Novel cytotoxic diterpenes from the stem of dysoxylum kuskusense

Three novel diterpenes, dysokusones A (1), B (2), and C (3), were isolated from the stem of Dysoxylum kuskusense as cytotoxic substances. The structures were established by spectroscopic examinations. Compounds 1, 2, and 3 were cytotoxic toward HL-60(TB) cells with EC₅₀ values of 2.25, 6.35, and 2.37 μM, respectively. Compound 1 also displayed cytotoxicity against K-562 and NCI-H522 cells with EC₅₀ values of 5.04 and 4.80 μM, respectively.     

Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells

Abstract: Metabotropic glutamate receptor (type 1; mGluR1 ) is expressed predominantly in the hippocampus and the cerebellum. Using cultured cerebellar granule cells, we investigated the regulation of the mGluR1 mRNA expression. Levels of mGluR1 mRNA were decreased to less than half by high potassium stimulation and by glutamate and quisqualate. Although these glutamate receptor agonists tested are also known to cause neuronal cell death in culture, the effect of cell death cannot explain the observed reduction in mGluR1 mRNA because of the following reasons: (a) antagonists of N -methyl-D-aspartate and non- N -methyl-D-aspartate receptors inhibited cell death, but not the reduction of the level of mGluR1 mRNA; (b) mGluR1 mRNA returned to its initial level 48 h after the agonist application; and (c) the mRNA level of one of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptors (GluR1) was not altered by these conditions. Therefore, we conclude that the glutamate or quisqualate stimulation can specifically inhibit the expression of mGluR1 mRNA. The dose response of quisqualate for the reduction in mGluR1 mRNA is consistent with that for inositol phosphate formation stimulated through the cloned mGluR1 . The mRNA reduction did not require extracellular calcium. Desensitization of mGluR1 with phorbol ester abolished the mRNA reduction. These results suggest that the reduction in mGluR1 mRNA is mediated by the activation of the metabotropic receptor itself.     

High-level expression of recombinant human soluble thrombomodulin in serum-free medium by CHO-K1 cells

Cultivation of gene-engineered Chinese hamster ovary (CHO-K1) cells that produce recombinant human soluble thrombomodulin (rsTM) was investigated to optimize conditions for high-level expression of the protein in a serum-free medium. For economic protein production, oxygenation of cultures with pure O₂ permitted sufficient cell growth for high rsTM production with only 1 g/l of microcarriers and a low foetal bovine serum concentration. A longer growth phase (over 5 days) with serum was important to establish sufficient growth of this cell line on the microcarriers for subsequent serum-free culture, and to support a long-term production phase (about 2 months). In the production phase, a high glucose concentration (6.15 g/l) in the serum-free medium was very effective for prolonging the harvest cycle interval. Under these conditions, up to 100 mg/l rsTM was expressed in the conditioned medium. The rates of glucose consumption (G) and lactae production (L) were measured periodically and their ratio (L/G ratio) correlated with rsTM productivity. When the average L/G ratio was lower, reflecting a lower lactate production rate due to appropriate oxygenation of the culture, the specific rsTM production rate increased. Thus it may be possible to estimate protein productivity from L/G ratios calculated from the glucose and lactate measurements. Correspondence to: M. Ogata     

Blockage of polymerase-catalyzed DNA chain elongation by chemically modified cytosine residues in templates and the release of blockage for readthrough.

The Klenow fragment-mediated in vitro DNA elongation was inhibited by the presence of a class of modified cytosines in the template DNA, i.e., the N4-amino(and -methoxy)-5,6-dihydrocytosine-6-sulfonate residues. We have studied the mechanism of the blockage, using as templates bisulfite-hydrazine (and -methoxyamine)- modified single strand phage-M13mp2 DNA and synthetic oligonucleotides. Both N4-amino-5,6-dihydrocytosine-6-sulfonate and N4-methoxy-5,6-dihydrocytosine-6-sulfonate residues blocked the elongation at one nucleotide before these sites. In this blockage, the idling of polymerase at the lesion site due to its 3'-5' exonuclease action appears not to play a major role, because Sequenase that lacks the 3'-5' exonuclease activity still could not readthrough these sites. It seems possible that conformational distortion of the template near these sites is responsible for the blockage, because on conversion of this 5,6-dihydropyrimidine-6-sulfonate structure into a planar pyrimidine, a complete restoration of polymerase-readthrough resulted. In the presence of RecA and SSB proteins, the Klenow fragment was able to partially readthrough these sites. Since there was no decrease in the 3'-5' exonuclease activity during this readthrough, it seems that the binding of these proteins relaxes the distortion in the modified template to allow the polymerase to readthrough the lesion site. These sites on phage DNA can be lethal but also are capable of inducing C-to-T transitions. This observation suggests that these sites can be read by E. coli DNA polymerases in vivo with accompanying errors.     

Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk

We described genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence, GlyLeuGlyGlyGlnGlyGlyGlyAlaGlyGlnGlyGlyTyrGly, designated SCAP(1), in spidroin I of spider dragline silk from Nephila clavipes and the secondary conformational analyses in the solid state by Fourier transform ir measurements. The polypeptides composed of 4, 5, 6, 7, 11, 12, or 13 repeats of SCAP(1) were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and then cleaved with cyanogen bromide to release N- and C-terminal extensions. Typical yields were from 1.2 to 5.2 mg of lyophilized uncleaved polypeptides per liter of fermentation medium at an absorbance of 2.0 at 600 nm, and the production levels increased with decreasing the molecular weight of the expressed polypeptides. The lyophilized powder of cleaved SCAP(13) adopted the random coil, whereas the cast film from formic acid formed the β-sheet structure. The conformational results might indicate that the glycine-rich sequence formed β-sheet structure in spidroin I. Cleaved SCAP(13) started to decompose under nitrogen at ca. 230°C, which was in agreement with the decomposition temperature of the spider dragline silk from N. clavipes. © 1998 John Wiley & Sons, Inc. Biopoly 45: 269–279, 1998     

Dynamic structure of subtilisin-eglin c complex studied by normal mode analysis

Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding. Proteins 32:324–333, 1998. © 1998 Wiley-Liss, Inc.     

3-O-Glutaryl-dihydrobetulin and related monoacyl derivatives as potent anti-HIV agents

3-O-Acyl-betulin and -dihydrobetulin derivatives were prepared and evaluated for anti-HIV activity. 3-O-Glutaryl-dihydrobetulin (17) demonstrated extremely potent anti-HIV activity with an EC(50) value of 2 x 10(-5) microM and a TI value of 1.12 x 10(6). 3-O-(3',3'-Dimethylsuccinyl)- and 3-O-(3',3'-dimethylglutaryl)-dihydrobetulins (15, 16) were also potent anti-HIV compounds with EC(50) values of 0.0017 and 0.0013 microM, respectively, and TI values of 16,160 and 19,530, respectively.     

Susceptibility alleles and haplotypes of human leukocyte antigen DRB1, DQA1, and DQB1 in autoimmune polyglandular syndrome type III in Japanese population

BACKGROUND: It has been reported that HLA class II haplotypes DRB1*0405-DQA1*0303-DQB1*0401 and DRB1*0901-DQA1*0302-DQB1*0303 are major susceptibility haplotypes for type 1 diabetes mellitus (DM) in Japanese population. However, little has been reported on the susceptibility HLA class II haplotypes in Japanese patients with autoimmune polyglandular syndrome type II and type III (APS III). PATIENTS AND METHODS: HLA class II haplotypes of DRB1-DQA1-DQB1 in 31 patients with APS III, 14 patients with Hashimoto's thyroiditis alone, and 15 patients with Graves' disease alone were examined in Japanese population. APS III patients were divided into three groups (A, B, and C) depending on the combination of autoimmune endocrine diseases. RESULTS: In 13 APS III patients with both Hashimoto's thyroiditis and type 1 DM (group A), the haplotype frequencies of the HLA DRB1*0802-DQA1*0401-DQB1*0402 and DRB1*0901-DQA1*0302-DQB1*0303 were significantly higher than in the controls. In patients with Hashimoto's thyroiditis alone, the haplotype frequency of DRB1*0901-DQA1*0302-DQB1*0303 was significantly higher than in controls, whereas the frequency of DRB1*0802-DQA1*0401-DQB1*0402 did not differ significantly from those in the controls. In 11 APS III patients with both Graves' disease and type 1 DM (group B), the haplotype frequencies of HLA DRB1*0405-DQA1*0303-DQB1*0401 and DRB1*0802-DQA1*0301-DQB1*0302 were significantly higher than in controls. In patients with Graves' disease alone, the haplotype frequency of DRB1*0803-DQA1*0103-DQB1*0601 were significantly higher than those in controls, suggesting that the susceptibility haplotypes for group B APS III differed from those for Graves' disease alone. In 7 APS III patients with both autoimmune thyroid diseases and pituitary disorders (group C), the haplotype frequency of HLA DRB1*0405-DQA1*0303-DQB1*0401 was significantly higher than in controls. CONCLUSIONS: Susceptible HLA class II haplotypes of DRB1-DQA1-DQB1 for APS III differ between the Japanese and Caucasian populations. More interestingly, the susceptible HLA class II haplotypes differ among the three types of Japanese APS III and are not merely a combination of susceptibility haplotypes of each endocrine disease.     

Xrs2p regulates Mre11p translocation to the nucleus and plays a role in telomere elongation and meiotic recombination

The Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in meiotic recombination, repair of damaged DNA, telomere elongation, and cell cycle checkpoint control. Xrs2p is known to be essential for all the functions of the complex, but its role in the complex has not been clearly elucidated. A 32-amino acid region near the C terminus of Xrs2p was identified as an Mre11p-binding site. No more function of Xrs2p than translocation of Mre11p from the cytoplasm to the nucleus is necessary for response to DNA damage. However, domains in Xrs2p located both 49 amino acids upstream and 104 amino acids downstream of the Mre11p binding site are required for meiotic recombination and telomere elongation, respectively, in addition to the 32-amino acid region. These findings demonstrate that Xrs2p acts as a specificity factor that allows the MRX complex to function in meiotic recombination and in telomere elongation.