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1.
Biochemistry ; 50(40): 8674-81, 2011 Oct 11.
Article in English | MEDLINE | ID: mdl-21892826

ABSTRACT

We previously identified Asp(340) in transmembrane segment 7 (TM7) as a key determinant of substrate affinity in Hxt7, a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae. To gain further insight into the structural basis of substrate recognition by Hxt7, we performed cysteine-scanning mutagenesis of 21 residues in TM5 of a Cys-less form of Hxt7. Four residues were sensitive to Cys replacement, among which Gln(209) was found to be essential for high-affinity glucose transport activity. The 17 remaining sites were examined further for the accessibility of cysteine to the hydrophilic sulfhydryl reagent p-chloromercuribenzenesulfonate (pCMBS). Among the Cys mutants, T213C was the only one whose transport activity was completely inhibited by 0.5 mM pCMBS. Moreover, this mutant was protected from pCMBS inhibition by the substrate d-glucose and by 2-deoxy-D-glucose but not by L-glucose, indicating that Thr(213) is situated at or close to a substrate recognition site. The functional role of Thr(213) was further examined with its replacement with each of the other 19 amino acids in wild-type Hxt7. Such replacement generated seven functional transporters with various affinities for glucose. Only three mutants, those with Val, Cys, and Ser at position 213, exhibited high-affinity glucose transport activity. All of these residues possess a side chain length similar to that of Thr, indicating that side chain length at this position is a key determinant of substrate affinity. A working homology model of Hxt7 indicated that Gln(209) and Thr(213) face the central cavity and that Thr(213) is located within van der Waals distance of Asp(340) (TM7).


Subject(s)
Glucose/metabolism , Monosaccharide Transport Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/metabolism , Amino Acid Sequence , Biological Transport , Glucose/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Monosaccharide Transport Proteins/genetics , Monosaccharide Transport Proteins/metabolism , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequence Alignment , Stereoisomerism , Substrate Specificity
2.
J Antimicrob Chemother ; 66(3): 626-34, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21172782

ABSTRACT

OBJECTIVES: To examine whether in vitro antifungal susceptibility test results correlate with in vivo efficacy of two cyclodextrin-solubilized itraconazole formulations (intravenous and oral) against Candida in a murine model of invasive infection. METHODS: A selected set of 12 Candida spp. strains with various itraconazole susceptibilities were tested. We studied the efficacy of intravenous and oral itraconazole administered once daily at dosages of 0.63, 2.5, 10 and 40 mg/kg body weight in mice lethally infected with each tested strain. Survival of mice in each treated group was monitored daily until the death of all control mice and compared between groups. RESULTS: Survival of mice infected with 9 of 12 Candida strains with itraconazole MICs of ≤0.016-2.0 mg/L was significantly prolonged by treatment with intravenous itraconazole at dosages of 2.5 or 10 mg/kg and above. In contrast, the other three strains resistant to 8 mg/L itraconazole in vitro were refractory to the therapy, even at the highest itraconazole dosage (40 mg/kg). Closely similar in vivo data were obtained with the oral itraconazole therapy. The effective doses of the two itraconazole formulations increased with increasing itraconazole MICs for the infecting strains. CONCLUSIONS: The in vivo efficacy of intravenous and oral itraconazole correlated with the in vitro susceptibility data.


Subject(s)
Antifungal Agents/administration & dosage , Antifungal Agents/pharmacology , Candida/drug effects , Candidiasis/drug therapy , Itraconazole/administration & dosage , Itraconazole/pharmacology , Administration, Oral , Animals , Disease Models, Animal , Injections, Intravenous , Male , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Rodent Diseases/drug therapy , Survival Analysis , Treatment Outcome
3.
Genes Dev ; 22(7): 918-30, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18334618

ABSTRACT

The molecular bases of circadian clocks have been studied in animals, fungi, bacteria, and plants, but not in eukaryotic algae. To establish a new model for molecular analysis of the circadian clock, here we identified a large number of components of the circadian system in the eukaryotic unicellular alga Chlamydomonas reinhardtii by a systematic forward genetic approach. We isolated 105 insertional mutants that exhibited defects in period, phase angle, and/or amplitude of circadian rhythms in bioluminescence derived from a luciferase reporter gene in their chloroplast genome. Simultaneous measurement of circadian rhythms in bioluminescence and growth rate revealed that some of these mutants had defects in the circadian clock itself, whereas one mutant had a defect in a specific process for the chloroplast bioluminescence rhythm. We identified 30 genes (or gene loci) that would be responsible for rhythm defects in 37 mutants. Classification of these genes revealed that various biological processes are involved in regulation of the chloroplast rhythmicity. Amino acid sequences of six genes that would have crucial roles in the circadian clock revealed features of the Chlamydomonas clock that have both partially plant-like and original components.


Subject(s)
Algal Proteins/genetics , Chlamydomonas reinhardtii/genetics , Circadian Rhythm/genetics , Mutation , Algal Proteins/physiology , Amino Acid Sequence , Animals , Blotting, Northern , Chlamydomonas reinhardtii/physiology , Chloroplasts/metabolism , Circadian Rhythm/physiology , Cloning, Molecular , DNA, Chloroplast/genetics , Gene Expression Profiling , Molecular Sequence Data , Mutagenesis, Insertional , Protein Isoforms/genetics , Protein Isoforms/physiology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid
4.
Plant Cell Physiol ; 46(3): 489-96, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15695451

ABSTRACT

Pi in the medium relieved the toxicity of arsenate against cellular growth of Chlamydomonas reinhardtii. To investigate the relationship between intracellular P contents and arsenate resistance, we determined the intracellular P contents of arsenate-sensitive and arsenate-resistant mutants, which had been generated by random insertional mutagenesis. All 13 arsenate-resistant mutants showed higher P contents than the parent strain, while arsenate-sensitive mutants with high P contents were not found. In one of the arsenate-resistant mutants, AR3, the intracellular P content was about twice that in the wild type during growth in the absence of arsenate. Arsenate incorporation in AR3 was suppressed within 10 min after the addition of 1 mM arsenate, while Pi incorporation continued even after arsenate uptake ceased. Whereas the P content of the wild type decreased to half in the presence of 0.5 mM arsenate, almost the same degree (about 50%) of decrease was observed in AR3 cells grown in the presence of as much as 3 mM arsenate. AR3, in which PTB1, a homolog of a Pi transporter gene, had been disrupted, exhibited a higher activity of a high-affinity Pi transporter, suggesting that it may be due to a compensatory transport activity. These data suggest that the intracellular level of P is one of the important factors of arsenate resistance.


Subject(s)
Arsenates/toxicity , Chlamydomonas reinhardtii/drug effects , Chlamydomonas reinhardtii/metabolism , Drug Resistance/physiology , Intracellular Fluid/metabolism , Phosphorus/metabolism , Animals , Arsenates/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Dose-Response Relationship, Drug , Molecular Sequence Data , Mutation/genetics , Sequence Homology, Amino Acid
5.
Genetics ; 168(2): 809-16, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15514055

ABSTRACT

In the green alga, Chlamydomonas, chloroplast DNA is maternally transmitted to the offspring. We previously hypothesized that the underlying molecular mechanism involves specific methylation of maternal gamete DNA before mating, protecting against degradation. To obtain direct evidence for this, we focused on a DNA methyltransferase, DMT1, which was previously shown to be localized in chloroplasts. The full-length DMT1 protein with a molecular mass of 150 kD was expressed in insect cells, and its catalytic activity was determined. In vitro assays using synthetic DNA indicated methylation of all cytosine residues, with no clear selectivity in terms of the neighboring nucleotides. Subsequently, transgenic paternal cells constitutively expressing DMT1 were constructed and direct methylation mapping assays of their DNA showed a clear nonselective methylation of chloroplast DNA. When transgenic paternal cells were crossed with wild-type maternal cells, the frequency of biparental and paternal offspring of chloroplasts increased up to 23% while between wild-type strains it was approximately 3%. The results indicate that DMT1 is a novel type of DNA methyltransferase with a nonselective cytosine methylation activity, and that chloroplast DNA methylation by DMT1 is one of factors influencing maternal inheritance of chloroplast genes.


Subject(s)
Chlamydomonas reinhardtii/enzymology , Chloroplasts/metabolism , Cytosine/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methylation , DNA, Chloroplast/genetics , Animals , Crosses, Genetic , Molecular Sequence Data , Plants, Genetically Modified , Quantitative Trait, Heritable , Transgenes/genetics , Zygote
6.
Plant Cell Physiol ; 45(9): 1313-9, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15509855

ABSTRACT

By searching a Chlamydomonas expressed sequence tag database and by comparing the retrieved data with homologous sequences from a DNA database, we identified an expressed Chlamydomonas reinhardtii putative major intrinsic protein (MIP) gene. The nucleotide sequence, consisting of 1,631 bp, contains an open reading frame coding for a 300-amino-acid protein named CrMIP1. It possesses conserved NPA motifs, but is not highly homologous to known aquaporins. CrMIP1 was expressed in Saccharomyces cerevisiae and assayed for water and glycerol transport activity. By the stopped-flow spectrophotometric assay, CrMIP1 did not enhance the osmotic water permeability of membrane vesicles of the yeast transformant. However, the transformant cells showed glycerol transport activity in the in vivo assay using [14C]glycerol. This is the first report on the isolation and functional identification of a MIP member from algae.


Subject(s)
Chlamydomonas reinhardtii/metabolism , Glycerol/metabolism , Protozoan Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , DNA Primers , Molecular Sequence Data , Open Reading Frames , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid
7.
Plant Cell Physiol ; 44(6): 597-606, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12826625

ABSTRACT

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.


Subject(s)
Algal Proteins/isolation & purification , Arsenates/pharmacology , Carrier Proteins/genetics , Chlamydomonas/drug effects , Chlamydomonas/genetics , Drug Resistance/genetics , Membrane Proteins/genetics , Mutagenesis, Insertional/genetics , Mutation/genetics , Algal Proteins/genetics , Animals , Cells, Cultured , Chlamydomonas/metabolism , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Molecular Sequence Data , Phospholipid Transfer Proteins , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
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