (AGE10) has recently been identified in human serum using synthetic melibiose-derived AGE (MAGE). We aimed at developing ELISA for AGE10 quantification, determining whether AGE10 is present in diabetic patients (n = 82), and evaluating its association with diabetic complications. In a competitive ELISA developed, the reaction of synthetic MAGE with anti-MAGE was inhibited by physiological AGE10 present in serum. In this assay, new murine IgE anti-MAGE monoclonal antibodies, which do not recognize conventional AGEs, a synthetic MAGE used to coat the plate, and LMW-MAGE (low molecular mass MAGE) necessary to plot a standard curve were used. AGE10 was significantly higher in patients with microangiopathy, in whom it depended on treatment, being lower in patients treated with aspirin. AGE10 levels were positively correlated with estimated glomerular filtration rate (eGFR) and negatively with creatinine. As a marker of stage ≥3 chronic kidney disease or microangiopathy, AGE10 displayed moderate overall accuracy (respectively, 69% and 71%) and good sensitivity (82.6% and 83.3%) but poor specificity (58.1% and 57.8%). In conclusion, newly developed immunoassay allows for AGE10 quantification. AGE10 elevation is associated with microangiopathy while its decrease accompanies stage ≥3 chronic kidney disease.

Klebsiella pneumoniae enolase-like membrane protein interacts with human plasminogen.

Many models assessing the risk of sepsis utilize the knowledge of the constituents of the plasminogen system, as it is proven that some species of bacteria can activate plasminogen, as a result of interactions with bacterial outer membrane proteins. However, much is yet to be discovered about this interaction since there is little information regarding some bacterial species. This study is aimed to check if Klebsiella pneumoniae, one of the major factors of nosocomial pneumonia and a factor for severe sepsis, has the ability to bind to human plasminogen. The strain used in this study, PCM 2713, acted as a typical representative of the species. With use of various methods, including: electron microscopy, 2-dimensional electrophoresis, immunoblotting and peptide fragmentation fingerprinting, it is shown that Klebsiella pneumoniae binds to human plasminogen, among others, due to plasminogen-bacterial enolase-like protein interaction, occurring on the outer membrane of the bacterium. Moreover, the study reveals, that other proteins, such as: phosphoglucomutase, and phosphoenolpyruvate carboxykinase act as putative plasminogen-binding factors. These information may virtually act as a foundation for future studies investigating: the: pathogenicity of Klebsiella pneumoniae and means for prevention from the outcomes of Klebsiella-derived sepsis.

Influence of Water Polarization Caused by Phonon Resonance on Catalytic Activity of Enolase.

Enolase is a conservative protein. Its cellular enzymatic activity catalyzes the conversion of 2-phospho-d-glycerate (2-PGA) to a phosphoenolpyruvate (PEP) product in the glycolysis pathway. This enzyme also has a multifunctional nature participating in several biological processes. This work aims to determine the effect of water polarization on the catalytic activity of enolase. The experiments have been set based on the concept that water, a polar dielectric, may undergo the phenomenon of electric polarization, decreasing its configurational and vibrational entropy. Prior to the reaction, the 2-PGA substrate was incubated for 5 h in the glass cuvette with an attached chip-inductor. The latter device was designed to transfer quantum information about a given quantum state from the quantum state generator to water by a phonon resonance. Then, such substrate samples preincubated with the chip-inductor were removed every hour in a separate quartz cuvette with the enzyme to determine its catalytic activity. The influence of the chip-inductor on the preincubated substrate resulted in an increase in the catalytic activity of enolase by 30% compared to the control substrate, not preincubated with the chip-inductor. This suggests that the catalytic activity of the enzyme is augmented when the substrate was primed by chip-inductors. In another kind of experiment, wherein enolase was exposed to methylglyoxal modification, the catalytic activity of the enzyme dropped to 71.7%, while the same enzyme glycated with methylglyoxal primed by chip-inductors restored its activity by 8.4%. This shows the protective effect of chip-inductors on enolase activity despite the harmful effect of methylglyoxal on the protein.

The melibiose-derived glycation product mimics a unique epitope present in human and animal tissues.

Non-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

Salmonella Typhimurium enolase-like membrane protein is recognized by antibodies against human enolase and interacts with plasminogen.

BACKGROUND: Enolase is generally known as the glycolytic pathway enzyme present in the cytoplasm of eukaryotic cells and in some microorganisms. In human cells, it is also a component of cell surface membranes, where it functions as a human plasminogen receptor. OBJECTIVES: The study aimed to purify Salmonella enterica serovar Typhimurium cytosolic enolase and obtain the antibodies against this protein; to identify enolase on the surface of bacteria; and to find cross-reactivity and plasminogen binding properties. MATERIAL AND METHODS: Cytosolic enolase from S. Typhimurium was purified using a five-step preparation method. Anti-cytosolic enolase antibodies combined with scanning electron microscopy (SEM) allowed us to detect enolase on the surface of intact S. Typhimurium cells. The binding of plasminogen to surface enolase and the cross-reactivity of this protein with antibodies against human enolases were tested with western blot. RESULTS: Antibodies against human α- and ß-enolases cross-reacted with S. Typhimurium membrane protein, the identity of which was further confirmed using a mass spectrometry analysis of enolase tryptic peptides. The enolase form bacterial membrane also bound plasminogen. CONCLUSIONS: The cross-reactivity of membrane enolase with antibodies against human enolases suggests that this bacterium shares epitopes with human proteins. Surface exposition of enolase and the demonstrated affinity for human plasminogen indicates that Salmonella membrane enolase could play a role in the interaction of S. Typhimurium with host cells.

Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and ß-enolases.

Human α- and ß-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-ß-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (ß-enolase). Selective anti-α- and anti-ß-enolase antibodies were obtained by affinity chromatography on either α- or ß-enolase-Sepharose columns. On Western blots, antibodies directed against human ß-enolase, did not react with human α-isoenzyme, but recognized pig and rat ß-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and ß-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - S262PDDPSRYISPDQ273) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - N193VIKEKYGKDATN205) was recognized by anti-ß-enolase antibodies. Interestingly, neither anti-α- nor anti-ß-antibody reacted with a peptide corresponding to the epitope 2 in ß-enolase (G194VIKAKYGKDATN206). Further analysis showed that substitution of E197 with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A198 with E in peptide representing ß-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E197 is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native ß-enolase.

Polyelectrolyte Oil-Core Nanocarriers for Localized and Sustained Delivery of Daunorubicin to Colon Carcinoma MC38 Cells: The Case of Polysaccharide Multilayer Film in Relation to PEG-ylated Shell.

The authors examine properties of daunorubicin (DNR)-loaded oil-core multilayer nanocapsules prepared via layer-by-layer approach with different polyelectrolyte (PE) coatings such as a standard one (containing polysodium 4-styrenesulphonate/poly(diallyldimethyl-ammonium) chloride) and a polysaccharide-based shell (dextran/chitosan), in regard to the outer layer of poly-l-glutamic acid (PGA) grafted with polyethylene glycol (PGA-g-PEG). The nanocarriers are obtained on a cationic nanoemulsion template (stabilized by dicephalic-type surfactant, N,N-bis[3,30-(trimethylammonio)propyl]-dodecanamide dimethylsulfate) and layered with the PE shell of different thicknesses resulting in average size of 150 nm in diameter (as shown by dynamic light scattering, scanning electron microscopy and cryogenic-transmission electron microscopy, and atomic force microscopy). The nanocapsules demonstrate efficient DNR encapsulation and its sustained release under physiological conditions or in the attendance of human serum albumin. The biocompatibility studies using colon carcinoma MC38 and macrophage P388D1 cell lines as well as human erythrocytes reveal that surface charge and outer PE layer type determine nanocarrier features that control their biological activity: protein adsorption, cellular internalization and localization, induction of apoptosis, and hemolytic activity. The investigations indicate that polysaccharide-coated nanocapsules present a considerable potential for application as efficient DNR delivery systems in chemotherapy of colon cancer as an alternative to nanocarriers with PEG-ylated shell.

Preparation and characterization of new zinc(II) phthalocyanine - Containing poly(l-lactide)-b-poly(ethylene glycol) copolymer micelles for photodynamic therapy.

Poly(l-lactide)-b-poly(ethylene oxide) block copolymer (mPEG-b-PLLA) micelles were fabricated and applied as a new biodegradable and biocompatible nanocarrier for solubilization of hydrophobic zinc (II) phthalocyanine (ZnPc). The nanocarrier demonstrated a good colloidal stability and its in vitro sustained cargo release profile was assessed. Photobleaching of ZnPc, both in its native form and encapsulated in the obtained polymeric micelles, was studied by means of spectroscopic measurements. The photodynamic reaction (PDR) protocol for cyto- and photocytotoxicity was performed on metastatic melanoma cells (Me45), normal human keratinocytes (HaCaT) being used for comparison. The intracellular accumulation of free and encapsulated ZnPc was visualized at various time periods (1, 3 and 24h). The proapoptotic potential of the encapsulated phthalocyanine was evaluated by monitoring DNA double strand break damage fragmentation (TUNEL assay) and caspase 3/7 activity. In addition, in vitro biocompatibility studies were conducted by determining hemolytic activity of Zn-Pc-loaded mPEG-b-PLLA micelles and their lack of cytotoxicity against macrophages (P388/D1) and endothelial cells (HUV-EC-C). Our results suggest that the PDR using Zn-Pc-loaded mPEG-b-PLLA micelles can be effective in inhibiting tumor cell growth and apoptosis induction with higher responses, observed for Me45 cells. Additionally, the ZnPc-loaded micelles appear to be hemato-biocompatible and safe for normal keratinocytes, macrophages and endothelial cells.

In vitro studies of serum albumin interaction with poly(D,L-lactide) nanospheres loaded by hydrophobic cargo.

The various polymer-based nanocarriers are very attractive for in vitro and in vivo bioapplications. A new type of a promising drug delivery systems for cancer tissues-poly(D,L-lactide) nanospheres stabilized with Cremophor EL and loaded with hydrophobic cyanines (IR-780 or ZnPc) or curcumin (CUR) were fabricated by the nanoprecipitation method. The Cremophor EL/PLA/water nanospheres demonstrated regular shape, low polydispersity (PdI<0.3) and high entrapment efficiency of selected cargo (over 90%). The size of those nanoconstructs below 130 nm are in the desired nanocarriers size range for tumor delivery. Low level of in vitro drug release from loaded nanospheres after long-time storage indicates their good stability. The half-life of nanocarriers in the circulation, and their biodistribution after parenteral administration are associated with the ability of plasma proteins adsorption. For these reasons the affinity of obtained nanospheres for albumin as a major plasma protein was in vitro investigated. The binding of nanocarrier containing cyanine IR-780 with albumin immobilized in the wells of polystyrene plate occurred with lower efficiency than analogs loaded with ZnPc or CUR. Similar relationships were observed after UV-vis spectra analysis of nanospheres in the presence of albumin at various protein concentrations.

Biocompatible long-sustained release oil-core polyelectrolyte nanocarriers: From controlling physical state and stability to biological impact.

It has been generally expected that the most applicable drug delivery system (DDS) should be biodegradable, biocompatible and with incidental adverse effects. Among many micellar aggregates and their mediated polymeric systems, polyelectrolyte oil-core nanocarriers have been found to successfully encapsulate hydrophobic drugs in order to target cells and avoid drug degradation and toxicity as well as to improve drug efficacy, its stability, and better intracellular penetration. This paper reviews recent developments in the formation of polyelectrolyte oil-core nanocarriers by subsequent multilayer adsorption at micellar structures, their imaging, physical state and stability, drug encapsulation and applications, in vitro release profiles and in vitro biological evaluation (cellular uptake and internalization, biocompatibility). We summarize the recent results concerning polyelectrolyte/surfactant interactions at interfaces, fundamental to understand the mechanisms of formation of stable polyelectrolyte layered structures on liquid cores. The fabrication of emulsion droplets stabilized by synergetic surfactant/polyelectrolyte complexes, properties, and potential applications of each type of polyelectrolyte oil-core nanocarriers, including stealth nanocapsules with pegylated shell, are discussed and evaluated.

Polymeric micelles for enhanced Photofrin II ® delivery, cytotoxicity and pro-apoptotic activity in human breast and ovarian cancer cells.

BACKGROUND: Searching for photodynamic therapy (PDT) - effective nanocarriers which enable a photosensitizer to be selectively delivered to tumor cells with enhanced bioavailability and diminished dark cytotoxicity is of current interest. The main objective of this study is to evaluate newly designed mixed polymeric micelles based on Pluronics P123 and F127 for the improved delivery of Photofrin II(®) (Ph II(®)) to circumvent unfavorable effects overcoming multidrug resistance (MDR) in tumor cells - in breast MCF-7/WT (caspase-3 deficient) and ovarian SKOV-3 (resistant to chemotherapy). METHODS: Ph II(®)-loaded micelles were obtained and analyzed for size and morphology, solubilization efficiency, physical stability and in vitro drug release. Intracellular uptake, reactive oxygen species (ROS) generation, mitochondrial oxidoreductive potential and proapoptotic activity (TUNEL assay) studies were evaluated in the examined cancer cells. The preliminary biocompatibility characteristics of all nanocarriers was determined by assessment of their hemolytic activity in human erythrocytes and dark toxicity in cancer cells. RESULTS: Dynamic light scattering (DLS) and atomic force microscopy (AFM) confirmed that almost monodisperse, sphere-shaped and nanosized (DH<20 nm) carriers were developed. Biological studies after photodynamic reaction (PDR) with encapsulated Ph II(®) revealed increased ROS level, malondialdehyde (MDA) concentration and protein damage in SKOV-3 and MCF-7/WT cells in comparison to treatment with free Ph II(®). Numerous apoptotic cells were detected after nano-therapy in both cell lines, with observed significant morphological disorders in ovarian cancer cells. In the case of encapsulated Ph II(®) only negligible disruption of human erythrocytes and cancer cells was observed. CONCLUSIONS: The obtained biocompatible long-lasting nanocarriers significantly enhance the Photofrin II(®) photodynamic effect and apoptosis in both SKOV-3 and MCF-7/WT cell lines.

Enolase-like protein present on the outer membrane of Pseudomonas aeruginosa binds plasminogen.

Pseudomonas aeruginosa is one of the pathogenic bacteria which utilize binding of the host plasminogen (Plg) to promote their invasion throughout the host tissues. In the present study, we confirmed that P. aeruginosa exhibits binding affinity for human plasminogen. Furthermore, we showed that the protein detected on the cell wall of P. aeruginosa and binding human plasminogen is an enolase-like protein. The hypothesis that alpha-enolase, a cytoplasmatic glycolytic enzyme, resides also on the cell surface of the bacterium was supported by electron microscopy analysis. The plasminogen-binding activity of bacterial cell wall outer membrane enolase-like protein was examined by immunoblotting assay.

Studies on the O-antigen of Hafnia alvei PCM 1224 structurally and serologically related to the O-antigen of H. alvei 481-L.

Mild hydrolysis of the lipopolysaccharide of Hafnia alvei PCM 1224 at pH 4.2 cleaved partially the O-polysaccharide chain by the glycosyl phosphate linkages to yield a phosphorylated hexasaccharide representing the repeating unit of the O-polysaccharide and higher oligosaccharides consisting of two or more repeating units. Studies of the degradation products before and after dephosphorylation by sugar and methylation analyses along with 1D and 2D (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, (1)H,(13)C HSQC, HSQC-TOCSY, and (1)H,(31)P HMBC experiments, enabled elucidation of the following structure of the O-polysaccharide: [formula - see text]. This structure is similar to that of the O-polysaccharide of H. alvei 481-L established earlier (Kubler-Kielb, J.; Vinogradov, E.; Garcia Fernandez, J. M.; Szostko, B.; Zwiefka, A.; Gamian, A. Carbohydr. Res.2006, 341, 2980-2985), which has the same sugar composition and the same main chain structure and differs in the site of attachment of α-d-Glcp only. A two-way serological cross-reactivity of the lipopolysaccharides of H. alvei PCM 1224 and 481-L with polyclonal rabbit antisera indicated the expediency of classification of both strains to the same O-serogroup.

Nanoemulsion-templated multilayer nanocapsules for cyanine-type photosensitizer delivery to human breast carcinoma cells.

There is great clinical interest in developing novel nanocarriers for hydrophobic cyanine dyes used as photosensitizing agents in photodynamic therapy (PDT). In the present study we have employed nanoemulsion-templated oil-core multilayer nanocapsules as robust nanocarriers for a cyanine-type photosensitizer IR-786. These nanoproducts were fabricated via layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes (PEs), i.e., anionic PSS and cationic PDADMAC on nanoemulsion liquid cores created by dicephalic or bulky saccharide-derived cationic surfactants. All nanocapsules, with different thicknesses of the PE shell and average size <200 nm (measured by DLS) demonstrated good capacity for IR-786 encapsulation. The nanocarriers were visualized by SEM and AFM and their photo-induced anticancer effect and cellular internalization in human breast carcinoma MCF-7/WT cells were determined. Biological response of the cell culture, expressed as dark and photocytotoxicity as well as fluorescence of drug molecules loaded in the multilayer vehicles, analyzed by the FACS and CLSM techniques, have indicated that the delivered IR-786 did not aggregate inside the cells and could, therefore, act as an effective third-generation photosensitizing agent. In vitro biological experiments demonstrated that the properties of studied nanostructures depended upon the PE type and the envelope thickness as well as on the surfactant architecture in the nanoemulsion-based templates employed for the nanocapsule fabrication. Similarity of results obtained for stored (three weeks in the dark at room temperature) and freshly-prepared nanocapsules, attests to viability of this stable, promising drug delivery system for poorly water-soluble cyanines useful in PDT.

Photo-oxidative action in MCF-7 cancer cells induced by hydrophobic cyanines loaded in biodegradable microemulsion-templated nanocapsules.

Searching for photodynamic therapy-effective nanocarriers which enable a photosensitizer to be selectively delivered to tumor cells with enhanced bioavailability and diminished dark cytotoxicity is of current interest. We have employed a polymer-based nanoparticle approach to encapsulate the cyanine-type photosensitizer IR-780 in poly(n-butyl cyanoacrylate) (PBCA) nanocapsules. The latter were fabricated by interfacial polymerization in oil-in-water (o/w) microemulsions formed by dicephalic and gemini saccharide-derived surfactants. Nanocarriers were characterized by SEM, AFM and DLS. The efficiency of PBCA nanocapsules as a potential system of photosensitizer delivery to human breast cancer cells was established by dark and photocytotoxicity as the function of the cellular mitochondria. The photodynamic effect of cyanine IR-780 was determined by investigation of oxidative stress markers. The nanocapsules were the main focus of our studies to examine their cellular uptake and dark and photocytotoxicity as the function of the cellular mitochondria as well as oxidative stress markers (i.e., lipid peroxidation and protein damage) in MCF-7/WT cancer cells. The effects of encapsulated IR-780 were compared with those of native photosensitizer. The penetration of the nanocapsules into cancer cells was visualized by CLSM and their uptake was estimated by FACS analysis. Cyanine IR-780 delivered in PBCA nanocapsules to MCF-7/WT cells retains its sensitivity upon photoirradiation and it is regularly distributed in the cell cytoplasm. The intensity of the photosensitizer-generated oxidative stress depends on IR-780 release from the effective uptake of polymeric nanocapsules and seems to remain dependent upon the surfactant structure in o/w microemulsion-based templates applied to nanocapsule fabrication.

Synthesis of glycinated glycoconjugates based on 1-thioglycosides and their preliminary studies as potential immunomodulatory factor.

The biological importance of lipopolysaccharides (LPS), components of bacterial cell wall has not been explained sufficiently. The glycine present in these structures could play an important role in the immunological response after bacterial infections and during sepsis. In our studies we obtained synthetic and stable substituted glycinated 1-thioglycosides derivatives of monosaccharides, e.g., D-glucose or D-galactose as well as disaccharides, e.g., melibiose and lactose. The conditions of acylation reactions were validated and specific products were separated by using chromatography methods. Their structures were confirmed by NMR. These compounds were conjugated with carrier proteins e.g., bovine serum albumin and horse myoglobin. Prior to conjugation proteins were modified with glycidol to create the protein-diol intermediates and subsequent periodate oxidation of the glycol moieties to generate the reactive aldehyde functionalities. Modified and formylated carrier proteins were conjugated with acylated thioglycosides in the presence of sodium cyanoborohydride. Subsequently, the products obtained were analyzed in SDS-PAGE and separated by using HW-55S gel-filtration chromatography. The immunoreactivity of selected glycinated glycoconjugates were studied in ELISA assays with specific anti-aminoacylated glyconjugate antibodies obtained after rabbit immunization with Escherichia coli K12 C600 core oligosaccharide glycine-containing glycoconjugate. The differences in the immunoreactivity of different glycinated 1-thioglycosides were observed. The received glycine-acylated glycoconjugates could mimic the non-sugar substituents localized in various bacterial LPS. These synthetic compounds could be candidates for their use as glycoconjugate vaccines in protection against serious bacterial infections, e.g.. sepsis.

O-aminoacylation of bacterial glycoconjugates: from native structure to vaccine design.

The aminoacylation of bacterial polysaccharide antigens and its biological role are poorly understood, although it might be relevant in infection and immunity. Due to the lability of ester-linked substituents on glycoconjugate antigens, such groups usually escape detection during routine structural investigation. Of the few data available, those on the occurrence of glycine in the endotoxic lipopolysaccharides of Gram-negative bacteria are well documented. This article summarizes these data on glycine as an integral constituent of bacterial LPS and also some other amino-acid esters in the teichoic acids and phosphatidylglycerol of Gram-positive bacteria. The possible functions of such noncarbohydrate ester-linked substituents in bacterial antigens are discussed. Because glycine, an inherent component of bacterial lipopolysaccharides in the core region, is supposed to participate in epitope formation, such a structure may be considered for potential use in the construction of a vaccine with broad specificity.

Glycation of the muscle-specific enolase by reactive carbonyls: effect of temperature and the protection role of carnosine, pyridoxamine and phosphatidylserine.

Reactive carbonyls such as 4-hydroxy-2-nonenal (4-HNE), trans-2-nonenal (T2 N), acrolein (ACR) can react readily with nucleophilic protein sites forming of advanced glycation end-products (AGE). In this study, the human and pig muscle-specific enolase was used as a protein model for in vitro modification by 4-HNE, T2 N and ACR. While the human enolase interaction with reactive α-oxoaldehyde methylglyoxal (MOG) was demonstrated previously, the effect of 4-HNE, T2N and ACR has not been identified yet. Altering in catalytic function were observed after the enzyme incubation with these active compounds for 1-24 h at 25, 37 and 45 °C. The inhibition degree of enolase activity occurred in following order: 4-HNE > ACR > MOG > T2N and inactivation of pig muscle-specific enolase was more effective relatively to human enzyme. The efficiency of AGE formation depends on time and incubation temperature with glycating agent. More amounts of insoluble AGE were formed at 45 °C. We found that pyridoxamine and natural dipeptide carnosine counteracted AGE formation and protected enolase against the total loss of catalytic activity. Moreover, we demonstrated for the first time that phosphatidylserine may significantly protect enolase against decrease of catalytic activity in spite of AGE production.