Search Results (13)

Dietary intervention is the basis for the treatment of diabetes mellitus. This study employed Ganoderma lucidum (GL) mycelium to ferment a compound medium of oat and purple potato (OPP), optimized fermentation conditions to increase the triterpene content in the resulting product (F-OPPF), and systematically investigated the impact of fermentation on the nutritional quality, structural characteristics, and functional properties of OPP. The results indicated that the triterpene content in F-OPPF significantly increased from 8.53 mg/g to 17.23 mg/g under optimal conditions (temperature: 28 °C, inoculum size: 10%, material quantity: 36 g/250 mL, and fermentation time: day 13). Fermentation resulted in enhanced nutritional quality, with increased contents of protein, soluble protein, crude fiber, ash, mineral elements, essential amino acids, polysaccharides, flavonoids, and total phenols. Mycelium not only enveloped the OPP surface but also penetrated its interior, forming a porous honeycomb-like structure. The types of reactive groups and crystals (C + V-type) were not changed after fermentation, while the crystallinity increased. F-OPPF exhibited positive changes in thermogravimetric properties, antioxidant and hypoglycemic activities, and adsorption capacity of insoluble dietary fiber. Additionally, incorporating F-OPPF into the diet markedly reduced fasting blood glucose levels and promoted weight gain in T2DM rats induced by a high-fat diet and streptozotocin. The fermented groups exhibited improvements in glyco- and lipo-metabolism, oxidative stress, and the function and pathological morphology of the pancreas, liver, and kidneys compared to the unfermented group. Collectively, these findings suggested that GL mycelium fermentation enhanced the nutritional and functional values of OPP, and F-OPPF holds potential as a raw material for developing diabetic-friendly foods. Full article

Background: Studies have shown that the chronic use of cannabis is associated with a decrease in blood pressure. Our previous studies prove that activating the cannabinoid type 2 (CB2) receptor in the brain can effectively reduce blood pressure in spontaneously hypertensive rats; however, the exact mechanism has not been clarified. The objective of this study is to demonstrate that activation of microglial CB2 receptors can effectively reduce the levels of TNF-α, IL-1β, and IL-6 in the paraventricular nucleus (PVN) through inhibiting aerobic glycolysis, thereby relieving hypertension. Methods: AngiotensinII (AngII) was administered to BV2 cells and C57 mice to induce hypertension and the release of proinflammatory cytokines. The mRNA and protein expression of the CB2 receptor, TNF-α, IL-1β, IL-6, and the PFK and LDHa enzymes were detected using RT-qPCR and Western blotting. The Seahorse XF Energy Metabolism Analyzer was used to measure the oxidative phosphorylation and aerobic glycolysis metabolic pathways in BV2 cells. The long-term effects of injecting JWH133, a selective CB2 receptor agonist, intraperitoneally on blood pressure were ascertained. ELISA was used to measure norepinephrine and lactic acid levels while immunofluorescence labeling was used to locate the CB2 receptor and c-Fos. By injecting pAAV-F4/80-GFP-mir30shRNA (AAV2-r-CB2shRNA) into the lateral cerebral ventricle, the CB2 receptor in microglia was specifically knocked down. Results: Activation of CB2 receptors by the agonist JWH133 suppressed TNF-α, IL-1β, and IL-6 by inhibiting PFK and LDHa enzymes involved in glycolysis, as well as lactic acid accumulation, along with a reduction in glycoPER levels (marks of aerobic glycolysis) in AngII-treated BV2 cells. In AngII-treated mice, the administration of JWH133 specifically activated CB2 receptors on microglia, resulting in decreased expression levels of PFK, LDHa, TNF-α, IL-1β, and IL-6, subsequently leading to a decrease in c-Fos protein expression within PVN neurons as well as reduced norepinephrine levels in plasma, ultimately contributing to blood pressure reduction. Conclusion: The results suggest that activation of the microglia CB2 receptor decreases the neuroinflammation to relieve hypertension; the underlying mechanism is related to inhibiting aerobic glycolysis of microglia. Full article

Avastin^® is a humanized recombinant monoclonal antibody used to treat cancer by targeting VEGF-A to inhibit angiogenesis. SIMAB054, an Avastin^® biosimilar candidate developed in this study, showed a different charge variant profile than its innovator. Thus, it is fractionated into acidic, main, and basic isoforms and collected physically by Cation Exchange Chromatography (CEX) for a comprehensive structural and functional analysis. The innovator product, fractionated into the same species and collected by the same method, is used as a reference for comparative analysis. Ultra-Performance Liquid Chromatography (UPLC) ESI-QToF was used to analyze the modifications leading to charge heterogeneities at intact protein and peptide levels. The C-terminal lysine clipping and glycosylation profiles of the samples were monitored by intact mAb analysis. The post-translational modifications, including oxidation, deamidation, and N-terminal pyroglutamic acid formation, were determined by peptide mapping analysis in the selected signal peptides. The relative binding affinities of the fractionated charge isoforms against the antigen, VEGF-A, and the neonatal receptor, FcRn, were revealed by Surface Plasmon Resonance (SPR) studies. The results show that all CEX fractions from the innovator product and the SIMAB054 shared the same structural variants, albeit in different ratios. Common glycoforms and post-translational modifications were the same, but at different percentages for some samples. The dissimilarities were mostly originating from the presence of extra C-term Lysin residues, which are prone to enzymatic degradation in the body, and thus they were previously assessed as clinically irrelevant. Another critical finding was the presence of different glyco proteoforms in different charge species, such as increased galactosylation in the acidic and afucosylation in the basic species. SPR characterization of the isolated charge variants further confirmed that basic species found in the CEX analyses of the biosimilar candidate were also present in the innovator product, although at lower amounts. The charge variants’ in vitro antigen- and neonatal receptor-binding activities varied amongst the samples, which could be further investigated in vivo with a larger sample set to reveal the impact on the pharmacokinetics of drug candidates. Minor structural differences may explain antigen-binding differences in the isolated charge variants, which is a key parameter in a comparability exercise. Consequently, such a biosimilar candidate may not comply with high regulatory standards unless the binding differences observed are justified and demonstrated not to have any clinical impact. Full article

Three murine glioma cell lines (GL261, CT2A, and ALTS1C1) were modified to downregulate the expression of the murine LDH-A gene using shRNA, and compared to shRNA scrambled control (NC) cell lines. Differences in the expression of LDH-A and LDH-B mRNA, protein and enzymatic activity, as well as their LDH isoenzyme profiles, were observed in the six cell lines, and confirmed successful LDH-A KD. LDH-A KD (knock-down) resulted in metabolic changes in cells with a reduction in glycolysis (GlycoPER) and an increase in basal respiratory rate (mitoOCR). GL261 cells had a more limited ATP production capacity compared to CT2A and ALTS1C1 cells. An analysis of mRNA expression data indicated that: (i) GL261 LDH-A KD cells may have an improved ability to metabolize lactate into the TCA cycle; and (ii) that GL261 LDH-A KD cells can upregulate lipid metabolism/fatty acid oxidation pathways, whereas the other glioma cell lines do not have this capacity. These two observations suggest that GL261 LDH-A KD cells can develop/activate alternative metabolic pathways for enhanced survival in a nutrient-limited environment, and that specific nutrient limitations have a variable impact on tumor cell metabolism and proliferation. The phenotypic effects of LDH-A KD were compared to those in control (NC) cells and tumors. LDH-A KD prolonged the doubling time of GL261 cells in culture and prevented the formation of subcutaneous flank tumors in immune-competent C57BL/6 mice, whereas GL261 NC tumors had a prolonged growth delay in C57BL/6 mice. In nude mice, both LDH-A KD and NC GL261 tumors grew rapidly (more rapidly than GL261 NC tumors in C57BL/6 mice), demonstrating the impact of an intact immune system on GL261 tumor growth. No differences between NC and KD cell proliferation (in vitro) or tumor growth in C57BL/6 mice (doubling time) were observed for CT2A and ALTS1C1 cells and tumors, despite the small changes to their LDH isoenzyme profiles. These results suggest that GL261 glioma cells (but not CT2A and ALTS1C1 cells) are pre-programmed to have the capacity for activating different metabolic pathways with higher TCA cycle activity, and that this capacity is enhanced by LDH-A depletion. We observed that the combined impact of LDH-A depletion and the immune system had a significant impact on the growth of subcutaneous-located GL261 tumors. Full article

Primary biliary cholangitis (PBC) is a rare chronic cholestatic and immune-mediated liver disease of unknown aetiology that targets intrahepatic bile duct cells (cholangiocytes) and primarily affects postmenopausal women, when their estrogen levels sharply decrease. An impaired cholangiocyte response to estrogen characterizes the terminal stage of the disease, as this is when an inefficiency of cholangiocyte proliferation, in balancing the loss of intrahepatic bile ducts, is observed. Here, we report that the estrogen precursor dehydroepiandrosterone (DHEA) and its sulfate metabolites, DHEA-S and 17 β-estradiol, enhance the proliferation of cholangiocytes and hepatocytes in vitro. Flow cytometry analysis showed that DHEA and DHEA-S decreased glyco-chenodeoxycholic acid (GCDC)-driven apoptosis in cholangiocytes. Cell viability assay (MTT) indicated that ER-α, -β, and the G-protein-coupled estrogen receptor, are involved in the protection of DHEA against oxidative stress in cholangiocytes. Finally, immunoblot analysis showed an elevated level of steroid sulfatase and a reduced level of sulfotransferase 1E1 enzymes, involved in the desulfation/sulfation process of estrogens in cirrhotic PBC, and primary sclerosis cholangitis (PSC) liver tissues, another type of chronic cholestatic and immune-mediated liver disease. Taken together, these results suggest that DHEA can prevent the deleterious effects of certain potentially toxic bile acids and reactive oxygen species, delaying the onset of liver disease. Full article

Hyperglycemia, when sustained over a long time in diabetes mellitus (DM), leads to biochemical and cellular abnormalities, primarily through the formation of advanced glycation end-products (AGEs). In the treatment of diabetes, beside blood-sugar-lowering medications, a consumption of herbal products that can inhibit the AGEs’ formation is recommended. This study investigated the in vitro antiglycoxidative potential of extracts and fractions from the rhizomes of Japanese, Giant, and Bohemian knotweeds (Reynoutria japonica (Houtt.), R. sachalinensis (F. Schmidt) Nakai, and R.× bohemica Chrtek et Chrtkova). Their effects on glycooxidation of bovine and human serum albumin were evaluated by incubation of the proteins with a mixture of glucose and fructose (0.5 M) and 150 µg/mL of extract for 28 days at 37 °C, followed by measuring early and late glycation products, albumin oxidation (carbonyl and free thiol groups), and amyloid-β aggregation (thioflavin T and Congo red assays). The highest antiglycoxidative activity, comparable or stronger than the reference drug (aminoguanidine), was observed for ethyl acetate and diethyl ether fractions, enriched in polyphenols (stilbenes, phenylpropanoid disaccharide esters, and free and oligomeric flavan-3-ols). In conclusion, the antiglycoxidative compounds from these three species should be further studied for potential use in the prevention and complementary treatment of DM. Full article

Understanding the molecular basis of the disease is of the utmost scientific interest as it contributes to the development of targeted strategies of prevention, diagnosis, and therapy. Protein carbonylation is a typical feature of glyco-oxidative stress and takes place in health disorders such as diabetes. Allysine as well as its oxidation product, the α-amino adipic acid (α-AA) have been found to be markers of diabetes risk whereas little is known about the chemistry involved in its formation under hyperglycemic conditions. To provide insight into this issue, human serum albumin was incubated in the presence of FeCl3 (25 μM) and increasing glucose concentrations for 32 h at 37 °C. These concentrations were selected to simulate (i) physiological fasting plasma concentration (4 mM), (ii) pathological pre-diabetes fasting plasma concentration (8 mM), and pathological diabetes fasting plasma concentration (12 mM) of glucose. While both allysine and α-AA were found to increase with increasing glucose concentrations, the carboxylic acid was only detected at pathological glucose concentrations and appeared to be a more reliable indicator of glyco-oxidative stress. The underlying chemical mechanisms of lysine glycation as well as of the depletion of tryptophan and formation of fluorescent and colored advanced glycation products are discussed. Full article

The results of recent studies indicate the key role of nitrosative stress and protein oxidative damage in the development of morbid obesity. Nevertheless, the effect of bariatric surgery on protein oxidation/glycation and nitrosative/nitrative stress is not yet known. This is the first study evaluating protein glycoxidation and protein nitrosative damage in morbidly obese patients before and after (one, three, six and twelve months) laparoscopic sleeve gastrectomy. The study included 50 women with morbid obesity as well as 50 age- and gender-matched healthy controls. We demonstrated significant increases in serum myeloperoxidase, plasma glycooxidative products (dityrosine, kynurenine, N-formyl-kynurenine, amyloid, Amadori products, glycophore), protein oxidative damage (ischemia modified albumin) and nitrosative/nitrative stress (nitric oxide, peroxy-nitrite, S-nitrosothiols and nitro-tyrosine) in morbidly obese subjects as compared to lean controls, whereas plasma tryptophan and total thiols were statistically decreased. Bariatric surgery generally reduces the abnormalities in the glycoxidation of proteins and nitrosative/nitrative stress. Noteworthily, in the patients with metabolic syndrome (MS+), we showed no differences in most redox biomarkers, as compared to morbidly obese patients without MS (MS−). However, two markers: were able to differentiate MS+ and MS− with high specificity and sensitivity: peroxy-nitrite (>70%) and S-nitrosothiols (>60%). Further studies are required to confirm the diagnostic usefulness of such biomarkers. Full article

Meloxicam is a non-steroidal anti-inflammatory drug, which has a preferential inhibitory effect to cyclooxyganase-2 (COX-2). Although the drug inhibits prostaglandin synthesis, the exact mechanism of meloxicam is still unknown. This is the first study to assess the effect of meloxicam on protein glyco-oxidation as well as antioxidant activity. For this purpose, we used an in vitro model of oxidized bovine serum albumin (BSA). Glucose, fructose, ribose, glyoxal and methylglyoxal were used as glycating agents, while chloramine T was used as an oxidant. We evaluated the antioxidant properties of albumin (2,2-di-phenyl-1-picrylhydrazyl radical scavenging capacity, total antioxidant capacity and ferric reducing antioxidant power), the intensity of protein glycation (Amadori products, advanced glycation end products) and glyco-oxidation (dityrosine, kynurenine, N-formylkynurenine, tryptophan and amyloid-β) as well as the content of protein oxidation products (advanced oxidation protein products, carbonyl groups and thiol groups). We have demonstrated that meloxicam enhances the antioxidant properties of albumin and prevents the protein oxidation and glycation under the influence of various factors such as sugars, aldehydes and oxidants. Importantly, the antioxidant and anti-glycating activity is similar to that of routinely used antioxidants such as captopril, Trolox, reduced glutathione and lipoic acid as well as protein glycation inhibitors (aminoguanidine). Pleiotropic action of meloxicam may increase the effectiveness of anti-inflammatory treatment in diseases with oxidative stress etiology. Full article

This is the first study to assess, comprehensively, the oral health status; salivary glands’ function and enzymatic and non-enzymatic antioxidant defense; and oxidative damage to proteins and lipids in the non-stimulated (NWS) and stimulated (SWS) whole saliva of stroke patients. The study included 30 patients in the subacute phase of the stroke and an age and gender-matched control group. We showed that the activity of antioxidant enzymes (catalase and salivary peroxidase) was significantly higher in both NWS and SWS of stroke patients, similarly to uric acid concentration. However, in the study group, the reduced glutathione (GSH) concentration in SWS decreased. The contents of protein glycooxidation products (advanced glycation end products (AGE) and protein oxidation products (AOPP)) and lipid hydroperoxides were significantly higher in NWS and SWS of stroke patients. In the study group there was also a decrease in stimulated saliva secretion and total protein content. Interestingly, products of protein and lipid oxidation correlate negatively with SWS flow. The ROC analysis showed that salivary GSH with 100% specificity and 100% sensitivity differentiates the analyzed groups (AUC = 1.0). To sum up, in subacute stroke patients there are redox imbalances and oxidative damage to proteins and lipids in non-stimulated and stimulated saliva. Stroke patients also suffer from salivary gland dysfunction. Full article

This study is the first to evaluate protein glycooxidation products, lipid oxidative damage and nitrosative stress in non-stimulated (NWS) and stimulated whole saliva (SWS) of children with chronic kidney disease (CKD) divided into two subgroups: normal salivary secretion (n = 18) and hyposalivation (NWS flow < 0.2 mL min⁻¹; n = 12). Hyposalivation was observed in all patients with severe renal failure (4–5 stage CKD), while saliva secretion > 0.2 mL/min in children with mild-moderate CKD (1–3 stage) and controls. Salivary amylase activity and total protein content were significantly lower in CKD children with hyposalivation compared to CKD patients with normal saliva secretion and control group. The fluorescence of protein glycooxidation products (kynurenine, N-formylkynurenine, advanced glycation end products), the content of oxidative damage to lipids (4-hydroxynonneal, 8-isoprostanes) and nitrosative stress (peroxynitrite, nitrotyrosine) were significantly higher in NWS, SWS, and plasma of CKD children with hyposalivation compared to patients with normal salivary secretion and healthy controls. In CKD group, salivary oxidation products correlated negatively with salivary flow rate, α-amylase activity and total protein content; however, salivary oxidation products do not reflect their plasma level. In conclusion, children with CKD suffer from salivary gland dysfunction. Oxidation of salivary proteins and lipids increases with CKD progression and deterioration of salivary gland function. Full article

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure–function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels. Full article

The aim of this study was to evaluate the effect of a food supplement containing α-lipoic acid and of a placebo on glyco-metabolic control and on oxidative stress markers in type 2 diabetics. We randomized 105 diabetics to either a supplementation containing 600 mg of α-lipoic acid, 165 mg of L-carnosin, 7.5 mg of zinc, and vitamins of group B, or a placebo, for three months. We evaluated body mass index, fasting plasma glucose (FPG), post-prandial-glucose (PPG), glycated hemoglobin (HbA_1c), fasting plasma insulin (FPI), HOMA-index (HOMA-IR), lipid profile, high sensitivity C-reactive protein (Hs-CRP), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA). There was a reduction of FPG, PPG, and HbA_1c with the food supplement containing α-lipoic acid compared with a baseline, and with the placebo. Concerning lipid profile, we observed a reduction of LDL-C, and Tg with the food supplement, compared with both the baseline, and the placebo. There was a reduction of Hs-CRP with the food supplement containing α-lipoic acid, both compared with the baseline and the placebo. An increase of SOD, and GSH-Px, and a decrease of MDA were reached by the food supplement containing α-lipoic acid, both compared with the baseline and the placebo. We can conclude that the food supplement containing α-lipoic acid, L-carnosin, zinc, and vitamins of group B improved glycemic control, lipid profile, and anti-oxidative stress markers. Full article