Human erythrocyte glutathione reductase. I. Purification and properties.

1. Glutathione reductase (NAD(P)H:oxidized-glutathione oxidoreductase, EC. 1.6.4.2) from human erythrocytes was purified 49 000-fold with an overall yield of 15% and a 280/460 nm absorbance ratio of 6.03. The procedure used was the method of Worthington and Rosemeyer modified by addition of heating and recrystallization. 2. It was concluded from the results of purification, electrofocusing and inhibition studies that glutathione reductase is a single enzyme which used both NADPH and NADH as hydrogen donors. 3. Apoenzyme cross-reacts with the antibody to the holoenzyme but has a slightly reduced affinity to the antibody. Apoenzyme can be removed from the hemolysate by heating and centrifugation without loss of holoenzyme. 4. Indirect immunological assay of the specific activity of the erythrocyte glutathione reductase is possible in the enzyme saturated with FAD.     

Maintenance and amplification of cell-associated immunological memory in in vivo culture system

By employing bovine serum albumin as antigen and the capsular polysaccharide of Klebsiella pneumoniae as adjuvant, maintenance and amplification of immunological memory were analyzed in an in vivo culture system in mice. For this purpose, the double cell transfer technique was employed to minimize the influence of regulatory factors on memory expression. Memory associated with primed cells is maintained at the original level during in vivo culture for at least a month in the absence of antigen. In contrast, memory is amplified more than 30 times during this period by stimulation with antigen. This secondary increase in memory does not require the action of adjuvant. Neither the residual primary antigen nor preformed primary antibody seems to play a significant role in the maintenance and amplification of memory of the primed cells. From these results it is probable that the enduring immunological memory in actively immunized mice is conveyed by long-lived memory cells, and additional antigenic stimulating on once-established memory cells serve to amplify (not simply to maintain) memory in a secondary fashion.     

The activation of coagulation factor X. Identity of cleavage sites in the alternative activation pathways and characterization of the COOH-terminal peptide.

Bovine Factor X can be activated by two alternative pathways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH2 terminus of the heavy chain, to produce alpha-Xa. This is then converted autocatalytically to another form of Factor Xa, beta-Xa, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent reaction. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor Xa on Factor X, in the presence of lipid, to release the same COOH-terminal peptide as is produced in the conversion of alpha-Xa to beta-Xa. The intermediate produced by the loss of this peptide from Factor X,I1, can be activated directly to beta-Xa by the tissue factor-Factor VII complex, with the loss of the same NH2-terminal peptide as is produced in the conversion of Factor X to alpha-Xa. The autocatalytic activation of Factor X by Factor Xa described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of I1-Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways. The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been determined. The carbohydrate moiety is attached via an O-glycosidic linkage at a threonine residue, and contains galactosamine but no glucosamine.     

Targeting selective autophagy by AUTAC degraders

ABSTRACT

Targeted degradation is a promising new modality in drug discovery that makes it possible to reduce intracellular protein levels with small molecules. It is a complementary approach to the conventional protein knockdown typically used in laboratories and may offer a way to approach the currently undruggable human proteome. Recently, the first autophagy-mediated degraders, called AUTACs, were developed based on observations in a xenophagy study.     

Fatty acid composition in fetal, neonatal, and cultured cardiomyocytes in rats

Reconstructed myocardial tissue still does not have enough pulsatile contraction. It is well known that fetal and mature neonatal cardiomyocytes utilize glucose and lipid, respectively, as their energy substrates, and that cultured ones mainly use glucose in spite of their age comparable to neonate ones, probably due to insufficient supply of lipids from culture medium. In the present study, we compared 7 saturated, 6 monounsaturated, and 11 polyunsaturated fatty acid contents in cultured cardiomyocytes (Cul group) with those in fetal (Fet group, approximately 17 d after impregnation) and neonatal (Neo group, 9 d old) rats, where the age of the Cul cells were set nearly equal to the Neo ones. Saturated fatty acid contents in the Cul group were generally lower than those in the Fet group and were close to those in the Neo group, except for C12:0 of which content was highest in the Neo group. Monounsaturated fatty acid contents in the Cul group were generally lower than those in the Fet group but similar to or higher than those in the Neo group, except for C24:1n-9 of which content was again highest in the Neo group. In contrast, most of polyunsaturated fatty acid (PUFA) contents in the Cul group appeared lower than those in both the Fet and Neo groups, and differences in 5 of 10 detected PUFAs were significant between the Cul and Neo groups. The results suggest that PUFA contents in cultured cardiomyocytes might be insufficient to exert enough contractile ability. In conclusion, it could be necessary for cultured cardiomyocytes to uptake more lipid; PUFAs in particular.     

Inhibition of Influenza Virus Replication by Phosphorothioate and Liposomally Endocapsulated Oligonucleotides

Abstract

We have demonstrated that antisense phosphorothioate oligonucleotides (S-ODNs) inhibit influenza virus A replication in MDCK cells. Phosphorothioate and liposomally encapsulated oligonucleotides with two target sites (PB1 and PB2) were synthesized and tested for virus-induced cytopathogenicity effects by a MTT assay using MDCK cells. The liposomally encapsulated S-ODNs complementary to the sites of the PB2-AUG initiation codon showed highly inhibitory effects. On the other hand, the inhibitory effect of the liposomally encapsulated S-ODNs targeted to PB1 was considerably decreased in comparison with the PB2 target sites. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than the free oligonucleotides. The activities of the modified oligonucleotides are effectively enhanced by using the liposomal carrier.     

HLA-DQB1*03 Confers Susceptibility to Chronic Hepatitis C in Japanese: A Genome-Wide Association Study

Hepatitis C virus (HCV) establishes a chronic infection in 70-80% of infected individuals. Many researchers have examined the effect of human leukocyte antigen (HLA) on viral persistence because of its critical role in the immune response against exposure to HCV, but almost all studies have proven to be inconclusive. To identify genetic risk factors for chronic HCV infection, we analyzed 458,207 single nucleotide polymorphisms (SNPs) in 481 chronic HCV patients and 2,963 controls in a Japanese cohort. Next, we performed a replication study with an independent panel of 4,358 cases and 1,114 controls. We further confirmed the association in 1,379 cases and 25,817 controls. In the GWAS phase, we found 17 SNPs that showed suggestive association (P < 1 × 10^-5). After the first replication study, we found one intronic SNP in the HLA-DQ locus associated with chronic HCV infection, and when we combined the two studies, the association reached the level of genome-wide significance. In the second replication study, we again confirmed the association (P _combined = 3.59 × 10⁻¹⁶, odds ratio [OR] = 0.79). Subsequent analysis revealed another SNP, rs1130380, with a stronger association (OR=0.72). This nucleotide substitution causes an amino acid substitution (R55P) in the HLA-DQB1 protein specific to the DQB1*03 allele, which is common worldwide. In addition, we confirmed an association with the previously reported IFNL3-IFNL4 locus and propose that the effect of DQB1*03 on HCV persistence might be affected by the IFNL4 polymorphism. Our findings suggest that a common amino acid substitution in HLA-DQB1 affects susceptibility to chronic infection with HCV in the Japanese population and may not be independent of the IFNL4 genotype.