Successive Isolation of Proteinase Hyperproductive Mutants of Aspergillus sojae

Hydrochloric acid treatment of methyl 3-(4-isobutylphenyl)-3-methylglycidate and methyl 2-hydroxy-3-(4-isobutylphenyl)-3-butenoate, a rearrangement product of the former, in acetic acid gave 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-isobutylphenyl)-pro-panal. The same treatment of 2-hydroxy-3-(4-isobutylphenyl)-3-butenoic acid gave 2-(4-isobutylphenyl)-propanal. Both 3-(4-isobutylphenyl)-3-methylpyruvic acid and 2-(4-iso-butylphenyl)-propanal were oxidized to 2-(4-isobutylphenyl)-propionic acid.     

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula)

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by ¹H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3’-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3”, 3’-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3’-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(?3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.     

Molecular basis of the isoform-specific ligand-binding affinity of inositol 1,4,5-trisphosphate receptors

Three isoforms of the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), IP(3)R1, IP(3)R2, and IP(3)R3, have different IP(3)-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP(3)R types exhibited K(d) values of 49.5 +/- 10.5, 14.0 +/- 3.5, and 163.0 +/- 44.4 nm, which are close to the intrinsic IP(3)-binding affinity previously estimated from the analysis of full-length IP(3)Rs. In contrast, residues 224-604 of IP(3)R1 and IP(3)R2 and residues 225-604 of IP(3)R3, which contain the IP(3)-binding core domain but not the suppressor domain, displayed an almost identical IP(3)-binding affinity with a K(d) value of approximately 2 nm. Addition of 100-fold excess of the suppressor domain did not alter the IP(3)-binding affinity of the IP(3)-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP(3)-binding core domain from different isoforms exhibited IP(3)-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP(3)-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP(3)-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP(3)-binding affinity of IP(3)Rs is specifically tuned through the intramolecular attenuation of IP(3)-binding affinity of the IP(3)-binding core domain by the amino-terminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP(3)R isoforms originates from at least the structural difference identified on the suppressor domain.     

Chemical characterization of acidic oligosaccharides in milk of the red kangaroo (Macropus rufus)

In the milk of marsupials, oligosaccharides usually predominate over lactose during early to mid lactation. Studies have shown that tammar wallaby milk contains a major series of neutral galactosyllactose oligosaccharides ranging in size from tri- to at least octasaccharides, as well as β(1-6) linked N-acetylglucosamine-containing oligosaccharides as a minor series. In this study, acidic oligosaccharides were purified from red kangaroo milk and characterized by (1)H-nuclear magnetic resonance spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be as follows: Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. These acidic oligosaccharides were shown to be sialylated or sulfated in the non-reducing ends to the major linear and the minor branched series of neutral oligosaccharides of tammar wallaby milk.     

Regulation of the phosphorylation of the inositol 1,4,5-trisphosphate receptor by protein kinase C

The various inositol 1,4,5-trisphosphate receptor (IP(3)R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that of IP(3)R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca(2+) inhibited its phosphorylation (IC(50)相似文献   

A simple procedure for synthesis of diastereoisomers of thymidine cyclic 3',5'-phosphate derivatives

Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.     

Analysis of the roles of 14-3-3 in the platelet glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3) using a reconstituted mammalian cell expression model

We have reconstituted the platelet glycoprotein (GP) Ib-IX-mediated activation of the integrin alpha(IIb)beta(3) in a recombinant DNA expression model, and show that 14-3-3 is important in GPIb-IX signaling. CHO cells expressing alpha(IIb)beta(3) adhere poorly to vWF. Cells expressing GPIb-IX adhere to vWF in the presence of botrocetin but spread poorly. Cells coexpressing integrin alpha(IIb)beta(3) and GPIb-IX adhere and spread on vWF, which is inhibited by RGDS peptides and antibodies against alpha(IIb)beta(3). vWF binding to GPIb-IX also activates soluble fibrinogen binding to alpha(IIb)beta(3) indicating that GPIb-IX mediates a cellular signal leading to alpha(IIb)beta(3) activation. Deletion of the 14-3-3-binding site in GPIbalpha inhibited GPIb-IX-mediated fibrinogen binding to alpha(IIb)beta(3) and cell spreading on vWF. Thus, 14-3-3 binding to GPIb-IX is important in GPIb-IX signaling. Expression of a dominant negative 14-3-3 mutant inhibited cell spreading on vWF, suggesting an important role for 14-3-3. Deleting both the 14-3-3 and filamin-binding sites of GPIbalpha induced an endogenous integrin-dependent cell spreading on vWF without requiring alpha(IIb)beta(3), but inhibited vWF-induced fibrinogen binding to alpha(IIb)beta(3). Thus, while different activation mechanisms may be responsible for vWF interaction with different integrins, GPIb-IX-mediated activation of alpha(IIb)beta(3) requires 14-3-3 interaction with GPIbalpha.     

Synthesis of structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B

O-alpha-d-Glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose (15), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)-al pha, beta-L-rhamnopyranose (17), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----3)-D-ribitol (23), and O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----4)-D-ribitol (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)- D-Rib-ol-(5-P----]n; 6A X = 3, 6B X = 4), have been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (3) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (4), and subsequent deallylation (----14) and debenzylation gave 15. Condensation of 14 with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside (2) followed by debenzylation gave 17. Acetylation of 17 followed by removal of AcO-1, conversion into the imidate, coupling with 1,2,4,5-tetra-O-benzyl-D-ribitol (11), deacetylation, and debenzylation gave 23. Coupling of the imidate with 1-O-allyloxycarbonyl-2,3,5-tri-O-benzyl-D-ribitol (12) followed by deallyloxycarbonylation, deacetylation, and debenzylation yielded 27.     

Stereochemical aspects of the formation of double bonds in abscisic acid

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.     

Diarylheptanoids from the rhizomes of Zingiber officinale

Seven new diarylheptanoids, i.e., (3S,5S)-3,5-diacetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane, (3R,5S)-3-acetoxy-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptane, (3R,5S)-3,5-dihydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptane, (5S)-5-acetoxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3-one, 5-hydroxy-1-(3,4-dihydroxy-5-methoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptan-3-one, 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(3,4-dihydroxy-5-methoxy-phenyl)heptan-3-one and 1,5-epoxy-3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)heptane were isolated from the rhizomes of Chinese ginger (Zingiber officinale Roscoe), along with 25 known compounds, i.e., 8 diarylheptanoids, 14 gingerol analogs, a diterpene and 2 steroids. Their structures were elucidated by spectroscopic and chemical methods.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by ¹H-NMR as follows: the saccharides from one animal: Gal(α1-3)Gal(β1-4)Glc (α3′-galactosyllactose), Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (A-tetrasaccharide), Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc; the saccharides from another animal: α3′-galactosyllactose, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]Glc, A-tetrasaccharide, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (A-pentasaccharide), Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc (difucosylheptasaccharide) and Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had α-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

The role of long-chain fatty-acid-CoA ligase 3 in vitamin D3 and androgen control of prostate cancer LNCaP cell growth

The antiproliferative effect of 1alpha,25(OH)(2)D(3) on human prostate cancer cells is well known, but the mechanism is still not fully understood, especially its androgen-dependent action. Based on cDNA microarray results, we found that long-chain fatty-acid-CoA ligase 3 (FACL3/ACS3) might play an important role in vitamin D(3) and androgen regulation of LNCaP cell growth. The expression of FACL3/ACS3 was found to be significantly upregulated by 1alpha,25(OH)(2)D(3) and the regulation was shown to be time-dependent, with the maximal regulation over 3.5-fold at 96h. FACL3/ACS3 was a dominant isoform of FACL/ACS expressed in LNCaP cells as indicated by measuring the relative expression of each isoform. 1alpha,25(OH)(2)D(3) had no significant effect on the expression of FACL1(FACL2), FACL4 and FACL6 except for its downregulation of FACL5 at 24 and 48h by around twofold. The upregulation of FACL3/ACS3 expression by 1alpha,25(OH)(2)D(3) was accompanied with increased activity of FACL/ACS as demonstrated by enzyme activity assay using a (14)C-labeled substrate preferential for FACL3/ACS3. The growth inhibitory effect of 1alpha,25(OH)(2)D(3) on LNCaP cells was significantly attenuated by FACL3/ACS3 activity inhibitor. Androgen withdrawal (DCC-serum), in the presence of antiandrogen Casodex or in AR-negative prostate cancer cells (PC3 and DU145), vitamin D(3) failed to regulate FACL3/ACS3 expression. The upregulation of FACL3/ACS3 expression by vitamin D(3) was recovered by the addition of DHT in DCC-serum medium. Western blot analysis showed that the expression of androgen receptor (AR) protein was consistent with vitamin D(3) regulation of FACL3/ACS3 expression. Taken together, the data suggest that the upregulation of FACL3/ACS3 expression by vitamin D(3) is through an androgen/AR-mediated pathway and might be one of the contributions of the vitamin D(3) antiproliferative effect in prostate cancer LNCaP cells.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

The ratio of serum 24,25-dihydroxyvitamin D(3) to 25-hydroxyvitamin D(3) is predictive of 25-hydroxyvitamin D(3) response to vitamin D(3) supplementation

24,25-Dihydroxyvitamin D (24,25VD) is a major catabolite of 25-hydroxyvitamin D (25VD) metabolism, and may be physiologically active. Our objectives were to: (1) characterize the response of serum 24,25VD(3) to vitamin D(3) (VD(3)) supplementation; (2) test the hypothesis that a higher 24,25VD(3) to 25VD(3) ratio (24,25:25VD(3)) predicts 25VD(3) response. Serum samples (n=160) from wk 2 and wk 6 of a placebo-controlled, randomized clinical trial of VD(3) (28,000IU/wk) were analyzed for serum 24,25VD(3) and 25VD(3) by mass spectrometry. Serum 24,25VD(3) was highly correlated with 25VD(3) in placebo- and VD(3)-treated subjects at each time point (p<0.0001). At wk 2, the 24,25:25VD(3) ratio was lower with VD(3) than with placebo (p=0.035). From wk 2 to wk 6, the 24,25:25VD(3) ratio increased with the VD(3) supplement (p<0.001) but not with placebo, such that at wk 6 this ratio did not significantly differ between groups. After correcting for potential confounders, we found that 24,25:25VD(3) at wk 2 was inversely correlated to the 25VD(3) increment by wk 6 in the supplemented group (r=-0.32, p=0.02) but not the controls. There is a strong correlation between 24,25VD(3) and 25VD(3) that is only modestly affected by VD(3) supplementation. This indicates that the catabolism of 25VD(3) to 24,25VD(3) rises with increasing 25VD(3). Furthermore, the initial ratio of serum 24,25VD(3) to 25VD(3) predicted the increase in 25VD(3). The 24,25:25VD(3) ratio may therefore have clinical utility as a marker for VD(3) catabolism and a predictor of serum 25VD(3) response to VD(3) supplementation.     

Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3).

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.     

Metabolism of three stereoisomers of astaxanthin in the fish, rainbow trout and tilapia

In rainbow trout (Salmo gairdneri), the dietary astaxanthin diesters were mostly absorbed and accumulated in their integuments keeping their configurations, and partially metabolized to (3R, 3'R)-zeaxanthin (major) and/or (3R, 3'S)-zeaxanthin (medium) and/or (3S,3'S)-zeaxanthin (minor). In tilapia (Tilapia nilotica), the three stereoisomers of astaxanthin diesters were promptly metabolized to only (3S,3'S)-astaxanthin, and subsequently to (3R,3'R)-zeaxanthin and/or (3R,3'S)-zeaxanthin and/or (3S,3'S)-zeaxanthin at an invariable ratio, 4:1:0.3. The above facts indicate that the conversion from 3S- to 3R-configuration was carried out in vivo, and vice versa, and that astaxanthins were reductively metabolized to zeaxanthins in both the fish.     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

Induction of B-A transitions of deoxyoligonucleotides by multivalent cations in dilute aqueous solution.

Circular dichroism (CD) spectra of d(CCCCGGGG) in the presence of Co(NH3)6(3+) are very similar to spectra of r(CCCCGGGG). In contrast, B-form characteristics are observed for d(CCCCGGGG) in the presence of Na+ and Mg2+, even at high salt concentrations. Spermidine induces modest changes of the CD of d(CCCCGGGG). The NMR chemical shifts of the nonexchangeable protons of d(CCCCGGGG) in the absence and presence of Co(NH3)6(3+) were assigned by proton two-dimensional (2D) NOESY and COSY measurements. The chemical shifts of the GH8 protons of d(CCCCGGGG) move upfield upon titration with Co(NH3)6Cl3. The sums of the sugar H1' coupling constants decrease with added Co(NH3)6Cl3. Cross peak intensities in the 2D proton NOESY spectra show a transformation from B-DNA to A-DNA characteristics upon the addition of Co(NH3)6Cl3. The temperature-dependent 59Co transverse and longitudinal relaxation rates demonstrate that Co(NH3)6(3+) is site-bound to the oligomer. Such localization is not a general feature of Co(NH3)6(3+) binding to oligonucleotides. 59Co NMR relaxation and CD measurements demonstrate chiral discrimination by d(CCCCGGGG) for the two stereoisomers of Co(en)3(3+). Both stereoisomers bind tightly as judged by 59Co NMR, and both cause large (but nonequivalent) changes in the CD of this oligomer.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: the saccharides from one animal: Gal(alpha1-3)Gal(beta1-4)Glc (alpha3'-galactosyllactose), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)Glc NAc(beta1-6)]Gal(beta1-4)Glc; the saccharides from another animal: alpha3'-galactosyllactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, A-tetrasaccharide, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[F uc(alpha1-3)]Glc (difucosylheptasaccharide) and Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)?Gal(alpha1-3) Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)?Gal(beta1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had alpha-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.