An innovative method used for the identification of N-glycans on soybean allergenβ-conglycinins

β-Conglycinin is one of the major allergens existed in soybean.N-Glycans attached to theβ-conglycinin influenced the immunoreactivity and antigen presenting efficiency ofβ-conglycinin.In this study,we described a new method used to release and collect the N-glycans fromβ-conglycinin,and the N-glycans existed in linear epitopes ofβ-conglycinin were identified.Glycopeptides hydrolyzed fromβ-conglycinin were purified by cotton hydrophilic chromatography.Trifluoromethylsulfonic acid was then used to release glycans from glycopeptides,and new glycopeptides containing one single N-acety1-D-glucosamine(G1 cNAc)moiety were then utilized for mass spectrometry.Five glycosylation sites(Asn-199,Asn-455,Asn-215,Asn-489 and Asn-326)and 22 kinds of glycopeptides were identified.It is noteworthy that the peptide VVN^(#)ATSNL(where^(#)represents for the glycosylation site)was analyzed to be both glycopeptide and linear epitope.Our results provided a new method for the N-glycoform analysis of food allergens,and laid a foundation for understanding the relationship between glyco sylation and food allergy.

A cross sectional study on antibiotic resistance pattern of Salmonella typhi clinical isolates from Bangladesh

Objective:To investigate and compare the resistance and sensitivity of Salmonella typhi samples to commonly used antibiotics in three major divisions of Bangladesh and to evaluate the gradually developing resistance pattern.Methods:The antibiotic susceptibility of 70 clinical isolates collected from blood,sputum,urine and pus samples were identified by specific antisera and with standard biochemical tests.The patients were divided into 5 age groups.Susceptibility and resistance was also tested by KirbyBauer disc-diffusion method using 12 regularly used antibiotics.Results:Antibiotic susceptibility test demonstrated that 64.28%isolates of Salmonella typhi were multidrug resistant.Present study suggests that the clinical samples were mostly resistant against nalidixic acid with all age groups and in all three divisions with similar resistance pattern.Resistance is more common among adult people(30-40 years)and children(0-10 years).Salmonella typhi was mostly sensitive against gentamycin,chloramphenicol and ciprofloxacin.Conclusions:Although the population density of Dhaka region is markedly higher than Rajshahi and Chittagong regions,no significant difference in resistance pattern was found.The rate of multidrug resistance is a matter of concern.Physicians should reconsider before prescribing nalidixic acid and cefixime.Further molecular study is needed to reveal the genomic and proteomic basis of resistance.

Glycosylation and secretion of human α-amylases

Three human α-amylases exist: Amy1 (salivary amylase), Amy2A (pancreatic amylase), and Amy2B (expressed in various tissues). These amylases share a 97% - 99% amino acid sequence identity, and two potential N-glycosylation sites (N427 and N476) are commonly found in the C-terminal region. In general, salivary amylase is more frequently glycosylated than pancreatic amylase, and it is still uncertain why differences in the glycosylation pattern among human amylase iso-zymes occur. In this study, we found that there was no significant change of ratio of glycosylated molecules among isozymes produced by the same cultured cells, indicating that glycosylation of amylase is influenced by the type of cell producing the enzyme rather than being an inherent property of the amylase isozymes. We analyzed the glycosylation efficiency of N-glycosylation sites in recombinant Amy2A mutants produced by HEK293 cells and found that glycosylation efficiencies of N427 and N476 were 3% - 18% and 40% - 52%, respectively, indicating that the major N-glycosylation site of glycosylated Amy2A produced by HEK293 cells is N476. The difference in the glycosylation efficiency of each N-glycosylation site also seemed to contribute in part to generate different glycosylation patterns of human amylases. We also confirmed that the C-terminal region of human amylase plays a critical role in secretion, although glycosylation does not play a part in this effect.

Chemically Modified Uridine Molecules Incorporating Acyl Residues to Enhance Antibacterial and Cytotoxic Activities

A new N-acetylsulfanilylation series of uridine have been synthesized in good yield using direct acylation method and afforded the 5’-O-N-acetylsulfanilyluridine. In order to obtain newer products, the 5’-O-N-acetylsulfanilyluridine derivative was further transformed to a series of 2’,3’-di-O-acyl derivatives containing a wide variety of functionalities in a single molecular framework. The chemical structures of the newly synthesized compounds were confirmed on the basis of their FTIR, 1H-NMR spectroscopy, physicochemical properties and elemental analysis. All the synthesized uridine derivatives were tested for their in vitro antibacterial activity against six human pathogenic bacterial strains and for comparison standard antibiotic Ampicillin was also determined. The study revealed that the selectively acylated deriva-tives 5’-O-N-acetylsulfanilyl-2’,3’-di-O-lauroyluridine and 5’-O-N-acetylsulfanilyl-2’,3’-di-O-pivaloyluridine showed highest inhibition against Staphylococcus aureus and Bacillus cereus, respectively. We also observed that the introduction of hexanoyl, decanoyl, lauroyl, myristoyl and pivaloyl groups, the antibacterial functionality of the compound uridine increases. Another noteworthy observation was that the uridine derivatives were found comparatively more effective against Gram-positive microorganisms than those of Gram-negative microorganisms. In addition, the test chemicals were also tested for cyto-toxicity by brine shrimp lethality bioassay and compounds showed different rate mortality with different concentrations.

Two-step derivatization and mass spectral distinction of α2,3 and α2,6sialic acid linkages on N-glycans by MALDI-TOF

Sialylation is one of important glycosylation in human beings and plays an important role in cancer development. α2,3-Linked and α2,6-linked sialic acids are normally observed on the end of N-glycans and have different functions. Derivatization on sialic acid was designed to detect the different linkages by MALDI-TOF MS. In this study, a two-step derivatization by dimethylamine and ammonium hydroxide was improved to modify the sialic acid and made it easier to detect the different linkages of sialic acids on MALDI-TOF MS. Using this derivatization method, specific sialic acids linkages on N-glycans of protein samples such as fetuin and lactoferrin were detected. For complex cell samples, increased a2,3-linked and a2,6-linked sialic acids on bi-antennary and tri-antennary N-glycans were observed in A549 cells induced by hypoxia environment. Taken together, our two-step derivatization of sialic acids offers a simple and accurate way to detect specific linkages on N-glycans with MALDI-TOF mass spectrometer.

Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry

Bisecting N-acetylglucosamine(GlcNAc),a GlcNAc linked to the coreβ-mannose residue via aβ1,4 linkage,is a special type of N-glycosylation that has been reported to be involved in various biological processes,such as cell adhesion and fetal development.This N-glycan structure is abundant in human trophoblasts,which is postulated to be resistant to natural killer cellmediated cytotoxicity,enabling a mother to nourish a fetus without rejection.In this study,we hypothesized that the human amniotic membrane,which serves as the last barrier for the fetus,may also express bisected-type glycans.To test this hypothesis,glycomic analysis of the human amniotic membrane was performed,and bisected N-glycans were detected.Furthermore,our proteomic data,which have been previously employed to explore human missing proteins,were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed.A total of 41glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc,and 25 of these glycoproteins were reported to exhibit this type of modification for the first time.These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane,and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.