Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (Ϸ1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu 393 , Phe 397 , Phe 415 , and His 440 ) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Tryptophan can be metabolized via 5-hydroxytryptamineϭserotonin to melatonin by a series of 4 enzymes in pineal body. Lack of serotonin in body fluid in the brain during daytime can lead to several psychiatric disorders, while shortage of plasma-melatonin at night can be related to sleep disorders. The Morningness-Eveningness (M-E) questionnaire and the original questionnaire including questions on sleep habits, mental symptoms, and contents of meals were administered to 1055 infants aged 0-6 yrs, 751 students attending an elementary school, and 473 students attending junior high school in Kochi City (33°N). The index of tryptophan taken at breakfast (Trp-Index) was calculated as tryptophan amount per one meal based on the tryptophan included in each 100 g of the foods and a standard amount of food per one meal. A significant positive-correlation between M-E scores and Trp-Index was not shown by relatively older students, aged 9-15 yrs (Pearson's test, rϭ0. 044-0.123, pϭ0.071-0.505), whereas a significant positive correlation was shown by infants and young elementary school students aged 0-8 yrs (rϭ0.180, 0.258, pϽ0.001). The more frequently the infants had difficulty falling asleep at bedtime and waking up in the morning, the less the Trp-Indices taken at breakfast were (Kruskall-Wallis-test, pϭ0.027 for difficulty falling asleep; pϭ0.008 for difficulty waking up). The more frequently infants became angry even by a little trigger, or depressed, the lower (more evening-typed) the M-E scores were (Kruskal-Wallis test: pՅ0.001). Tryptophan ingested at breakfast is very important for children to keep a morning-type diurnal rhythm, high quality of sleep, and indirectly good mental health, presumably, through the metabolism of tryptophan to serotonin in daytime and further to melatonin at night.

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The present findings suggest that factor V is strongly expressed in mesangial cells in active IgAN accompanied with mesangial proliferation and may exert procoagulant activity, leading to intramesangial coagulation.

The Na(+)/glucose cotransporter gene SGLT1 was analyzed in a Japanese patient with congenital glucose-galactose malabsorption. Genomic DNA was used as a template for amplification by the polymerase chain reaction of each of the 15 exons of SGLT1. The amplification products were cloned and sequenced. About half of the exon 5 clones of the patient contained a C-->T transition, resulting in an Arg(135)-->Trp mutation, whereas the remaining clones contained the normal exon 5 sequence. In addition, whereas some exon 12 clones exhibited the normal sequence, others showed a CAgtaggtatcatc-->CAgacc mutation at the splice donor site of intron 12 that may result either in the skipping of exon 12 or in read-through of intron 12. Neither the Arg(135)-->Trp mutant nor either of the possible intron 12 mutant proteins exhibited Na(+)-dependent glucose transport activity when expressed in Xenopus oocytes. Immunocytochemical analysis indicated, however, that the Arg(135)-->Trp mutant was localized to the oocyte plasma membrane. DNA sequence analysis revealed that the missense mutation in exon 5 and the splice site mutation in intron 12 were inherited from the proband's father and mother, respectively. These results indicate that the patient is a compound heterozygote for this disease, and that the Arg(135)-->Trp mutant of SGLT1 undergoes normal trafficking to the plasma membrane but is non-functional.

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae. Site-directed mutagenesis has been extensively used in attempts to determine functional sites in transporters (1, 2). This approach is limited, however, by the fact that it is usually not possible to mutate every amino acid and replacements that are made often yield results that are negative in nature (3, 4). As an alternative method, the use of chimeras to identify functional domains of transporters has proved highly fruitful (5-9).We have used chimeras to analyze two homologous sugar transporters in the yeast Saccharomyces cerevisiae (3, 4): Gal2, a high affinity galactose transporter (10) that was unexpectedly found to transport glucose with nearly the same affinity (3), and Hxt2, a major glucose transporter that does not transport galactose (3, 10). These two transporters belong to the Glut transporter family, the largest known organic solute transporter family comprising more than 80 transporters found in prokaryotes through mammals (11,12). Creating chimeras between the Gal2 and Hxt2 transporters gave us an opportunity to study the galactose recognition site in Gal2 and to gain insights into the substrate recognition sites in Glut family transporters in general. To unequivocally determine the substrate recognition site, we have taken two steps. In the first step (3), three types of systematic chimeras were made using the Escherichia coli homologous recombination system. The site responsible for differentially recognizing galactose and glucose was localized to a 101-amino acid region that includes the transmembrane 10 (TM10), 1 TM11, and TM12 segments and the proximal half of the C-terminal hydrophilic tail. In the second step (4), the 101-amino acid region was subdivided into the above four regions by introducing five restriction enzyme sites into the corresponding segments of each gene without changing the amino acids encoded. By analyzing plasmids containing all the possible combinations of these segments inserted into the corresponding parts of Hxt2, we identified TM10 as the domain where galactose and glucose are differentially recognized. TM10 contains 35 amino acid residues, of which only 12 are different between Gal2 and Hxt2. Thus, it is reasonable to assume that the amino acid residue(s) essential for the substrate recognition can be found among these 12 residues. We employed a new comprehensive approach and found that 2 amino acid residues in TM10 are important for substrate recognition. EXPERIMENTAL PROCEDURESProduction of GAL2 and HXT2 Cassette Vectors-A DNA fragment containing GAL2 was cut out by PmaCI and EcoRI and ligated to SmaI and EcoRI sites in a multicloning site of pTV3, a YEp vector (3). The nucleotide sequence immediately following the initiation codon was modified from ATGGCAGTTGAG to ATGGCAGAATTC to create an EcoRI site, which changed the deduced amino acid sequence from Met-Ala-Val-...