The third chains of living organisms--a trail of glycobiology that started from the third floor of building 4 in NIH

Application of a finger-printing method to the analysis of human milk oligosaccharides led to the finding that several oligosaccharides were missing in the milk of non-secretor or Lewis-negative individuals. This finding helped us in opening the door of elucidating the enzymatic basis of blood types in human. Based on these successful studies, a strategy to establish reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins was devised. It was to contrive enzymatic and chemical means to release quantitatively the N-linked sugar chains as oligosaccharides, and finger-print them by using appropriate methods to demonstrate the sugar pattern of a glycoprotein. These methods enabled us to determine that the N-linked sugar chains of glycoproteins can be classified into three subgroups: high mannose-type, complex-type, and hybrid-type. By comparative studies of the sugar patterns of a glycoprotein produced by different organs and different animals, occurrences of organ- and species-specific glycosylation were found in many glycoproteins. By comparative studies of the glycosylation patterns of the subunits constructing human chorionic gonadotropin and other glycoproteins, occurrence of site-directed N-glycosylation was also found, indicating that the processing and maturation of the N-linked sugar chains of a glycoprotein might be controlled by the structure of polypeptide moiety. Furthermore, these methods enabled us to elucidate the structural alteration of the sugar chains of a glycoprotein induced by diseased state of the producing cells, such as rheumatoid arthritis and malignancy. Recent studies of glycoproteins in the brain-nervous system through aging revealed that N-glycosylation of P(0) in the rat spinal cord is induced by aging. Therefore, glycobiology is expanding tremendously into fields such as pathological and gerontological research.     

A new isolation procedure and further characterisation of the lectin from the red marine alga Ptilota serrata

Ptilota serrata has been shown previously to contain a lectin (PSL) which is non-specific for human blood groups. We report here a new isolation procedure for PSL using a single step affinity chromatography technique on cross-linked guar gum and further characterisation studies. PSL was inhibited by galactose and its derivatives. The carbohydrates o-nitrophenyl-N-acetyl-α-D-galactoside, p-nitrophenyl-N-acetyl-β-D-galactoside and lactose were strong inhibitors. The glycoproteins porcine stomach mucin, asialo bovine mucin and asialofetuin were also inhibitory. The Mr of PSL, determined by gel filtration, was 55,470. SDS-PAGE revealed one single protein band with Mr of 18,390, indicating the native protein to be a trimer of apparently identical subunits. PSL was shown to contain large amounts of acidic and hydroxyl amino acids but low in basic amino acids. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of clomiphene citrate and human chorionic gonadotropin on the expression of CatSper1, CatSper2, LHCGR,and SF1 genes,as well as the structural changes in testicular tissue of adult rats

The purpose of this study was to investigate the effect of clomiphene citrate and human chorionic gonadotropin (HCG) on the structural changes, as well as the evaluation of the expression of cation channel sperm‐associated protein 1 (CatSper1), cation channel sperm‐associated protein 2 (CatSper2), luteinizing hormone/choriogonadotropin receptor (LHCGR), and steroidogenic factor 1 (SF1) genes in testicular tissue of rats. All rats divided into five groups as follows; G1 as the control group that received normal saline, G2 received olive oil, G3 received 100 IU/kg HCG, G4 received 5 mg/kg clomiphene citrate, and G5 received 5 mg/kg clomiphene citrate and 100 IU/kg HCG. At the end of the experiment period, Day 56, blood samples were taken and the serum was isolated. Then, histomorphometric analysis, hormonal assess, and real‐time polymerase chain reaction to measure the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were performed. The results showed that the concentrations of testosterone, follicle‐stimulating hormone, and luteinizing hormone were decreased in the G4 group, whereas these parameters were increased in the G3 group. A comparison of the sperm quality indicated a significant reduction in the quality of sperm cells in the G4 group compared with other groups. The quality of sperm was significantly enhanced in the G3 and G5 groups in comparison with the G1 group. Also, our findings demonstrated that the expression of CatSper1, CatSper2, LHCGR, and SF1 genes were significantly elevated in the G3 group when compared with other experimental groups. According to the obtained results, it seems that clomiphene citrate reduces the process of spermatogenesis and the detrimental impacts of this compound would be neutralized by the administration of HCG.     

Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane

Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium.