Occurrence of terminal {alpha}2 ->8-linked disialylated poly-N-acetyllactosamine chains with Lex and I antigenic glycotopes in tetraantennary arms of an N-linked glycoprotein isolated from rainbow trout ovarian fluid

The Pronase digestion of a 54K glycoprotein present in ovarianfluid of rainbow trout yielded a major glycopeptide. Carbohydratecompositional analysis revealed that this glycopeptide was likelyto possess a single large N-glycan chain having low molecularweight oligomers of N-acetylneuraminic acid (oligoNeu5Ac). Structuralstudies of this glycopeptide revealed novel     

Resistin concentrations in murine adipose tissue and serum measured by a new enzyme immunoassay

Objective: In an attempt to clarify the conflicting data on resistin mRNA expression and protein analysis by western blotting in adipose tissue and serum, we developed a sensitive enzyme‐linked immunosorbent assay (ELISA) for direct measurement of mouse resistin. Research Methods and Procedures: We developed polyclonal antibodies directed to the N (21 to 40) and C (79 to 91) termini of mouse resistin. Then, affinity‐purified anti‐C‐terminal resistin immunoglobin G (IgG) was biotinylated. ELISA was based on the sandwiching of antigen between antibody IgG coated on polystyrene plates and biotinylated antibody IgG. The bound biotinylated antibody was quantified with streptavidin‐linked horseradish peroxidase. Results: New ELISA can measure a concentration as low as 0.5 ng/mL of recombinant mouse resistin and is sensitive and specific enough to measure resistin protein in various adipose tissues and in sera. In normal mice, decreases in resistin concentrations in both white adipose tissue and serum were age dependent during 6 to 24 weeks of development. Resistin concentrations were significantly higher in omental adipose tissue in comparison with perirenal and abdominal adipose tissues and were 2‐ to 5‐fold higher in females than males during the growth period. ob/ob mice had significantly lower resistin concentrations than the control mice in both sera and the white adipose tissues, particularly in the omental fat. The treatment by testosterone, but not progesterone or β‐estradiol, in cultured adipocytes reduces resistin protein levels in a dose‐dependent manner. Discussion: New sensitive ELISA for mouse resistin clarified that the resistin concentrations in normal mice were markedly elevated in the omental adipose depots as compared with the perirenal and abdominal adipocyte depots and significantly elevated compared with adipose tissues in genetically obese mice.     

Amantadine for dyskinesias in Parkinson's disease: a randomized controlled trial

Background

Dyskinesias are some of the major motor complications that impair quality of life for patients with Parkinson''s disease. The purpose of the present study was to investigate the efficacy of amantadine in Parkinson''s disease patients suffering from dyskinesias.

Methods

In this multi-center, double-blind, randomized, placebo-controlled, cross-over trial, 36 patients with Parkinson''s disease and dyskinesias were randomized, and 62 interventions, which included amantadine (300 mg /day) or placebo treatment for 27 days, were analyzed. At 15 days after washout, the treatments were crossed over. The primary outcome measure was the changes in the Rush Dyskinesia Rating Scale (RDRS) during each treatment period. The secondary outcome measures were changes in the Unified Parkinson''s Disease Rating Scale part IVa (UPDRS-IVa, dyskinesias), part IVb (motor fluctuations), and part III (motor function).

Results

RDRS improved in 64% and 16% of patients treated with amantadine or placebo, respectively, with significant differences between treatments. The adjusted odds-ratio for improvement by amantadine was 6.7 (95% confidence interval, 1.4 to 31.5). UPDRS-IVa was improved to a significantly greater degree in amantadine-treated patients [mean (SD) of 1.83 (1.56)] compared with placebo-treated patients [0.03 (1.51)]. However, there were no significant effects on UPDRS-IVb or III scores.

Conclusions

Results from the present study demonstrated that amantadine exhibited efficacious effects against dyskinesias in 60–70% of patients.

Trial Registration

UMIN Clinical Trial Registry UMIN000000780     

Increase of Lipid Hydroperoxides in Liver Mitochondria and Inhibition of Cytochrome Oxidase by Carbon Tetrachloride -Intoxication in Rats

The cellular localization of lipid hydroperoxides was determined for the first time in mitochondria, microsomes and cytosol of rat liver using a specific method involving chemical derivatization and HPLC. Mitochondria contained the highest level of hydroperoxides. After 6h of intragastric administration of carbon tetrachloride (CCl₄) to rats (2 ml/kg body weight), the concentration of lipid hydroperoxides increased significantly in liver mitochondria and cytochrome oxidase activity was inhibited to 35% of the control rats. The mitochondrial content of haem a decreased to 60% of the control at 12 h of CCl₄ administration. In vitro reaction of mitochondria with CCl₄ caused inactivation of cytochrome oxidase. These observations suggested that cytochrome oxidase and haem a in mitochondria were targets of CCl₄.     

Identification,characterization, and developmental expression of a novel α2 → 8-KDN-transferase which terminates elongation of α2 → 8-linked oligo-polysialic acid chain synthesis in trout egg polysialoglycoproteins

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2–5mm Mg²⁺ or Mn²⁺. Expression of KDN-transferase was developmentally regulated in parallel with expression of the 2 8-polysialytransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in capping of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of 2 8-polysialylation in glycoproteins.Abbreviations KDN 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid or naturally occurring deaminated neuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ge N-glycolylneuraminic acid - CMP-KDN cytidine 5-(3-deoxy-d-glycero-d-galacto-2-nonulosonic phosphate) or cytidine 5-KDN phosphate - CMP-NeuAc cytidine 5-Neu5Ac phosphate; oligo-polySia, oligo- and/or polysialic acid - PSGP rainbow trout egg polysialoglycoprotein comprising 2 8-linked oligo- polyNeu5Gc - PSGP (low Sia) a precursor of PSGP present at early stages of oogenesis which contains mostly the disialyl group, Sia2 8Sia2 6- - *K-PSGP [¹⁴C]KDN-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]KDN with the immature cortical vesicle fraction P1 containing KDN-transferase - *A-PSGP [¹⁴C]Neu5Ac-labelled PSGP obtained by incubating PSGP and CMP-[¹⁴C]Neu5Ac with the P1 fraction - A-*K-PSGP andK-*K-PSGP the products obtained after incubating *K-PSGP with P1 fraction and unlabelled CMP-Neu5Ac or CMP-KDN, respectively - *K-PSGP _cho ,A-*K-PSGP _cho , andK-*K-PSGP _cho mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of *K-PSGP,A-*K-PSGP, and K-*K-PSGP, respectively - *A-PSGP _cho a mixture of oligosaccharide alditols obtained by alkaline borohydride treatment of [¹⁴C]Neu5Ac-labelled PSGP - Endo-N endo-N-acylneuraminidase - DP degree of polymerization - GLC gas-liquid chromatography - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Does an animal peptide:N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein?

Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide:N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994)J Biol Chem 269, 17611–18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g.K _m=114 µm for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase; the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man1 3(Man1 6)Man, but not by mannose and -methyl-d-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition proteinin vivo. Such dual properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases — PNGase A and PNGase F, respectively.Abbreviations GLC gas liquid chromatography - GlcNAc-Asn 2-acetamido-1--(l-aspartamido)-1,2-dideoxy-d-glucose - BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Man d-mannose; triomannose, Man1 3(Man1 6)Man; - MES 2-(N-morphorino)ethanesulfonic acid - NeuAc N-acetyl-neuraminic acid - PNGase peptide:N ⁴-(N-accetyl-glucosaminyl)asparagine amidase (peptide:N-glycanase,EC 3.5.1.52) - PNP p-nitrophenyl     

Gelatinases and metalloproteinase inhibitor secreted by murine colonic carcinoma cells with differing metastatic potential

Gelatinase activity and inhibitory activity against collagenase were measured in serum-free medium conditioned by murine colonic carcinoma cells with different spontaneous metastatic potentials to the lung. The medium conditioned with poorly metastatic NM11 cells gave higher inhibitory activity than that conditioned with highly metastatic LuM1 cells, while the level of secreted gelatinases in the same medium was lower in NM11 medium than in LuM1 case. Northern analysis showed the higher gene expression of both tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 in NM11 cells than in LuM1 cells, suggesting that both TIMPs are responsible for the increase of inhibitory activity in NM11 conditioned medium. Examination of the balance of gelatinases and inhibitor revealed that the amount of inhibitor exceeded that of gelatinases in the medium conditioned with NM11 cells. In contrast, the medium conditioned with LuM1 cells contained excess amounts of gelatinases. The results indicated a close correlation between the balance of gelatinases and inhibitors and the metastatic behavior of murine tumor cells.     

Stability of N-acyl groups in methylated α2 → 8-linked oligosialosyl chains toward methanolysis. Analysis by chemical ionization-mass spectrometry

The stability of N-acetyl group of methylated trisaccharide of N-acetylneuraminic acid toward methanolysis under conditions used in methylation analysis was investigated. The analysis of the products obtained after a reaction sequence, methylation-methanolysis-deuterioacetylation, by chemical ionization-mass spectrometry has led to unequivocal conclusion that N-acetyl group of internal 8-O-substituted residue of the methylated oligosialosyl compound is de-N-acetylated under conditions sufficient to cleave glycosidic linkages, whereas the fully methylated nonreducing terminal residue of neuraminic acid is completely resistant to de-N-acetylation. The reaction mechanism to explain these observations is presented.