Overt nephrogenic diabetes insipidus in mice lacking the CLC-K1 chloride channel.

CLC-K1 is a kidney-specific chloride channel that mediates transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) in the inner medulla. Transport of NaCl in the tAL is thought to be a component of urinary concentration in a passive model of the countercurrent multiplication system, but there has been no direct evidence that CLC-K1 is involved in urine concentration. To analyse the physiological function of CLC-K1 in vivo, we generated mice lacking CLC-K1 by targeted gene disruption. Clcnk1-/- mice were physically normal appearance, but produced approximately five times more urine than Clcnk1+/- and Clcnk1+/+ mice. After 24 hours of water deprivation, Clcnk1-/- mice were severely dehydrated and lethargic, with a decrease of approximately 27% in body weight. Intraperitoneal injection of the V2 agonist 1-deamino-8-D-arginine vasopressin (dDAVP) induced a threefold increase in urine osmolarity in Clcnk1+/- and Clcnk1+/+ mice, whereas only a minimal increase was seen in Clcnk1-/- mice, indicating nephrogenic diabetes insipidus. After in vitro perfusion of the tAL, the lumen-to-bath chloride gradient did not produce a diffusion potential in Clcnk1-/- mice in contrast to Clcnk1+/+ and Clcnk1+/- mice. These results establish that CLC-K1 has a role in urine concentration, and that the countercurrent system in the inner medulla is involved in the generation and maintenance of hypertonic medullary interstitium.

Identification of yeast Rho1p GTPase as a regulatory subunit of 1,3-beta-glucan synthase.

1,3-beta-D-glucan synthase [also known as beta(1-->3) glucan synthase] is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase (GTPase), Rho1p, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1p. Glucan synthase from mutants expressing constitutively active Rho1p did not require exogenous guanosine triphosphate for activity. Rho1p copurified with beta(1-->3)glucan synthase and associated with the Fks1p subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Therefore, it appears that Rho1p is a regulatory subunit of beta(1-->3)glucan synthase.

ROM7/BEM4 encodes a novel protein that interacts with the Rho1p small GTP-binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP-binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. The RHO1(G22S, D125N) mutation is a temperature-sensitive and dominant negative mutation of RHO1, and a multicopy suppressor of RHO1(G22S, D125N), ROM7, was isolated. Nucleotide sequencing of ROM7 revealed that it is identical to the BEM4 gene (GenBank accession number L27816), although its physiological function has not yet been reported. Disruption of BEM4 resulted in the cold- and temperature-sensitive growth phenotypes, and cells of the deltabem4 mutant showed abnormal morphology, suggesting that BEM4 is involved in the budding process. The temperature-sensitive growth phenotype was suppressed by overexpression of RHO1, ROM2, which encodes a Rho1p-specific GDP/GTP exchange factor, or PKC1, which encodes a target of Rho1p. Moreover, glucan synthase activity, which is activated by Rho1p, was significantly reduced in the deltabem4 mutant. Two-hybrid and biochemical experiments revealed that Bem4p directly interacts with the nucleotide-free form of Rho1p and, to lesser extents, with the GDP- and GTP-bound forms of Rho1p, although Bem4p showed neither GDP/GTP exchange factor, GDP dissociation inhibitor, nor GTPase-activating protein activity toward Rho1p. These results indicate that Bem4p is a novel protein directly interacting with Rho1p and is involved in the RHO1-mediated signaling pathway.

Regulation of catalytic activity of a multifunctional polyketide biosynthetic enzyme, 6-hydroxymellein synthase, by interaction between NADPH and phenylglyoxal-sensitive amino acid residue at the reaction center.

Treatment of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme in carrot cells, with phenylglyoxal yielded a chemically modified protein in which approximately two moles of the reagent were covalently attached to each subunit of the enzyme. Only NADH- but not NADPH-associated form of native 6-hydroxymellein synthase was inhibited by cerulenin; however, the NADPH-synthase complex lost the insensitivity by the chemical modification of the enzyme protein with phenylglyoxal. Appreciable differences in K(m) values observed between the NADPH- and NADH-associated enzymes were greatly reduced by the treatment with phenylglyoxal. Although the catalytic activity of the NADPH-associated synthase was enhanced by the addition of free CoA, the compound exhibited a significant inhibitory activity to the phenylglyoxal-modified enzyme. A marked deuterium isotope effect in the catalytic reaction of the native synthase-NADPH complex was appreciably decreased in the chemically modified enzyme. These results strongly suggest that an electrostatic interaction between the phosphate group attached to the 2'-position of adenosyl moiety of NADPH and the phenylglyoxal-sensitive amino acid residue, probably arginine, at the reaction center of 6-hydroxymellein synthase regulates several biochemical properties of this multifunctional enzyme.

The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase.

We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation. The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae. The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S. cerevisiae GDP-mannose pyrophosphorylase. We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system. We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.

Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis.

In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1. The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S. cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted. When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase. Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms. Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S. cerevisiae agm1Delta null mutant. Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not. These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis. Because Ser(64) corresponds to the phosphorylating serine in the E. coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64). Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.

Phenoxazinone synthesis by human hemoglobin.

We found that 2-amino-5-methylphenol was converted to the dihydrophenoxazinone with a reddish brown color by purified human hemoglobin, lysates of human erythrocytes, and human erythrocytes. The reddish brown compound was identified as 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazin-3-one by the measurement of NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in this phenoxazinone were studied under various conditions after mixing 2-amino-5-methylphenol with purified oxy- or methemoglobin, or with human erythrocytes. The production of 2-amino-4,4 alpha-dihydro-4 alpha,7-dimethyl-3H-phenoxazine-3-one from 2-amino-5-methylphenol was found to be tightly coupled with the oxidation of ferrous hemoglobin and reduction of ferric hemoglobin under aerobic conditions. By studying the production rates of the dihydrophenoxazinone and the oxido-reduction rates of ferrous and ferric hemoglobins during the reactions of ferrous or ferric hemoglobin with 2-amino-5-methylphenol under aerobic and anaerobic conditions, the reaction mechanism was extensively proposed.

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.

A point mutation within each of two ATP-binding motifs inactivates the functions of elongation factor 3.

We have investigated how point mutations in the two ATP-binding motifs (G(463)PNGCGK(469)ST and G(701)PNGAGK(707)ST) of elongation factor 3 (EF-3) affect ribosome-activated ATPase activity of EF-3, polyphenylalanine synthesis, and growth of Saccharomyces cerevisiae. The point mutation impaired the ribosome-activated ATPase activity of EF-3, when glycine(463 and 701) and lysine(469 and 707) were replaced with valine and arginine, respectively. Thus, each glycine and lysine residue in both ATP-binding motifs is indispensable for EF-3's binding with ATP and the ensuing generation of ribosome-activated ATPase activity. Additionally, the mutant EF-3s did not catalyze polyphenylalanine synthesis in vitro when each glycine(463 and 701) was replaced with valine. The mutant EF-3s did not support cell growth in TEF3-disrupted S. cerevisiae, when each lysine(469 and 707) and glycine(463) was replaced with arginine and valine, respectively. Thus, each of the two ATP-binding motifs of EF-3 is indispensable for the ribosome-activated ATPase activity of EF-3, which is required for protein synthesis and cell growth in S. cerevisiae.

Cloning and sequencing of sulfite reductase alpha subunit gene from Saccharomyces cerevisiae.

A DNA fragment of 2.1 kb was specifically amplified by PCR with primers based on the amino acid sequences obtained from the N-terminal region and the cyanogen bromide-derived peptide of the sulfite reductase alpha subunit in Saccharomyces cerevisiae. With this fragment as a probe, the gene coding for the sulfite reductase alpha subunit was isolated from a genomic library of S. cerevisiae. Sequencing analysis revealed that the gene contains a 3105-bp open reading frame, which is large enough to code for a protein of 1035 amino acid residues. The transcript of the sulfite reductase alpha subunit gene was detected by Northern analysis after methionine deprivation, but the amount of the transcript did not directly correlate with the enzyme activity. The DNA fragment containing the sulfite reductase alpha subunit gene rescued the met10 phenotype by complementation.

Cloning of the Candida glabrata TRP1 and HIS3 genes, and construction of their disruptant strains by sequential integrative transformation.

The Candida glabrata (Cg) TRP1 and HIS3 genes have been isolated by complementation of the Saccharomyces cerevisiae (Sc) trp1 and his3 mutants, respectively. Cg TRP1 encodes a polypeptide of 217 amino acids (aa), whose aa sequence is 58% identical to that of Sc TRP1. Cg HIS3 encodes a polypeptide of 210 aa, whose aa sequence is 73% identical to that of the Sc HIS3. Both Cg TRP1 and HIS3 were disrupted by sequential integrative transformation where the Sc URA3 was used as a selection marker for transformation. The resulting auxotrophic strain of his3- and trp1- was used to examine the ability of the Sc genes to complement the Cg mutations; Sc HIS3 and TRP1 complemented the Cg his3- and trp1- mutations, respectively.

Isolation of a Candida glabrata centromere and its use in construction of plasmid vectors.

A centromere has been isolated from Candida glabrata by functional selection based on the lethality of the SUP11 gene at high copy number. Nucleotide sequence analysis revealed a centromeric structure similar to that of Saccharomyces cerevisiae: the two highly conserved elements CDEI (8 bp) and CDEIII (26 bp) are separated by a 79-bp A+T-rich element, CDEII. Three centromere-bearing plasmid vectors with different selection markers have been constructed. These plasmids were highly stable in mitosis (< 1% loss rate per generation) and exist in one or two copies per cell.

Inhibition of HIV-reverse transcriptase activity by some phloroglucinol derivatives.

Four phloroglucinol derivatives, named mallotophenone (5-methylene-bis-2,6-dihydroxy-3-methyl-4-methoxyacetophenone), mallotochromene (8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-2,2-dimethylchromene), mallotojaponin (3-(3,3(dimethylallyl)5-(3(acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone) and mallotolerin (3-(3-methyl-2-hydroxybut-3-enyl)-5(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)-reverse transcriptase. Under the reaction conditions with (rA)n.(dT)12-18 as the template.primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 micrograms/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS-2 phage RNA as the template.primer. The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template.primer, (rA)n.(dT)12-18, and non-competitive with respect to the triphosphate substrate, dTTP. The Ki value of mallotojaponin for HIV-reverse transcriptase was determined to be 6.1 microM.

Isolation and characterization of the Candida albicans gene for mRNA 5'-triphosphatase: association of mRNA 5'-triphosphatase and mRNA 5'-guanylyltransferase activities is essential for the function of mRNA 5'-capping enzyme in vivo.

The amino acid sequence of the Saccharomyces cerevisiae mRNA 5'-triphosphatase (TPase) diverges from those of higher eukaryotes. In order to confirm the sequence divergence of TPases in lower and higher eukaryotes, the Candida albicans gene for TPase was identified and characterized. This gene designated CaCET1 (C. albicans mRNA 5'-capping enzyme triphosphatase 1) has an open reading frame of 1.5 kb, which can encode a 59-kDa protein. Although the N-terminal one-fifth of S. cerevisiae TPase (ScCet1p) is missing in CaCet1p, CaCet1p shares significant sequence similarity with ScCet1p over the entire region of the protein; the recombinant CaCet1p, which was expressed as a fusion protein with glutathione S-transferase (GST), displayed TPase activity in vitro. CaCET1 rescued CET1-deficient S. cerevisiae cells when expressed under the control of the ADH1 promoter, whereas the human capping enzyme derivatives that are active for TPase activity but defective in mRNA 5'-guanylyltransferase (GTase) activity did not. Yeast two-hybrid analysis revealed that C. albicans Cet1p can bind to the S. cerevisiae GTase in addition to its own partner, the C. albicans GTase. In contrast, neither the full-length human capping enzyme nor its TPase domain interacted with the yeast GTase. These results indicate that the failure of the human TPase activity to complement an S. cerevisiae cet1delta null mutation is attributable, at least in part, to the inability of the human capping enzyme to associate with the yeast GTase, and that the physical association of GTase and TPase is essential for the function of the capping enzyme in vivo.

Discovery of novel aldose reductase inhibitors using a protein structure-based approach: 3D-database search followed by design and synthesis.

Aldose reductase (AR) has been implicated in the etiology of diabetic complications. Due to the limited number of currently available drugs for the treatment of diabetic complications, we have carried out structure-based drug design and synthesis in an attempt to find new types of AR inhibitors. With the ADAM&EVE program, a three-dimensional database (ACD3D) was searched using the ligand binding site of the AR crystal structure. Out of 179 compounds selected through this search followed by visual inspection, 36 compounds were purchased and subjected to a biological assay. Ten compounds showed more than 40% inhibition of AR at a 15 microg/mL concentration. In a subsequent lead optimization, a series of analogues of the most active compound were synthesized based on the docking mode derived by ADAM&EVE. Many of these congeners exhibited higher activities compared to the mother compound. Indeed, the most potent, synthesized compound showed an approximately 20-fold increase in inhibitory activity (IC(50) = 0.21 vs 4.3 microM). Furthermore, a hydrophobic subsite was newly inferred, which would be useful for the design of inhibitors with improved affinity for AR.

Oxidative condensation of 2-amino-4-methylphenol to dihydrophenoxazinone compound by human hemoglobin.

2-Amino-4-methylphenol was converted to a brownish yellow material by the lysates of human erythrocytes or purified human hemoglobin. The reaction proceeded oxidatively, coupled with the oxidation of hemoglobin. The major component of the brownish yellow material produced by oxidative condensation of 2-amino-4-methylphenol was identified as 3-amino-1,4 alpha-dihydro-4 alpha, 8-dimethyl-2H-phenoxazin-2-one on the basis of its spectral data including NMR spectra, IR spectra, EI mass spectra, and absorption spectra. The changes in 3-amino-1,4 alpha-dihydro-4 alpha,8-dimethyl-2H-phenoxazin-2-one during incubation of purified human hemoglobin and 2-amino-4-methylphenol were analyzed spectrophotometrically and by using HPLC. The reaction mechanism involved may be similar to that of actinomycin synthase, which oxidizes 2-amino-5-methylphenol to the dihydrophenoxazinone.

Polyamino acids that inhibit the interaction of yeast translational elongation factor-3 (EF-3) with ribosomes.

EF-3 is a translational elongation factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Addition reaction of dialkyl disulfides to terminal alkynes catalyzed by a rhodium complex and trifluoromethanesulfonic acid.

[reaction: see text]. Addition of dialkyl disulfides to terminal alkynes is catalyzed by a rhodium-phosphine complex and trifluoromethanesulfonic acid giving (Z)-bis(alkylthio)olefins stereoselectively.

Rhodium-catalyzed Beckmann rearrangement.

[figure: see text] Beckmann rearrangement of oxime is catalyzed by [RhCl(cod)]2, trifluoromethanesulfonic acid, and tris(p-tolyl)phosphine in refluxing dichloroethane, giving the corresponding amide in good yield. Product/acid ratios of 10:20 can be attained in the reaction of benzophenone oximes.