Synthesis of enantiopure 2-aryl-2-methoxypropionic acids and determination of their absolute configurations by X-ray crystallography

Racemic 2-aryl-2-methoxypropionic acids were enantioresolved by the use of (S)-(-)-phenylalaninol 4. For instance, racemic 2-methoxy-2-phenylpropionic acid (+/-)-7 was condensed with phenylalaninol (S)-(-)-4 yielding a diastereomeric mixture of amides, which was easily separated by HPLC on silica gel affording the first-eluted amide (-)-13a and the second-eluted amide (+)-13b: alpha = 3.19, Rs = 3.49. The absolute configuration of amide (-)-13a was determined to be (R;S) by X-ray crystallography by reference to the S configuration of the phenylalaninol moiety. Amide (R;S)-(-)-13a was converted to oxazoline (R;S)-(-)-14a, from which enantiopure 2-methoxy-2-phenylpropionic acid (R)-(-)-7 was recovered. Other 2-aryl-2-methoxypropionic acids, (R)-(-)-8, (R)-(-)-9, (R)-(+)-10, (R)-(-)-11, and (R)-(-)-12, were similarly prepared in enantiopure forms with the use of phenylalaninol (S)-(-)-4, and their absolute configurations were clearly determined by X-ray crystallography or by chemical correlation.     

Identification of a Hypouricemia Patient with SLC2A9 R380W,A Pathogenic Mutation for Renal Hypouricemia Type 2

Hypouricemia is characterized by low serum uric acid (SUA) levels (≤3.0 mg/dL) with complications such as urolithiasis and exercise-induced acute renal failure. We have previously reported that urate transporter 1 (URAT1/SLC22A12) and glucose transporter 9 (GLUT9/SLC2A9) are causative genes for renal hypouricemia type 1 (RHUC1) and renal hypouricemia type 2 (RHUC2), respectively. In the series of experiments, two families have been revealed to have RHUC2 due to GLUT9 missense mutations R198C or R380W, respectively. Thus far, however, no studies have reported other RHUC2 families or patients with these pathogenic mutations. This study is aimed to find other cases of RHUC2.

We performed mutational analyses of GLUT9 exon 6 (for R198C) and exon 10 (for R380W) in 50 Japanese hypouricemia patients. Patients were analyzed out of a collection of more than 2000 samples from the Japan Multi-Institutional Collaborative Cohort Study (J-MICC Study).

We identified a novel male patient with heterogeneous RHUC2 mutation R380W. The SUA of this hypouricemia patient was 2.6 mg/dL, which is similar to that of our previous report (SUA: 2.7 mg/dL).

This is the second report indicating RHUC2 patient due to GLUT9 mutation R380W. This mutation occurs in highly conserved amino acid motifs and is reported to be an important membrane topology determinant. R380W is a dysfunctional mutation which completely diminishes the urate transport activities of GLUT9. Our study revealed a second hypouricemia patient with GLUT9 R380W, a pathogenic mutation of RHUC2, which may help to expand our understanding of RHUC pathogenesis.     

Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2), a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology) domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3)P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3)P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3)P.     

Diversity of DNA sequences among Vibrio cholerae O139 Bengal detected by PCR-based DNA fingerprinting

Abstract Vibrio cholerae O139, a causative agent of a large epidemic of cholera-like illness, has suddenly emerged and spread widely over several months. To investigate the characteristics unique to O139, traditional typing techniques for V. cholerae , such as biochemical characteristics, antibiotic susceptibility and detection of toxin production, were performed, with the result that 145 O139 strains, except for two O139 strains isolated from Argentina and Germany, were indistinguishable from O1 strains. Thus, in order to clarify the genetical relatedness among O139 strains, and between O139 and O1 strains, the RAPD (random amplified polymorphic DNA) DNA fingerprinting method was undertaken. Although the RAPD arrays in five O139 isolates from Vellore with one arbitrary primer were slightly different from the other O139 strains, the RAPD patterns of the 145 forty-five O139 strains except for two O139 strains from Argentina and Germany were quite similar to each other, but were different from those of O1 strains, indicating that those O139 epidemic strains are closely related to each other regardless of their place of isolation. Furthermore, the RAPD patterns of the O139 strains resembled those of E1 Tor strains rather than classical strain, and a small change in the RAPD pattern of O139 strains occurred during subculture for 200 generations. These results taken together suggested that O139 V. cholerae have emerged from a common origin associated with the E1 Tor strain.     

Par14 Protein Associates with Insulin Receptor Substrate 1 (IRS-1), Thereby Enhancing Insulin-induced IRS-1 Phosphorylation and Metabolic Actions

Pin1 and Par14 are parvulin-type peptidyl-prolyl cis/trans isomerases. Although numerous proteins have been identified as Pin1 substrates, the target proteins of Par14 remain largely unknown. Par14 expression levels are increased in the livers and embryonic fibroblasts of Pin1 KO mice, suggesting a compensatory relationship between the functions of Pin1 and Par14. In this study, the association of Par14 with insulin receptor substrate 1 (IRS-1) was demonstrated in HepG2 cells overexpressing both as well as endogenously in the mouse liver. The analysis using deletion-mutated Par14 and IRS-1 constructs revealed the N-terminal portion containing the basic domain of Par14 and the two relatively C-terminal portions of IRS-1 to be involved in these associations, in contrast to the WW domain of Pin1 and the SAIN domain of IRS-1. Par14 overexpression in HepG2 markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events, PI3K binding with IRS-1 and Akt phosphorylation. In contrast, treating HepG2 cells with Par14 siRNA suppressed these events. In addition, overexpression of Par14 in the insulin-resistant ob/ob mouse liver by adenoviral transfer significantly improved hyperglycemia with normalization of hepatic PEPCK and G6Pase mRNA levels, and gene suppression of Par14 using shRNA adenovirus significantly exacerbated the glucose intolerance in Pin1 KO mice. Therefore, although Pin1 and Par14 associate with different portions of IRS-1, the prolyl cis/trans isomerization in multiple sites of IRS-1 by these isomerases appears to be critical for efficient insulin receptor-induced IRS-1 phosphorylation. This process is likely to be one of the major mechanisms regulating insulin sensitivity and also constitutes a potential therapeutic target for novel insulin-sensitizing agents.     

Cha-Koji,comprising green tea leaves fermented with Aspergillus luchuensis var kawachii kitahara,increases regulatory T cell production in mice and humans

Green tea leaves fermented with Aspergillus luchuensis var kawachii kitahara (Cha-Koji) are a health food containing live A. luchuensis. In this study, we examined the effects of Cha-Koji on the immune system and the enteric environment. First, we designed a clinical trial; after ingesting Cha-Koji daily for 28 days, blood parameters and the fecal composition of the participants were analyzed. Similarly, mice were administered (oral administration) with Cha-Koji suspension or its vehicle for 14 days. Thereafter, both humans and mice were examined by analyzing their immune cell phenotypes and intestinal microbiota. Regulatory T cell (Treg) numbers were significantly increased after administering Cha-Koji. An increase of Clostridium subcluster XIVa, that were known to be rich in butyrate-producing bacterium, was observed in human feces, but not in mice. These results suggest that Cha-Koji has the ability to increase Treg production in both humans and mice, irrespective of the presence of enteric butyrate.     

Contribution of hyperglycemia on diabetic complications in obese type 2 diabetic SDT fatty rats: effects of SGLT inhibitor phlorizin

The spontaneously diabetic torii (SDT) fatty rat is a new model of type 2 diabetes showing overt obesity, hyperglycemia and hyperlipidemia. With early onset of diabetes mellitus, diabetic microvascular complications, including nephropathy, peripheral neuropathy and retinopathy, are observed at young ages. In the present study, blood glucose levels of female SDT fatty rats were controlled with phlorizin, a non-selective SGLT inhibitor, to examine whether and how these complications are caused by hyperglycemia. Phlorizin treatment adequately controlled plasma glucose levels during the experiment. At 29 weeks of age, urinary albumin excretion considerably increased in SDT fatty rats. Glomerulosclerosis and tubular pathological findings also indicate diabetic nephropathy. These renal parameters tended to decrease with phlorizin; however, effects were partial. Sciatic nerve conduction velocities were significantly delayed in SDT fatty rats compared with Sprague-Dawley (SD) rats. Intraepidermal nerve fiber density, an indicator of subclinical small nerve fiber neuropathy, significantly decreased in SDT fatty rats. Retinal dysfunction (prolongation of peak latency for oscillatory potential in electroretinograms) and histopathological eye abnormalities, including retinal folding and mature cataracts were also observed. Both nerve and eye disorders were prevented with phlorizin. These findings indicate that severe hyperglycemia mainly causes diabetic complications in SDT fatty rats. However, other factors, such as hyperlipidemia and hypertension, may affect diabetic nephropathy. These characteristics of diabetic complications will become helpful in evaluating new drugs for diabetic complications using SDT fatty rats.     

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships. Received: 20 March 1998 / Accepted: 30 April 1998