Notch信号通路——对健康和疾病的机械论观点

The Notch signaling pathway is evolutionarily conserved across metazoan species and plays key roles in many physiological processes.The Notch receptor is activated by two families of canonical ligands(Deltalike and Serrate/Jagged)where both ligands and receptors are single-pass transmembrane proteins usually with large extracellular domains,relative to their intracellular portions.Upon interaction of the core binding regions,presented on opposing cell surfaces,formation of the receptor/ligand complex initiates force-mediated proteolysis,ultimately releasing the transcriptionally-active Notch intracellular domain.This review focuses on structural features of the extracellular receptor/ligand complex,the role of posttranslational modifications in tuning this complex,the contribution of the cell membrane to ligand function,and insights from acquired and genetic diseases.

Serum tumor markers expression(CA199,CA242,and CEA)and its clinical implications in type 2 diabetes mellitus

BACKGROUND Glucose and lipid metabolic disorder in patients with type 2 diabetes mellitus(T2DM)is associated with the levels of serum tumor markers of the digestive tract,such as cancer antigen(CA)199.Therefore,tumor markers in T2DM are important.AIM To evaluate the expression of serum tumor markers[CA199,CA242,and carcinoembryonic antigen(CEA)]and the clinical implications of the expression in T2DM.METHODS For this observational study conducted at Hefei BOE Hospital,China,we enrolled 82 patients with first-onset T2DM and 51 controls between April 2019 and December 2020.Levels of fasting blood glucose(FBG),tumor markers(CA199,CEA,and CA242),glycosylated hemoglobin(HbA1c),etc.were measured and group index levels were compared.Moreover,FBG and HbA1c levels were correlated with tumor marker levels.Tumor markers were tested for diagnostic accuracy in patients with>9%HbA1c using the receiver operating curve(ROC)curve.RESULTS The T2DM group had high serum FBG,HbA1c,CA199,and CEA levels(P<0.05).A comparative analysis of the two groups based on HbA1c levels(Group A:HbA1c≤9%;Group B:HbA1c>9%)revealed significant differences in CEA and CA199 levels(P<0.05).The areas under the ROC curve for CEA and CA199 were 0.853 and 0.809,respectively.CA199,CEA,and CA242 levels positively correlated with HbA1c(r=0.308,0.426,and 0.551,respectively)and FBG levels(r=0.236,0.231,and 0.298,respectively).CONCLUSION As compared to controls,serum CEA and CA199 levels were higher in patients with T2DM.HbA1c and FBG levels correlated with CA199,CEA,and CA242 levels.Patients with poorly controlled blood sugar must be screened for tumor markers.

Insights into the history and tendency of glycosylation and digestive system tumor:A bibliometric-based visual analysis

BACKGROUND Glycosylation,a commonly occurring post-translational modification,is highly expressed in several tumors,specifically in those of the digestive system,and plays a role in various cellular pathophysiological mechanisms.Although the importance and detection methods of glycosylation in digestive system tumors have garnered increasing attention in recent years,bibliometric analysis of this field remains scarce.The present study aims to identify the developmental trends and research hotspots of glycosylation in digestive system tumors.AIM To find and identify the developmental trends and research hotspots of glycosylation in digestive system tumors.METHODS We obtained relevant literature from the Web of Science Core Collection and employed VOSviewer 1.6.19 and CiteSpace(version 6.1.R6)to perform bibliometric analysis.RESULTS A total of 2042 documents spanning from 1978 to the present were analyzed,with the research process divided into three phases:the period of obscurity(1978-1990),continuous development period(1991-2006),and the rapid outbreak period(2007-2023).These documents were authored by researchers from 66 countries or regions,with the United States and China leading in terms of publication output.Reis Celso A had the highest number of publications,while Pinho SS was the most cited author.Co-occurrence analysis revealed the most popular keywords in this field are glycosylation,expression,cancer,colorectal cancer,and pancreatic cancer.Furthermore,the Journal of Proteome Research was the most prolific journal in terms of publications,while the Journal of Biological Chemistry had the most citations.CONCLUSION The bibliometric analysis shows current research focus is primarily on basic research in this field.However,future research should aim to utilize glycosylation as a target for treating tumor patients.

Preparation,characterization and antioxidant activity analysis of three Maillard glycosylated bone collagen hydrolysates from chicken,porcine and bovine

Bone collagen hydrolysates(peptides)derived from byproduct of animal product processing have been used to produce commercially valuable products due to their potential antioxidant activity.Maillard glycosylated reaction is considered as a promising method to enhance the antioxidant activity of peptides.Hence,this research aims at investigating the Maillard glycosylation activity and antioxidant activity of bone collagen hydrolysates from different sources.In this study,3 glycosylated bone collagen hydrolysates were prepared and characterized,and cytotoxicity and antioxidant activity were analyzed and evaluated.The free amino groups loss,browning intensity,and fluorescence intensity of G-Cbcp(glycosylated chicken bone collagen hydrolysates(peptides))were the heaviest,followed by G-Pbcp(glycosylated porcine bone collagen hydrolysates(peptides))and G-Bbcp(glycosylated bovine bone collagen hydrolysates(peptides)).The results of amino acid analysis showed that amino acid composition of different bone collagen hydrolysates was significantly different and the amino acid decreased to different degrees after Maillard glycosylated reaction,which may lead to differences in Maillard glycosylated reaction activity.Furthermore,the 3 glycosylated hydrolysates showed no significant cytotoxicity.The results showed that glycosylation process significantly increased the antioxidant activity of bone collagen hydrolysates,and G-Cbcp showed the strongest antioxidant activity,followed by G-Pbcp and G-Bbcp.Therefore,compared with the bone collagen hydrolysates,3 glycosylated hydrolysates showed significant characteristic and structural changes,and higher antioxidant activity.

A MONOCLONAL ANTIBODY RECOGNIZING NON DERIVATIVE 13 HYDROXY GIBBERELLINS AND THEIR GLUCOSIDES *

The production and characterization of a monoclonal antibody (MAb AB10) against GA 3 glucoside as well as GA 3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA 3 at carbon 3. This antibody showed high affinity for GA 3 glucoside as well as for 13 hydroxy gibberellins (GA 1, GA 3, GA 5, etc). The affinity of MAb AB10 for 13 hydroxy GAs was significantly reduced by methylation of the 7 oic acid but not by glycosylation of 3 hydroxyl group. Based on this antibody, both of competitive enzyme linked immunosorbent assays (ELISAs) for GA 3 glucoside and for GA 3 were developed. These two ELISAs displayed linear detection ranges from 0 2 pmol to 20 pmol. Using these assays, the fluctuation of GA 3 like and GA 3 glucoside like substances in the leaves of Rumex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6 benzyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.

Use of Glucosyl 3,5-Dinitrobenzoate in Synthesis of Glucosides and Related Oligosaccharides

To investigate a new glycosylation method. Methods In the presence of TMSOTfas catalyst, 1-O-(3, 5-dinitrobenzoyl)-2, 3, 4, 6-tetra-O-benzyl-α-D-glucopyranose 1 reacted with aseries of carboxylic acid, phenols, alcohols and saccharides respectively to give the correspondingglycosylation products. The compounds were determined by ~1H NMR and ^(13)C NMR spectra. ResultsThe α-glu-co-pyranosides and related oligosaccharides were prepared in high yields. Conclusion The3, 5-dinitro-benzoyl group was found to be a good leaving group at the anomeric position andO-glucopyranosides and oligosaccharides were stereoselectively synthesized in good yield.

Clostridium clariflavum GH10木聚糖酶的克隆表达、酶学性质及位点功能分析

首次将来源于Clostridium clariflavum的木聚糖酶基因Clocl-2441中的糖苷水解酶家族10(glycosyl hydrolase families,GH)结构域基因连接到pET28a(+)载体上,在E.coli BL21(DE3)成功异源表达。经镍柱和脱盐分离纯化达到电泳纯,并对其酶学性质进行解析。结果表明,重组木聚糖酶rXyn2441GH10最适温度和pH分别为70℃和7.0,属于中性嗜热木聚糖酶。rXyn2441GH10在65℃以下和pH 4.0~9.0范围比较稳定,金属离子中5 mmol/L的Mg^(2+)可以提高79.2%的酶活。以榉木木聚糖为底物重组酶的动力学参数,V_(max)为1 691.5μmol/(mg·min),K_m值为2.5 mg/mL,k_(cat)为1 236.4/s,k_(cat)/K_m为494.6 mL/(mg·s)。定点饱和突变表明209位点的组氨酸对酶活有重要的作用。

Deglycosylation altered the gating properties of rNav1.3:glycosylation/deglycosylation homeostasis probably complicates the functional regulation of voltage-gated sodium channel

Objective To examine the effect of deglycosylation on gating properties of rNav1.3. Methods rNav1.3 was expressed in Xenopus oocyte, with glycosylation inhibition by using tunicamycin. Two-electrode voltage clamp was employed to record the whole-cell sodium current and data were analyzed by Origin software. Those of glycosylated rNav1.3 were kept as control. Results Compared with glycosylated ones, the steady-state activation curve of deglycosylated rNav1.3 was positively shifted by about 10 mV, while inactivation curve was negatively shifted by about 8 mV. Conclusion Glycosylation altered the gating properties of rNav 1.3 and contributed to the functional diversity.

Association of Dietary Pattern during Pregnancy and Gestational Diabetes Mellitus： A Prospective Cohort Study in Northern China

Objective To examine the association of maternal dietary patterns during pregnancy with gestationa diabetes mellitus （GDM） in northern China. Methods The dietary intakes of pregnant women were recorded twice by 24-hour dietary recalls for three days prior to having been diagnosed with GDM, at 5-15 and 24-28 gestational weeks, respectively. GDM was diagnosed, and serum glycosylated hemoglobin （HbAlc） was measured at 24-28 weeks. Dietary patterns were assessed by factor analysis. The association of the dietary pattern with GDM and HbAlc was examined by multiple logistic models. Results Of 753 participants, 64 （8.5%） were diagnosed with GDM. Four dietary patterns were identified： Western pattern （dairy, baked/fried food and white meat）, traditional pattern （light-colored vegetables, fine grain, red meat and tubers）, mixed pattern （edible fungi, shrimp/shellfish and red meat） and prudent pattern （dark-colored vegetables and deep-sea fish）. Compared with the prudent pattern, both the Western pattern and the traditional pattern were associated with an increased risk of GDM （aOR = 4.40, 95% CI： 1.58-12.22; aOR = 4.88, 95% Ch 1.79-13.32） and a high level of HbAlc （aOR = 12.37, 95% Ch 1.47-103.91; aOR = 26.23, 95% CI： 2.54-270.74）. Compared to the lowest quartile （Q）, 0.3 of the Western pattern scores and Q3-Q4 of the traditional pattern scores were associated with a higher risk of GDM. Conclusion The consumption of the Western pattern or the traditional pattern during pregnancy may increase the risk of GDM.

Recombinant Expression and Purification of Mouse Nectin-like 4 Glycoprotein in 293ET Cell Line

Objective To screen the transient and stable cell lines with high production of Nectin-like 4(Necl-4)protein.Methods First,c DNA sequences encoding the extracellular domain of Necls were cloned into the modified vector p APtag at the N terminus of alkaline phosphatase(AP)for fusion expression.Next,293ET cells stably expressed Necls-AP fusion protein and secreted it into the culture medium which were detected by the AP activity assay and Western blot analysis.Then,by adding N-glycosylation processing inhibitor kifunensine into the medium,complex glycan was inhibited to generate.The residual glycan of purified protein was removed by endoglycosidase H.Finally,AP protein was removed by using human rhinovirus protease and size exclusion chromatography.The concentration of purified Necl-4 protein was monitored by measuring the absorbance at280 nm and analyzed by SDS-PAGE.Results The transient and stable cell lines with high production of Necl-4 protein were screened by the color reaction with the AP-tag in the recombinant vector.The soluble and active form of purified Necl-4 protein was obtained after deglycosylation of native N-glycan protein with an expression level of 4 mg/L culture and purity of 95%.Conclusions By using modified AP mammalian protein expression system,we can easily screen the high productive stable cell lines by using AP activity assay.By adding mannosidase inhibitor kifunensine into the medium and cutting purified protein by using endoglycosidase H,we can obtain deglycosylated Necl-4 protein in milligram quantities.Our method might throw a light on the expression and purification of glycoprotein for structural and functional studies.

Dynamic Analyses of PrP and PrP^(Sc) in Brain Tissues of Golden Hamsters Infected With Scrapie Strain 263K Revealed Various PrP Forms

Objective To expatiate dynamic changes in hamsters infected with scrapie strain 263K, to observe the presence and aggravation of various forms of PrP and PrPSc during incubation period, and to probe primarily the relationship between the onset of clinic manifestations and the presence of different PrPSc forms. Methods Hamster-adapted scrapie strain 263K was intracerebrally inoculated into hamsters. Different forms of PrP and PrPSc were monitored dynamically by Western blot and immuno-histochemical assays. The presence of scrapie-associated fibril (SAF) was assayed by electron microscopy analysis (EM) and immune-golden EM. Results PrPSc was initially detected in the brain tissues of the animals in 20 days post-inoculation by immunohistochemistry and 40 days with Western blot. Quantitative evaluations revealed that the amounts of PrP and PrPSc in brain tissues increased along with the incubation. Several high and low molecular masses of PrP were seen in the brains of the long-life span infected animals. Deglycosylation assays identified that the truncated PrP in the infected brains showed similar glycosylation patterns as the full-length PrP. The presence of short fragments was seemed to relate with the onset of clinical conditions. Conclusion These results indicate that infectious agents exist and accumulate in central nerve system prior to the onset of the illness. Various molecular patterns of PrPSc may indwell in brain tissues during the infection.

Effect of icarisid II on diabetic rats with erectile dysfunction and its potential mechanism via assessment of AGEs, autophagy, roTOR and the NO-cGMP pathway

Erectile dysfunction （ED） is a major complication of diabetes mellitus. Icariin has been shown to enhance erectile function through its bioactive form, icarisid Ih This study investigates the effects of icarisid Ⅱ on diabetic rats with ED and its potential mechanism viathe assessment of advanced glycosylation end products （AGEs）, autophagy, mTOR and the NO-cGMP pathway. Icarisid Ⅱ was extracted from icariin by an enzymatic method. In the control and diabetic ED groups, rats were administered normal saline; in the icarisid Ⅱ group, rats were administered icarisid Ⅱ intragastrically. Erectile function was evaluated by measuring intracavernosal pressure/mean arterial pressure （ICP/MAP）. AGE concentrations, nitric oxide synthase （NOS） activity and cGMP concentration were assessed by enzyme immunoassay. Cell proliferation was analysed using methyl thiazolyl tetrazolium assay and flow cytometry. Autophagosomes were observed by transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 Iocalisation. The expression of NOS isoforms and key proteins in autophagy were examined by western blot. Our results have shown that Icarisid Ⅱ increased ICP/MAP values, the smooth muscle cell （SMC） growth curve, S phase and SMC/collagen fibril （SMC/CF） proportions and decreased Beclin 1 （P〈0.05）. Icarisid Ⅱ significantly increased the proliferative index and p-p70S6K（Thr389） levels and decreased the numbers of autophagosomes and the levels of LC3-11 （P〈0.01）. Icarisid Ⅱ decreased AGE concentrations and increased cGMP concentration, NOS activity （P〈0.05） and cNOS levels （P〈0.01） in the diabetic ED group. Therefore, Icarisid Ⅱ constitutes a promising compound for diabetic ED and might be involved in the upregulation of SMC proliferation and the NO-cGMP pathway and the downregulation of AGEs, autophagy and the mTOR pathway.

Recombinant human zona pellucida proteins ZP1, ZP2 and ZP3 co-expressed in a human cell line

Aim: To produce biologically active recombinant human (rh) ZP proteins in a human cell for use in sperm function tests. Methods: The human embryonic kidney cell line 293T was employed to produce rhZP1, rhZP2 and rhZP3 proteins individually and together by co-expression. Presence of these proteins in the culture medium and cell lysate was assessed by Western blotting analysis. The effect of the recombinant proteins on the human AR was assessed. Results: RhZP2 and rhZP3 were secreted into the culture medium, whereas rhZPl was found only in the cell lysate. Interestingly, when all zona pellucida proteins were co-expressed in the same cells, rhZPl was also secreted into the culture medium. However, despite the presence of all three ZP proteins in sufficient concentration and evidence of heavy glycosylation on gel electrophoresis, biological activity to induce the AR was not observed. Conclusion: RhZP1, rhZP2 and rhZP3 were successfully expressed in the human embryonic kidney cell line 293T. It appears that an interaction amongst these proteins may be required for release of rhZPl from the cell. Although this approach is not satisfactory for producing active human ZP proteins, it makes a significant contribution to the understanding of the structural and functional characteristics of the ZP proteins.

Relationship between diabetes and periodontal infection

Periodontal disease is a high prevalent disease.In the United States 47.2% of adults ≥ 30 years old have been diagnosed with some type of periodontitis.Longitudinal studies have demonstrated a two-way relationship between diabetes and periodontitis,with more severe periodontal tissue destruction in diabetic patients and poorer glycemic control in diabetic subjects with periodontal disease.Periodontal treatment can be successful in diabetic patients.Short term effects of periodontal treatment are similar in diabetic patients and healthy population but,more recurrence of periodontal disease can be expected in no well controlled diabetic individuals.However,effects of periodontitis and its treatment on diabetes metabolic control are not clearly defined and results of the studies remain controversial.

Post-translational modifications of prostaglandin-endoperoxide synthase 2 in colorectal cancer:An update

The biosynthesis of prostanoids is involved in both physiological and pathological processes. The expression of prostaglandin-endoperoxide synthase 2(PTGS2; also known as COX-2) has been traditionally associated to the onset of several pathologies, from inflammation to cardiovascular, gastrointestinal and oncologic events. For this reason, the search of selective PTGS2 inhibitors has been a focus for therapeutic interventions. In addition to the classic non-steroidal anti-inflammatory drugs, selective and specific PTGS2 inhibitors, termed coxibs, have been generated and widely used. PTGS2 activity is less restrictive in terms of substrate specificity than the homeostatic counterpart PTGS1, and it accounts for the elevated prostanoid synthesis that accompanies several pathologies. The main regulation of PTGS2 occurs at the transcription level. In addition to this, the stability of the mRNA is finely regulated through the interaction with several cytoplasmic elements, ranging from specificmicroR NAs to proteins that control mR NA degradation. Moreover, the protein has been recognized to be the substrate for several post-translational modifications that affect both the enzyme activity and the targeting for degradation via proteasomal and non-proteasomal mechanisms. Among these modifications, phosphorylation, glycosylation and covalent modifications by reactive lipidic intermediates and by free radicals associated to the proinflammatory condition appear to be the main changes. Identification of these post-translational modifications is relevant to better understand the role of PTGS2 in several pathologies and to establish a correct analysis of the potential function of this protein in diseases progress. Finally, these modifications can be used as biomarkers to establish correlations with other parameters, including the immunomodulation dependent on molecular pathological epidemiology determinants, which may provide a better frame for potential therapeutic interventions.

Serum angiotensin-converting enzyme level for evaluating significant fibrosis in chronic hepatitis B

AIM To evaluate the diagnostic performance of angiotensinconverting enzyme(ACE)on significant liver fibrosis in patients with chronic hepatitis B(CHB). METHODS In total,100 patients with CHB who underwent liver biopsy in our hospital were enrolled,and 70 patients except for 30 patients with hypertension,fatty liver or habitual alcoholic consumption were analyzed.We compared histological liver fibrosis and serum ACE levels and evaluated the predictive potential to diagnose significant liver fibrosis by comparison with several biochemical marker-based indexes such as the aspartate aminotransferase(AST)-to-platelet ratio index(APRI),the fibrosis index based on four factors(FIB-4),the Mac-2 binding protein glycosylation isomer(M2BPGi)level and the number of platelets(Plt). RESULTS Serum ACE levels showed moderately positive correlation with liver fibrotic stages(R2=0.181).Patients with significant,advanced fibrosis and cirrhosis(F2-4)had significantly higher serum ACE levels than those with early-stage fibrosis and cirrhosis(F0-1).For significant fibrosis(≥F2),the 12.8 U/L cut-off value of ACE showed 91.7%sensitivity and 75.0%specificity.The receiver-operating characteristic(ROC)curves analysis revealed that the area under the curve(AUC)value of ACE was 0.871,which was higher than that of APRI,FIB-4,M2BPGi and Plt. CONCLUSION The serum ACE level could be a novel noninvasive,easy,accurate,and inexpensive marker of significant fibrosis stage in patients with CHB.

Glycosyltransferases and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease(NAFLD) is the most common form of chronic liver disease and its incidence is increasing worldwide. However, the underlying mechanisms leading to the development of NAFLD are still not fully understood. Glycosyltransferases(GTs) are a diverse class of enzymes involved in catalyzing the transfer of one or multiple sugar residues to a wide range of acceptor molecules. GTs mediate a wide range of functions from structure and storage to signaling, and play a key role in many fundamental biological processes. Therefore, it is anticipated that GTs have a role in the pathogenesis of NAFLD. In this article, we present an overview of the basic information on NAFLD, particularly GTs and glycosylation modification of certain molecules and their association with NAFLD pathogenesis. In addition, the effects and mechanisms of some GTs in the development of NAFLD are summarized.

Design and Synthesis of Glycosylated Aromatic Nitrogen Mustard Derivatives

Antibody-directed enzyme prodrug therapy（ADEPT） is a new strategy for the treatment of cancer that has arisen in recent twenty years, the main merits of which are that it can improve the selectivity of anticancer drugs and reduce the side effects in remote tissue. In the present study, two prodrugs-glycosylated aromatic nitrogen mustard derivatives were synthesized. Glucose and lactose were converted into glycosyl donors-trichloroacetimidate; the obtained glycosyl donors were glycosylated with p-nitrophenol （ glycosyl donors） to form β-glucosyl p-nitrobenzene and β-lactosyl p-nitrobenzene that were protected by acetyl in a stereoselective manner; the two products were reduced by zinc dust and then treated with ethylene oxide, afforded two glycosylated nitrogen mustard derivatives that were protected by acetyl; the last step was to deacetylate and then afforded the two target compounds that could be used as prodrugs of ADEPT for further Anti-tumor research.

Membrane-bound mucins and mucin terminal glycans expression in idiopathic or Helicobacter pylori, NSAID associated peptic ulcers

AIM: To determine the expression of membrane-bound mucins and glycan side chain sialic acids in Helicobacter pylori (H. pylori)-associated, non-steroidal inflammatory drug (NSAID)-associated and idiopathic-gastric ulcers.

Role of serum β2-microglobulin, glycosylated hemoglobin, and vascular endothelial growth factor levels in diabetic nephropathy

BACKGROUND Diabetic nephropathy(DN)is a common complication of type 1 and type 2 diabetes that can lead to kidney damage and high blood pressure.Increasing evidence support the important roles of microproteins and cytokines,such asβ2-microglobulin(β2-MG),glycosylated hemoglobin(HbA1c),and vascular endothelial growth factor(VEGF),in the pathogenesis of this disease.In this study,we identified novel therapeutic options for this disease.AIM To analyze the guiding significance ofβ2-MG,HbA1c,and VEGF levels in patients with DN.METHODS A total of 107 patients with type 2 diabetes mellitus complicated with nephropathy and treated in our hospital from May 2018 to February 2021 were included in the study.Additionally,107 healthy individuals and 107 patients with simple diabetes mellitus were selected as the control groups.Changes inβ2-MG,HbA1c,and VEGF levels in the three groups as well as the different proteinuria exhibited by the three groups were examined.RESULTS Changes inβ2-MG,HbA1c,and VEGF levels in the disease,healthy,and simple diabetes groups were significantly different(P<0.05).The expression of these factors from high to low were evaluated in different groups by pairwise comparison.In the disease group,high to low changes inβ2-MG,HbA1c,and VEGF levels were noted in the massive proteinuria,microproteinuria,and normal urinary protein groups,respectively.Changes in these factors were positively correlated with disease progression.CONCLUSION The expression of serumβ2-MG,HbA1c,and VEGF was closely correlated with DN progression,and disease progression could be evaluated by these factors.