A case of renal hypouricemia caused by urate transporter 1 gene mutations.

Hypouricemia is a common disorder in the general population. Herein, renal hypouricemia caused by human urate transporter 1 (hURAT1) gene mutations in a Japanese patient with intellectual disability is reported. She had compound heterozygous mutations in this gene (W258X and IVS2+1G>A), nevertheless, she showed no clinical manifestations such as urolithiasis and exercise-induced acute renal failure. Restriction enzyme analysis with HphI was useful to screen the IVS2+1G>A mutation in hURAT1 gene.

Chain extension of sugar delta-lactones with the enolate of tert-butyl bromoacetate and elaboration into functionalized C-ketosides, C-glycosides, and C-glucosyl glycines.

[structure: see text] We describe the synthesis of a series of exocyclic sugar epoxides 1 prepared in a one-step procedure from sugar delta-lactones with the enolate of tert-butyl bromoacetate. Ring opening of the sugar oxiranes provides C-ketosides while reduction affords functionalized C-glycosides bearing an alpha-hydroxy ester moiety. The alpha-hydroxy ester can be converted into C-glucosyl glycine analogues 2.

Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1.

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

Fluorous oligosaccharide synthesis using a novel fluorous protective group.

The Bfp (bisfluorous chain type propanoyl) group, a novel fluorous protecting reagent, was able to be prepared easily. The Bfp group was readily introduced to carbohydrate, was removed in high yield, and was recyclable after cleavage. Use of the Bfp group made it possible to synthesize a tetrasaccharide by minimal column chromatography purification. Each synthetic intermediate was able to be easily purified by using only simple fluorous-organic solvent extraction and was monitored by NMR, mass spectroscopy, and TLC. [structure: see text]

A study of C-F...M(+) interaction: metal complexes of fluorine-containing cage compounds.

The C-F...M(+) interaction was investigated by observation of the NMR spectroscopic changes and complexation studies between metal cations and the cage compounds 1 and 2 which have fluorobenzene units as donor atoms. As a result, the interaction was detected with all of the metal cations which form complexes with 1 and 2. The stability of the complexes of 1 and 2 was determined by the properties of the metal cations (ionic radii and degree of hydrolysis), not by the hard-soft nature of the cations. Crystallographic analyses of Tl(+) subset 1 and La(3+) subset 2 provided structural information (interatomic distances and bond angles), and the bond strengths, C-F...M(+), O...M(+), and N...M(+), were estimated by Brown's equation based on the structural data. Short C-F...Tl(+) (2.952-3.048 A) distances were observed in the complex Tl(+) subset 1. The C-F bond lengths in the complexes, Tl(+) subset 1 and La(3+) subset 2, are elongated compared to those of the metal-free compounds. Interestingly, no solvent molecules including water molecules were coordinated to La(3+) in the La(3+) subset 2. The stabilization energy of cation-dipole interaction was calculated on the basis of the data from X-ray crystallographic analysis, and it is roughly consistent with the (-)Delta H values estimated in solution. Thus, the C-F...M(+) interaction can be expressed by the cation-dipole interaction. This result explains the fact that compound 1 which has fluorine atom as hard donor strongly binds soft metals such as Ag(+) and Tl(+). Furthermore, it was concluded that the fluorobenzene unit has a poor electron-donating ability compared to that of ether oxygen or amine nitrogen, and thus the ratio of the coordination bond in C-F...M(+) is small. The specific and remarkable changes in the (1)H, (13)C, and (19)F NMR spectra were observed accompanied by the complexation between M(+) and the hosts 1 and 2. These spectral features are important tools for the investigation of the C-F...M(+) interaction. Furthermore, F.Tl(+) spin couplings were observed at room temperature in the Tl(+) subset 1, 2 (J(F-Tl) = 2914 Hz for Tl(+) subset 1 and 4558 Hz for Tl(+) subset 2), and these are clear and definitive evidence of the interaction.

A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases.

A disaccharide substrate of Manbeta1-4GlcNAc-oxazoline 2 was designed and synthesized as a novel probe for detection of the transglycosylating activity of endoglycosidases. A regio- and stereoselective transglycosylation reaction of 2 to GlcNAcbeta1-O-pNP or Dns-Asn(GlcNAc)-OH catalyzed by endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) and endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has been demonstrated for the first time, resulting in the core trisaccharide derivative Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-O-pNP 8 (or -(Dns)Asn-OH). Interestingly, the transglycosylation proceeds irreversibly; the resulting trisaccharide 8 was not hydrolyzed by Endo-M and Endo-A. Based on these results, a new mechanism including an oxazolinium ion intermediate has been proposed for the endoglycosidase-catalyzed hydrolysis or transglycosylation.

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization.

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

Cloning and functional expression of an E box-binding protein from rat granulosa cells.

Ovarian granulosa cells undergo cell growth and cytodifferentiation during follicular maturation. In a number of tissues, the gene expression that is responsible for the cytodifferentiation is largely dependent on E box(es) located upstream of the responsible genes. In this study, we report on the cloning of cDNA(s) encoding E box (5'-CACGTG-3')-binding protein from a rat granulosa cell cDNA library using a yeast one-hybrid system. When multiple E box sequences were used as target, we obtained a positive clone that encodes the rat homologue of upstream stimulatory factor 2 (USF2). An analysis of the nucleotide sequence and its deduced amino acid sequence reveals that rat USF2 protein consists of 346 amino acid residues and belongs to the basic helix-loop-helix/leucine zipper protein family. Northern blot analysis shows that rat USF2 mRNA exists as multiple forms between 1.6 and 2.2 kilobases. The size of the cloned insert was identical to that of the transcript of maximal length. Electrophoretic mobility shift assays showed that in vitro-translated rat USF2 specifically binds to the E box. In addition, cotransfection experiments with luciferase-reporter constructs in HepG2 cells reveal that the overexpression of rat USF2 leads to an increase of luciferase activity in the E box sequence-dependent manner. Thus, we report molecular cloning, expression, and functional characterization of full-length rat USF2 cDNA.

A study of C-F.M+ interaction: alkali metal complexes of the fluorine-containing cage compound.

The C-F.M(+) interaction was investigated by employing a cage compound 1 that has four fluorobenzene units. The NMR ((1)H, (13)C, and (19)F) spectra and X-ray crystallographic analyses of 1 and its metal complexes showed clear evidence of the interaction. Short C-F.M(+) distances (C-F.K(+), 2.755 and 2.727 A; C-F.Cs(+), 2.944 and 2.954 A) were observed in the crystalline state of K(+) subset 1 and Cs(+) subset 1. Furthermore, the C-F bond lengths were elongated by the interaction with the metal cations. By calculating Brown's bond valence, it is shown that the contribution of the C-F unit to cation binding is comparable or greater than the ether oxygen in the crystalline state. Representative spectroscopic changes implying the C-F.M(+) interaction were observed in the NMR ((1)H, (13)C, and (19)F) spectra. In particular, (133)Cs-(19)F spin coupling (J = 54.9 Hz) was observed in the Cs(+) complex.

A new beta-1,2-N-acetylglucosaminyltransferase that may play a role in the biosynthesis of mammalian O-mannosyl glycans.

Recent studies have shown that O-mannosyl glycans are present in several mammalian glycoproteins. Although knowledge on the functional roles of these glycans is accumulating, their biosynthetic pathways are poorly understood. Here we report the identification and initial characterization of a novel enzyme capable of forming GlcNAc beta 1-2Man linkage, namely UDP-N-acetylglucosamine: O-linked mannose beta-1,2-N-acetylglucosaminyltransferase in the microsome fraction of newborn rat brains. The enzyme transfers GlcNAc to beta-linked mannose residues, and the formed linkage was confirmed to be beta 1-2 on the basis of diplococcal beta-N-acetylhexosaminidase susceptibility and by high-pH anion-exchange chromatography. Its activity is linearly dependent on time, protein concentration, and substrate concentration and is enhanced in the presence of manganese ion. Its activity is not due to UDP-N-acetylglucosamine: alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT-I) or UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,2-D-acetylglucosaminyltransferase II (GnT-II), which acts on the early steps of N-glycan biosynthesis, because GnT-I or GnT-II expressed in yeast cells did not show any GlcNAc transfer activity against a synthetic mannosyl peptide. Taken together, the results suggest that the GlcNAc transferase activity described here is relevant to the O-mannosyl glycan pathway in mammals.

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin.

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H(2)O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.

The C-F...cation interaction: an ammonium complex of a hexafluoro macrocyclic cage compound.

An ammonium complex of the hexafluoro cage compound 1 was isolated and its structure was elucidated by X-ray crystallographic analysis. The C-F bonds are elongated by the complexation, which is clear evidence of C-F...cation interaction. The driving force of NH4(+) inclusion is the C-F cation interaction, but the C-F...HN+ hydrogen bond does not contribute to this complexation. The crystal structure of the NH4+ complex 1 shows short C-F...HN+ contacts (2.286-2.662 A). Furthermore, it shows that closer F...H(+)(-N) distances give a larger F...H(+)-N angle. Although such structural features seem to indicate the existence of C-F...HN+ hydrogen bonds, the spectral data (1H NMR, 19F VT-NMR, and IR spectroscopy) did not support the existence of hydrogen bonds. Thermodynamic parameters, log K(s) (4.6 +/- 0.1, 298 K), deltaH (-5.3 +/- 0.1 kcal mol(-1)), and deltaS (3.2 +/- 0.3 cal mol(-1) K(-1)), of the complexation were obtained in CDCl3/CD3CN mixture.

Synthesis of macrocyclic cage compounds by diamine-dihalide one-step coupling reaction

Macropolycyclic cage compounds were synthesized by a direct reaction between diamines and bis(bromomethyl) compounds. The procedure for constructing the polycyclic cage structure is simple and straightforward. The macropolycyclic compounds obtainable from this cyclization procedure are three-dimensional cage compounds, and any other isomers were not obtained except for two examples. Benzene, pyridine, and aliphatic units could be introduced into the cage structure. The macrocycles that have strong cation affinity were obtained as their potassium complexes.

Cloning and expression of pleckstrin 2, a novel member of the pleckstrin family.

A novel member of the pleckstrin family has been identified and designated as mouse pleckstrin 2. The cDNA clone with an insert of 1588 bp contained a 1059-bp open reading frame encoding a polypeptide of 353 amino acid residues. The deduced amino acid sequence predicted that pleckstrin 2 has two pleckstrin homology (PH) domains at the N- and C-termini and a DEP (Dsh, egl-10, and pleckstrin) domain in the central region and showed 35% identity with the sequence of chicken pleckstrin. Northern blot and reverse-transcription polymerase chain reaction analysis revealed that pleckstrin 2 mRNA is ubiquitously expressed. Southern blot analysis indicated that the mouse pleckstrin 2 gene may consisit of two or more exons. To obtain information relative to natural ligand(s) for each of the PH domains in vivo, we employed the green fluorescent protein (GFP) tagged fusion protein system. Distributions of N-terminal and C-terminal PH domains of pleckstrin 2 were quite different from each other, suggesting that these PH domains may interact with distinct factor(s).

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.

Genomic organization and promoter analysis of a mouse homeobox gene, Hex.

A homeobox gene, Hex, is mainly expressed in haematopoietic cells and hepatocytes. It is assumed to play a role in the early stage of differentiation of these cells. To understand the mechanisms involved in the regulation of the Hex gene expression in hepatocytes, we cloned and characterized the mouse Hex gene. The gene consists of four exons and three introns, and spans about 5.7 kb. All the exon-intron boundaries are consistent with the "GT-AG" rule. A single transcription start site was identified by primer extension and S1 mapping analyses. Although the 5'-flanking region is G/C rich (69%), it contains probable "TATA and CCAAT" boxes. Potential binding sequences for transcriptional regulatory proteins including Sp1 and AP-2 are also present in this region. Functional analysis of the Hex promoter was performed by transfecting MH1C1, HeLa, COS-7, and Caco-2 cells with Hex promoter region-luciferase constructs. We found three possible positive regulatory regions, comprising of nucleotides -199 and -172, -154 and -133, and -105 and -68, respectively, required for Hex gene expression in MH1C1 cells by analyses of a series of 5'-deletion constructs of the fusion genes. The activities of these constructs were extremely low in HeLa, COS-7, and Caco-2 cells suggesting that they possess cell-type specificity. Further analysis revealed two GC boxes, GC box1 and GC box2, at nucleotides -197 to -188 and -176 to -167, respectively, necessary for Hex gene expression. Thus, multiple regulatory elements contribute to the Hex gene expression in hepatocytes.

cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein.

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of alpha-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

Chemo-enzymatic synthesis of a calcitonin derivative containing a high-mannose type oligosaccharide by endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae.

Chemo-enzymatic addition of a high-mannose type oligosaccharide to eel calcitonin (CT), a calcium-regulating hormone, was examined. The endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) transglycosylated the Man(6)-GlcNAc moiety from an ovalbumin-derived high-mannose type glycosyl asparagine, Asn(Man(6)-GlcNAc(2))-OH, to a calcitonin derivative, [Asn(GlcNAc)(3)]-CT, in which the N-acetyl-d-glucosamine (GlcNAc) is attached to the third l-asparagine (Asn) residue of the peptide, and a calcitonin derivative containing a high-mannose type oligosaccharide, [Asn(Man(6)-GlcNAc(2))(3)]-CT, was synthesized. The optimal reaction conditions for the synthesis of [Asn(Man(6)-GlcNAc(2))(3)]-CT from Asn(Man(6)-GlcNAc(2))-OH and [Asn(GlcNAc)(3)]-CT catalyzed by Endo-A were examined. The transglycosylation yield relative to the concentration of the [Asn(GlcNAc)(3)]-CT added was 32.7%, and 4.42 mg of [Asn(Man(6)-GlcNAc(2))(3)]-CT was prepared.

Effective Moisture Diffusivity of Fresh Japanese Noodle (udon) as a Function of Temperature.

The effect of moisture content and temperature on the effective moisture diffusivity was investigated to identify the optimal drying condition for Japanese noodle (udon) by using a diffusion model. The drying of fresh udon with different moisture contents was carried out under constant conditions of relative humidity and airflow at 20°C, 30°C and 40°C. The effective moisture diffusivity calculated from diffusion model was found to be constant at each temperature, and not to be influenced by the initial moisture content of the fresh udon. The moisture content calculated from the effective moisture diffusivity (2.1-3.7×10(-7) cm(2) s(-1)) agrees well with the experimental data. The effect of temperature on the effective moisture diffusivity was adequately modeled by the Arrhenius relationship. The activation energy was 21.3 kJ mol(-1).

Chemo-enzymatic synthesis of calcitonin derivatives containing N-linked oligosaccharides.

Eel calcitonin derivatives containing various N-linked oligosaccharides were chemo-enzymatically synthesized by the transglycosylation reaction of Mucor hiemalis endo-beta-N-acetylglucosaminidase (Endo-M) to a glycosylated calcitonin derivative [Asn(GlcNac)3]-CT in which N-acetyl-D-glycosamine (GlcNAc) is attached to the L-asparagine (Asn) residue of the peptide.