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Due to their pivotal role in orchestrating the immune response, HLA loci were recognized as candidates for genetic association studies related to the severity of COVID-19. Since the findings on the effects of HLA alleles on the outcome of SARS-CoV-2 infection remain inconclusive, we aimed to elucidate the potential involvement of genetic variability within HLA loci in the molecular genetics of COVID-19 by classifying the articles according to different disease severity/outcomes and by conducting a systematic review with meta-analysis. Potentially eligible studies were identified by searching PubMed, Scopus and Web of Science literature databases. A total of 28 studies with 13,073 participants were included in qualitative synthesis, while the results of 19 studies with 10,551 SARS-CoV-2-positive participants were pooled in the meta-analysis. According to the results of quantitative data synthesis, association with COVID-19 severity or with the lethal outcome was determined for the following alleles and allele families: HLA-A*01, HLA-A*03, HLA-A*11, HLA-A*23, HLA-A*31, HLA-A*68, HLA-A*68:02, HLA-B*07:02, HLA-B*14, HLA-B*15, HLA-B*40:02, HLA-B*51:01, HLA-B*53, HLA-B*54, HLA-B*54:01, HLA-C*04, HLA-C*04:01, HLA-C*06, HLA-C*07:02, HLA-DRB1*11, HLA-DRB1*15, HLA-DQB1*03 and HLA-DQB1*06 (assuming either allelic or dominant genetic model). We conclude that alleles of HLA-A, -B, -C, -DRB1 and -DQB1 loci may represent potential biomarkers of COVID-19 severity and/or mortality, which needs to be confirmed in a larger set of studies.
Assuntos
COVID-19 , Antígenos HLA-C , Humanos , Antígenos HLA-C/genética , Alelos , Cadeias HLA-DRB1/genética , Haplótipos , COVID-19/diagnóstico , COVID-19/genética , SARS-CoV-2/genética , Antígenos HLA-A/genética , Antígenos HLA-B/genéticaRESUMO
BACKGROUND: We previously demonstrated that insulin-like growth factor-1 (IGF-1) regulates sodium/potassium adenosine triphosphatase (Na+/K+-ATPase) in vascular smooth muscle cells (VSMC) via phosphatidylinositol-3 kinase (PI3K). Taking into account that others' work show that IGF-1 activates the PI3K/protein kinase B (Akt) signaling pathway in many different cells, we here further questioned if the Akt/mammalian target of rapamycin (mTOR)/ribosomal protein p70 S6 kinase (S6K) pathway stimulates Na+/K+-ATPase, an essential protein for maintaining normal heart function. METHODS AND RESULTS: There were 14 adult male Wistar rats, half of whom received bolus injections of IGF-1 (50 µg/kg) for 24 h. We evaluated cardiac Na+/K+-ATPase expression, activity, and serum IGF-1 levels. Additionally, we examined the phosphorylated forms of the following proteins: insulin receptor substrate (IRS), phosphoinositide-dependent kinase-1 (PDK-1), Akt, mTOR, S6K, and α subunit of Na+/K+-ATPase. Additionally, the mRNA expression of the Na+/K+-ATPase α1 subunit was evaluated. Treatment with IGF-1 increases levels of serum IGF-1 and stimulates Na+/K+-ATPase activity, phosphorylation of α subunit of Na+/K+-ATPase on Ser23, and protein expression of α2 subunit. Furthermore, IGF-1 treatment increased phosphorylation of IRS-1 on Tyr1222, Akt on Ser473, PDK-1 on Ser241, mTOR on Ser2481 and Ser2448, and S6K on Thr421/Ser424. The concentration of IGF-1 in serum positively correlates with Na+/K+-ATPase activity and the phosphorylated form of mTOR (Ser2448), while Na+/K+-ATPase activity positively correlates with the phosphorylated form of IRS-1 (Tyr1222) and mTOR (Ser2448). CONCLUSION: These results indicate that the Akt/mTOR/S6K signalling pathway may be involved in the IGF-1 regulating cardiac Na+/K+-ATPase expression and activity.
Assuntos
Fator de Crescimento Insulin-Like I , Proteínas Proto-Oncogênicas c-akt , Animais , Masculino , Ratos , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases S6 RibossômicasRESUMO
Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes.
Assuntos
Falência Renal Crônica , Receptores da Transferrina , Transferrina , Humanos , Transferrina/metabolismo , Glicosilação , Falência Renal Crônica/terapia , Falência Renal Crônica/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Receptores da Transferrina/metabolismo , Diálise Peritoneal , Idoso , Adulto , Ferro/metabolismoRESUMO
Blue C-phycocyanin (C-PC), the major Spirulina protein with innumerable health-promoting benefits, is an attractive colourant and food supplement. A crucial obstacle to its more extensive use is its relatively low stability. This study aimed to screen various food-derived ligands for their ability to bind and stabilise C-PC, utilising spectroscopic techniques and molecular docking. Among twelve examined ligands, the protein fluorescence quenching revealed that only quercetin, coenzyme Q10 and resveratrol had a moderate affinity to C-PC (Ka of 2.2 to 3.7 × 105 M-1). Docking revealed these three ligands bind more strongly to the C-PC hexamer than the trimer, with the binding sites located at the interface of two (αß)3 trimers. UV/VIS absorption spectroscopy demonstrated the changes in the C-PC absorption spectra in a complex with quercetin and resveratrol compared to the spectra of free protein and ligands. Selected ligands did not affect the secondary structure content, but they induced changes in the tertiary protein structure in the CD study. A fluorescence-based thermal stability assay demonstrated quercetin and coenzyme Q10 increased the C-PC melting point by nearly 5 °C. Our study identified food-derived ligands that interact with C-PC and improve its thermal stability, indicating their potential as stabilising agents for C-PC in the food industry.
Assuntos
Proteína C , Spirulina , Animais , Ubiquinona , Antioxidantes/farmacologia , Ficocianina , Simulação de Acoplamento Molecular , Quercetina , Resveratrol/farmacologia , Aditivos Alimentares , Decapodiformes , Suplementos NutricionaisRESUMO
Metal ions seem to play important roles in the pathogenesis of the novel coronavirus disease of 2019 (Covid-19) and are under investigation as potential prognostic markers and supplements in therapeutic procedures. The present study was aimed at assessing the relationship between the most abundant essential microelements (iron, zinc and copper) and their major binding proteins in the circulation in the early stage of infection. The concentration of zinc ions was measured to be higher in infected than in healthy persons, as well as ratios zinc/albumin and zinc/alpha-2-macroglobulin. Increased zinc levels could be attributed to cellular redistribution of zinc ions or to a use of zinc supplementation (zinc concentration was above the upper reference limit in one-third of infected individuals). Immunoblot analysis of protein molecular forms revealed that infected persons had greater amounts of proteinase-bound alpha-2-macroglobulin tetramer and albumin monomer than healthy individuals. The quantities of these forms were correlated with the concentration of zinc ions (r = 0.42 and 0.55, respectively) in healthy persons, but correlations were lost in infected individuals, most likely due to very high zinc concentrations in some participants which were not proportionally followed by changes in the distribution of protein species. Although we still have to wait for a firm confirmation of the involvement of zinc in beneficial defense mechanisms in patients with Covid-19, it seems that this ion may contribute to the existence of circulating protein forms which are the most optimal.
Assuntos
COVID-19 , Proteínas de Transporte/genética , Oligoelementos , Cobre , Humanos , Ferro , SARS-CoV-2 , ZincoRESUMO
Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.
Assuntos
Neoplasias Colorretais/sangue , Neoplasias Colorretais/metabolismo , Lectinas/metabolismo , Análise Serial de Proteínas/métodos , alfa-Macroglobulinas/metabolismo , Idoso , Estudos de Casos e Controles , Feminino , Glicosilação , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The intention of this work was to systematically study the presence of insulin-like growth factor-binding protein 1 and 2 (IGFBP-1 and IGFBP-2) complexes with alpha-2-macroglobulin (α2M) in the circulation of patients with various types of cancer. Serum samples were collected from patients diagnosed with cancer and divided into eight groups according to the tumor type: liver, pancreas, colon, lung, prostate, breast, cervix and ovary. Results obtained for each cancer group were compared with results obtained for the age and gender-matched controls. Electrophoretic separation and densitometric evaluation of immunoreactive protein bands were performed. According to our results, IGFBP-1/α2M and IGFBP-2/α2M complexes did not exhibit the same presence in different tumors and were also not uniformly related to the amount of monomer forms. Variations in relative quantities of IGFBP-1 monomers and complexes in different tumor types were much more pronounced than those of IGFBP-2. The amount of IGFBP-2 monomer increased in sera from all studied patients compared to controls, whereas the amount of IGFBP-2/α2M complexes most often decreased, being significantly reduced in patients with pancreas, colon, breast or ovary tumor. Although there is still no confirmation of the physiological role of IGFBP-2/α2M complexes, their decreased concentration in the circulation provides greater amount of free IGFBP-2.
Assuntos
Biomarcadores Tumorais/sangue , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Complexos Multiproteicos/sangue , alfa-Macroglobulinas/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Immunoblotting , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Masculino , Pessoa de Meia-Idade , NeoplasiasRESUMO
The components of the insulin-like growth factor (IGF) system and molecules with which they interact are associated with the neoplastic transformation of cells in colorectal cancer. The IGF-binding protein-2 (IGFBP-2) plays a significant role in mitotic stimulation of the cancer cells and its concentration is significantly elevated in tumor states. Little is known about IGFBP-2 at the molecular level and the purpose of this study was to examine the interactions between IGFBP-2 and some other proteins, the fragmentation pattern and posttranslational modifications that might have occurred due to a disease. Results have shown that the amount of monomer IGFBP-2 was 20-30% greater in patients with cancer and the amount of fragmented IGFBP-2 was doubled compared to healthy people, whereas the portion of IGFBP-2 in complex with α2 macroglobulin (α2M) was 2.5 times lower in cancer patients. According to this distribution, IGFBP-2 was not only increasingly synthetized in patients with cancer, but also the amount involved in complexes with α2M was reduced favoring the existence of binary IGFBP-2/IGF complexes, free to leave the circulation. Both IGFBP-2 and α2M were significantly more oxidized in patients with colon cancer than in healthy individuals and α2M was additionally sialylated. It can be speculated that the formation of IGFBP-2/α2M complexes is part of the control mechanism involved in the regulation of IGFBP-2 and, consequently, IGF availability. It also seems that posttranslational modifications are more important factors in determining the amount of IGFBP-2/α2M complexes than the actual quantity of these two proteins.
Assuntos
Neoplasias Colorretais/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , alfa-Macroglobulinas/metabolismo , Idoso , Western Blotting , Estudos de Casos e Controles , Neoplasias Colorretais/patologia , Feminino , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Several studies on biomarker properties of microRNAs from liquid biopsy in prostate cancer (PCa) identified miR-146a-5p as a potential novel diagnostic marker. However, other studies with the same or similar topic failed to confirm the supposed discriminatory ability of miR-146a-5p, for which reason we aimed at elucidating the potential biomarker role of circulatory/urinary miR-146a-5p in PCa by conducting a qualitative and quantitative data synthesis. METHODS: Eligible articles were identified by searching PubMed, Scopus and Web of Science databases. Open MetaAnalyst software was used for pooling data on sensitivity, specificity, likelihood ratio and diagnostic odds ratio (OR) of miR-146a-5p. RESULTS: A total of 15 articles were eligible for qualitative data synthesis, while the results from 13 studies with 2080 participants were included in the meta-analysis. The established between-study heterogeneity was high, while the expression of hsa-miR-146a was associated with a diagnostic OR of 3.544 (P < 0.001; 95â¯%CI 2.186-5.747). Pooled sensitivity was found to be lower than 70â¯% (0.655, 95â¯%CI 0.573-0.729, P < 0.001), while the obtained value for specificity was 65â¯% (95â¯%CI 0.583-0.709, P < 0.001). Segregating studies according to ethnicity, sample type or the type of controls did not result in significantly higher sensitivity and specificity in subgroups, compared to the overall pooled data. CONCLUSIONS: The resulting pooled sensitivity, specificity and diagnostic OR do not qualify miR-146a-5p for a reliable diagnostic biomarker of PCa.
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Under simulated physiological conditions, this study investigates the interaction between nutraceutical phycocyanobilin (PCB) and the universal anti-protease protein human alpha-2-macroglobulin (α2M). Extensive molecular docking analyses on multiple α2M conformations, spectroscopic techniques, and α2M activity assays were utilized to examine the complex formation. The results revealed that for every protein conformation, two high energy binding sites exist: the first, conformationally independent, at the interface region between two monomer chains and the second, conformationally dependent, in the pocket composed of amino acids from four distinct domains (TED, RBD, CUB, and MG2) of the single protein chain. Spectrofluorimetric measurements indicated a moderate affinity between α2M and PCB with a moderately high binding constant of 6.3 × 105 M-1 at 25 °C. The binding of PCB to α2M resulted in minor changes in the secondary structure content of α2M. Furthermore, PCB protected α2M from oxidation and preserved its anti-protease activity in the oxidative environment. These findings suggest that PCB binding could indirectly impact the body's response to oxidative stress by influencing α2M's role in controlling enzyme activity during the inflammatory process.Communicated by Ramaswamy H. Sarma.
RESUMO
In previous publications, we pointed out the importance of mannosylation of fibrinogen for the development of cardiovascular complications and fucosylation as a predictor of peritoneal membrane dysfunction in patients on peritoneal dialysis (PD). After a follow-up period of 30 months from the onset of the COVID-19 pandemic, we evaluated the significance of 1,25-dihydroxyvitamin D3 (calcitriol) therapy, primary disease, biochemical and hematologic analyzes, and previously performed glycan analysis by lectin-based microarray as predictors of mortality in this patient group. After univariate Cox regression analysis, diabetes mellitus (DM) and calcitriol therapy were found to be potential predictors of mortality. Additional multivariate Cox regression analysis confirmed that only DM was a predictor of mortality. Nevertheless, the use of calcitriol in therapy significantly reduced mortality in this patient group, as shown by the Kaplan-Meier survival curve. The presence of DM as a concomitant disease proved to be a strong predictor of fatal outcome in PD patients infected with SARS-CoV-2. This is the first study to indicate the importance and beneficial effect of calcitriol therapy on survival in PD patients with COVID-19 infection. In addition, this study points to the possibility that adverse thrombogenic events observed in PD patients during the pandemic may be caused by aberrant fibrinogen glycosylation.
Assuntos
COVID-19 , Hemostáticos , Diálise Peritoneal , Humanos , Calcitriol , Pandemias , SARS-CoV-2 , Diálise Peritoneal/efeitos adversos , FibrinogênioRESUMO
Lectin-based protein microarrays are used for glycoprofiling of various kinds of biological samples. Here we describe lectin-based microarray assay in the reverse-phase format where glycoprotein samples are spotted onto microarray slide and then are incubated with set of lectins. This configuration allows high-throughput screening of a large cohort of samples by a set of lectins without need of separation of glycans from glycoproteins. We applied the described method for glycan analysis of glycoprotein biomarkers of colorectal cancer associated with the insulin-like growth factor system.
Assuntos
Neoplasias Colorretais , Somatomedinas , Biomarcadores/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Glicosilação , Humanos , Lectinas/metabolismo , Análise em Microsséries/métodos , Polissacarídeos/análise , Análise Serial de Proteínas/métodos , Somatomedinas/metabolismoRESUMO
Insulin-like growth factors (IGFs) are peptides which exert mitogenic, endocrine and cytokine activities. Together with their receptors, binding proteins and associated molecules, they participate in numerous pathophysiological processes, including cancer development. Colorectal cancer (CRC) is a disease with high incidence and mortality rates worldwide, whose etiology usually represents a combination of the environmental and genetic factors. IGFs are most often increased in CRC, enabling excessive autocrine/paracrine stimulation of the cell growth. Overexpression or increased activation/accessibility of IGF receptors is a coinciding step which transmits IGF-related signals. A number of molecules and biochemical mechanisms exert modulatory effects shaping the final outcome of the IGF-stimulated processes, frequently leading to neoplastic transformation in the case of irreparable disbalance. The IGF system and related molecules and pathways which participate in the development of CRC are the focus of this review.
RESUMO
Genes involved in the regulation of viral recognition and its entry into a host cell have been identified as candidates for genetic association studies on COVID-19 severity. Published findings on the effects of polymorphisms within ACE1, ACE2, TMPRSS2, IFITM3 and VDR genes remained inconclusive, so we conducted a systematic review and meta-analysis in order to elucidate their potential involvement in the genetic basis underlying the severity of COVID-19 and/or an outcome of SARS-CoV-2 infection. Identification of potentially eligible studies was based on PubMed, Scopus and Web of Science database search. Relevant studies (n=29) with a total number of 8247 SARS-CoV-2-positive participants were included in qualitative synthesis, while results of 21 studies involving 5939 were pooled in meta-analysis. Minor allele I of rs1799752 located within ACE1 was identified as a protective variant against severe COVID-19, while its effect on mortality rate was opposite. Similarly, minor allele A of ACE2 polymorphism, rs2285666, was found to associate with a decreased risk of severe COVID-19 (P = 0.003, OR = 0.512, 95 % CI = 0.331-0.793). Statistical significance was also seen for the association between COVID-19 severity and rs12329760 located within TMPRSS2. Our results did not support the supposed association of rs12252 in IFITM3 and polymorphisms within VDR with disease severity. We conclude that genetic variants within ACE1, ACE2 and TMPRSS2 may be potential biomarkers of COVID-19 severity, which needs to be further confirmed in a larger set of studies.
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Albumin (HSA) is a multifunctional protein and due to its free Cys34 thiol group, represents a main source of free thiols in the circulation. This property of HSA, combined with its ability to sequester redox active Cu(II) ions, makes HSA a dominant circulatory antioxidant. End stage kidney disease (ESRD) is a condition accompanied by elevated oxidative stress. The aim of the present study was to examine changes in the antioxidative capacity of HSA and Cu(II) binding affinity in patients on peritoneal dialysis (PD), and relate it to the Cys34 thiol group content and other structural changes of this molecule. HSA molecules are modified in ESRD patients subjected to PD, having significantly lower thiol group and bound Cu(II) content, reduced antioxidant capacity, an increased content of advanced glycation end-products and altered conformation. Also, Cu(II) binding capacity of HSA in these patients is impaired, since a significant portion of the high-affinity metal-binding site is unable to interact with Cu(II). Taking into account that the concentration of Cu(II) in the circulation of ESRD patients is much higher than in healthy persons and that Cu(II) binding capacity of HSA in these patients is significantly impaired, HSA may be considered as a novel circulatory pro-oxidant, thus exacerbating oxidative stress.
Assuntos
Falência Renal Crônica , Diálise Peritoneal , Antioxidantes/metabolismo , Humanos , Falência Renal Crônica/terapia , Espécies Reativas de Oxigênio , Albumina Sérica/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
In this study, the interaction between clozapine, an atypical antipsychotic drug, and alpha-2-macroglobulin (α2M), a multipurpose anti-proteinase, was investigated under simulated (patho) physiological conditions using multiple spectroscopic techniques and molecular modeling. It was found that α2M binds clozapine with a moderate affinity (the binding constant of 0.9 × 105 M-1 at 37 °C). The preferable binding site for both clozapine's atropisomers was revealed to be a large pocket at the interface of C and D monomer subunits of the protein. Hydrogen bonds and the hydrophobic effect were proposed as dominant forces in complex formation. The binding of clozapine did not induce significant conformational change of the protein, as confirmed by virtually unaltered α2M secondary structure and anti-proteinase activity. However, both clozapine and α2M shielded each other from the deleterious influence of strong oxidants: sodium hypochlorite and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). Moreover, clozapine in a concentration range that is usually targeted in the plasma during patients' treatment effectively protected the anti-proteinase activity of α2M under AAPH-induced free radical overproduction. Our results suggest that the cooperation between α2M and clozapine may be a path by which these two molecules synergistically protect neural tissue against injury caused by disturbed proteostasis or oxidative stress.
Assuntos
Antipsicóticos/metabolismo , Clozapina/metabolismo , Estresse Oxidativo , alfa-Macroglobulinas/metabolismo , Antipsicóticos/química , Sítios de Ligação , Clozapina/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Oxirredução , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , alfa-Macroglobulinas/químicaRESUMO
Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.
Assuntos
Colo/patologia , Neoplasias Colorretais/metabolismo , Lectinas/metabolismo , Análise Serial de Proteínas , Receptores de Somatomedina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Polissacarídeos/metabolismoRESUMO
OBJECTIVE: Metabolic changes in type 1 diabetes mellitus (T1DM) impair vasodilation, and this leads to tissue hypoxia and microvascular pathology. Hyperbaric oxygen therapy (HBOT) can significantly improve the outcome of ischemic conditions in T1DM patients and reduce vascular complications. The aim of our study was to assess the effects of HBOT on plasma fatty acid (FA) composition, and expression of insulin-like growth factor binding protein 1 (IGFBP-1) in T1DM patients. METHODS: Our study included 24 adult T1DM patients diagnosed with peripheral vascular complications. The patients were exposed to 10 sessions of 100% oxygen inhalation at 2.4 atmosphere absolute for 1 hour. Blood samples were collected at admission and after HBOT for measurement of metabolic parameters, FA composition and IGFBP-1. Measurement of plasma FA composition was determined by gas chromatography. Expression of IGFBP-1 in the serum was estimated by Western blot analysis. RESULTS: HBOT decreased blood levels of total cholesterol (p<0.05), triglycerides (p<0.05) and low-density lipoprotein (p<0.05). HBOT increased plasma levels of individual FAs: palmitic acid (p<0.05), palmitoleic acid (p<0.05), docosapentaenoic acid (p<0.05) and docosahexaenoic acid (p<0.01), and decreased levels of stearic acid (p<0.05), alpha linolenic acid (p<0.05) and linoleic acid (p<0.01). Expression of IGFBP-1 (p<0.01) was increased, whereas the level of insulin (p<0.001) was decreased in the serum after HBOT. CONCLUSIONS: Our results indicate that HBOT exerts beneficial effects in T1DM patients by improving the lipid profile and altering FA composition.
Assuntos
Biomarcadores/sangue , Complicações do Diabetes/sangue , Diabetes Mellitus Tipo 1/sangue , Ácidos Graxos/sangue , Oxigenoterapia Hiperbárica/métodos , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Doenças Vasculares Periféricas/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Complicações do Diabetes/etiologia , Complicações do Diabetes/terapia , Diabetes Mellitus Tipo 1/complicações , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Vasculares Periféricas/etiologia , Doenças Vasculares Periféricas/terapia , Projetos Piloto , Prognóstico , Estudos ProspectivosRESUMO
Glycosylation may strongly affect protein structure and functions. A high risk of cardiovascular complications seen in patients with end-stage renal disease (ESRD) is, at least partly associated with delayed clot formation, increased clot strength, and delayed cloth lysis. Taking into consideration that fibrinogen mediates these processes, we isolated fibrinogen from the plasma from patients with ESRD on peritoneal dialysis (ESRD-PD), and examined glycosylation of native fibrinogen and its subunits by lectin-based microarray and lectin blotting. Compared to healthy controls, fibrinogen from patients had increased levels of A2BG2 and decreased levels of FA2 glycan. The distribution of glycans on individual chains was also affected, with the γ chain, responsible for physiological functions of fibrinogen (such as coagulation and platelet aggregation), being most prone to these alterations. Increased levels of multi-antennary N-glycans in ESRD-PD patients were also associated with the type of dialysis solutions, whereas an increase in the fucosylation levels was strongly related to the peritoneal membrane damage. Consequently, investigation of fibrinogen glycans can offer better insight into fibrinogen-related complications observed in ESRD-PD patients and, additionally, contribute to prognosis, choice of personalised therapy, determination of peritoneal membrane damage, and the length of utilization of peritoneum for dialysis.
Assuntos
Fibrinogênio/química , Fibrinogênio/metabolismo , Fucose/metabolismo , Falência Renal Crônica/sangue , Diálise Peritoneal , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Feminino , Glicosilação , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Lectinas/sangue , Lectinas/química , Masculino , Pessoa de Meia-Idade , Polissacarídeos/sangue , Polissacarídeos/química , Polissacarídeos/metabolismo , Prognóstico , Análise Serial de ProteínasRESUMO
Alpha-2-macroglobulin (α2M) is a molecule generally associated with inflammation, and chronic inflammation is associated with ageing and cancer. The degree of inflammation was recently proposed to be considered as a biomarker of biological ageing. In this study, glycans attached to α2M were analysed in a human population of different ages by lectin-based protein microarray. Higher reactivity of α2M with several lectins was detected in older individuals indicating an increased content of specific monosaccharides: α2,6 sialic acid, mannose and N-acetylglucosamine, and multiantennary complex type N-glycans. The increased glycosylation of α2M was accompanied by reduced binding of Zn ions and insulin-like growth factor-binding protein 2 (IGFBP-2). Glycosylation of α2M and its reactivity with IGFBP-2 is similarly affected by ageing and incidence of colon cancer, but the reactivity of α2M with Zn ions is differently affected, as the binding of Zn ions remains unaltered in patients with colon cancer compared to healthy middle-aged individuals. Thus, the binding of IGFBP-2 to α2M seems to be related to structural changes in the glycan moieties of α2M, whereas binding of Zn ions, most likely, is not.