Oxygen and sulfur esters of "inverse substrates": different responses of amidinophenol and amidinothiophenol in the activation of the rate of tryptic hydrolysis of the inverse esters

Kinetic parameters for the trypsin-catalyzed hydrolysis of the oxygen and sulfur "inverse substrates," p-amidinophenyl and p-amidinothiophenyl acetates and trimethylacetates, have been compared. The results suggest that both series of compounds are hydrolyzed via an identical pathway. Appreciable differences, however, were observed in the efficiency of the acylation process in both series, possibly reflecting the spatial requirements of the enzyme's active site toward these substrates. As reported previously, acceleration in deacylation by a positively charged molecule is a characteristic feature of trypsin-catalyzed hydrolysis of "inverse substrates." In the present investigation, it was shown that p-amidinothiophenol is ineffective as an activator, whereas its oxygen counterpart behaves as a potent activator toward oxygen and sulfur substrates. It is assumed that some ionic interaction between the enzyme and the ligand molecule could prevent the rate enhancement.     

Photolabeled sites with a tetrodotoxin derivative in the domain III and IV of the electroplax sodium channel.

Forty three percent of the labeled sites, at least, in the electroplax sodium channel with a photoactivable tetrodotoxin derivative were identified by probing protease-digested labeled fragments with several sequence-directed antibodies. They are located in the loop between segments S5 and S6 of domain IV, as well as the region containing transmembrane segment S6 and adjacent extracellular and cytoplasmic sequences in domain III. No photolabeled fragments were detected in the corresponding region of domain I. These results suggest that C-11 of tetrodotoxin where the photoreactive moiety is attached orients to the region between S5 and S6 in domain III and IV. Probable orientation of the tetrodotoxin molecule in sodium channels is considered by taking together with the recent report of the site-directed mutagenesis.     

Diazipine, a novel photoaffinity probe for dihydropyridine receptors of calcium channels.

A new 1,4-dihydropyridine photoaffinity ligand, [3H]diazipine, has been assessed by binding and photolabeling, and compared with a currently used [3H]azidopine. [3H]Diazipine reversibly binds to skeletal muscle Ca2+ channels with a similar affinity to [3H]azidopine, but [3H]diazipine labels the channel two times more efficiently and no release of the incorported amount is observed after dithiothreitol treatment.     

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.     

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.     

Effect of Caffeine-Containing Beverage Consumption on Serum Alanine Aminotransferase Levels in Patients with Chronic Hepatitis C Virus Infection: A Hospital-Based Cohort Study

Introduction

To date, there have been no prospective studies examining the effect of coffee consumption on serum alanine aminotransferase (ALT) level among individuals infected with the hepatitis C virus (HCV). We conducted a hospital-based cohort study among patients with chronic HCV infection to assess an association between baseline coffee consumption and subsequent ALT levels for 12 months.

Materials and Methods

From 1 August 2005 to 31 July 2006, total 376 HCV-RNA positive patients were recruited. A baseline questionnaire elicited information on the frequency of coffee consumption and other caffeine-containing beverages. ALT level as a study outcome was followed through the patients’ medical records during 12 months. The association between baseline beverage consumption and subsequent ALT levels was evaluated separately among patients with baseline ALT levels within normal range (≤45 IU/L) and among those with higher ALT levels (>45 IU/L).

Results

Among 229 patients with baseline ALT levels within normal range, 186 (81%) retained normal ALT levels at 12 months after recruitment. Daily drinkers of filtered coffee were three times more likely to preserve a normal ALT level than non-drinkers (OR=2.74; P=0.037). However, decaffeinated coffee drinkers had a somewhat inverse effect for sustained normal ALT levels, with marginal significance (OR=0.26; P=0.076). In addition, among 147 patients with higher baseline ALT levels, 39 patients (27%) had ALT reductions of ≥20 IU/L at 12 months after recruitment. Daily drinkers of filtered coffee had a significantly increased OR for ALT reduction (OR=3.79; P=0.034). However, in decaffeinated coffee drinkers, OR could not be calculated because no patients had ALT reduction.

Conclusion

Among patients with chronic HCV infection, daily consumption of filtered coffee may have a beneficial effect on the stabilization of ALT levels.