Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

Cilia sense and transduce extracellular signals via specific receptors. The intraflagellar transport (IFT) machinery mediates not only bidirectional protein trafficking within cilia but also the import/export of ciliary proteins across the ciliary gate. The IFT machinery is known to comprise two multisubunit complexes, namely, IFT-A and IFT-B; however, little is known about how the two complexes cooperate to mediate ciliary protein trafficking. We here show that IFT144–IFT122 from IFT-A and IFT88–IFT52 from IFT-B make major contributions to the interface between the two complexes. Exogenous expression of the IFT88(Δα) mutant, which has decreased binding to IFT-A, partially restores the ciliogenesis defect of IFT88-knockout (KO) cells. However, IFT88(Δα)-expressing IFT88-KO cells demonstrate a defect in IFT-A entry into cilia, aberrant accumulation of IFT-B proteins at the bulged ciliary tips, and impaired import of ciliary G protein–coupled receptors (GPCRs). Furthermore, overaccumulated IFT proteins at the bulged tips appeared to be released as extracellular vesicles. These phenotypes of IFT88(Δα)-expressing IFT88-KO cells resembled those of IFT144-KO cells. These observations together indicate that the IFT-A complex cooperates with the IFT-B complex to mediate the ciliary entry of GPCRs as well as retrograde trafficking of the IFT machinery from the ciliary tip.     

Microwave irradiation for fixation and immunostaining of endothelial cells in situ

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.     

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.     

Reaction of 2-Deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides with Oligonucleotide1

Abstract

Reaction of methyl 2-deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides, prepared from methyl 2,3-anhydro-α-D-ribofuranoside, with oligodeoxyribonucleotide (21mer) in acetonitrile-H₂O (pH 7) and subsequent treatment with piperidine resulted in the cleavage of the nucleotide chain at the position G, A, and C.     

Tailoring the Peptide-Binding Specificity Of An RNA by Combinations of Specificity-Altering Mutations

In this study, the ability to tailor the peptide-binding specificity of an RNA was investigated. First, variants of the Rev-response element (RRE) RNA with different specificities toward the natural binding partner, Rev, and two RRE-binding aptamers, the RSG-1.2 and the Kl peptides, were identified. Next, hybrid RRE mutants with combinations of two sets of specificity-altering substitutions were tested for peptide-binding specificity. It was shown that in most cases the results of the combination of individual mutations were of an additive nature, therefore providing a way to manipulate the peptide-binding specificity of an RNA in a predictable manner.     

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.     

A Reduction in Age-Enhanced Gluconeogenesis Extends Lifespan

The regulation of energy metabolism, such as calorie restriction (CR), is a major determinant of cellular longevity. Although augmented gluconeogenesis is known to occur in aged yeast cells, the role of enhanced gluconeogenesis in aged cells remains undefined. Here, we show that age-enhanced gluconeogenesis is suppressed by the deletion of the tdh2 gene, which encodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein that is involved in both glycolysis and gluconeogenesis in yeast cells. The deletion of TDH2 restores the chronological lifespan of cells with deletions of both the HST3 and HST4 genes, which encode yeast sirtuins, and represses the activation of gluconeogenesis. Furthermore, the tdh2 gene deletion can extend the replicative lifespan in a CR pathway-dependent manner. These findings demonstrate that the repression of enhanced gluconeogenesis effectively extends the cellular lifespan.