Mutational analyses of cysteine residues of bovine dihydrodiol dehydrogenase 3

The cloning, bacterial expression and purification of bovine liver cytosolic dihydrodiol dehydrogenase 3 (DD3) cDNA (1330 bp in full length) using the pKK223-3 expression vector has been reported previously. Recombinant DD3 (rDD3) was characterized in terms of its substrate specificity and inhibitor sensitivity [Terada et al., Adv. Exp. Biol. Res. 414 (1997) 543-553]. The nucleotide sequence of DD3 cDNA completely matched with that of bovine liver-type prostaglandin F synthase [Suzuki et al., J. Biol. Chem. 274 (1999) 241-248]. In the present study, we succeeded in high level expression of rDD3 in Escherichia coli BL21 (DE3) using the pET28a expression vector. rDD3 was easily and quickly purified to apparent homogeneity by one-step column chromatography using Ni(2+)-affinity resin. Furthermore, rDD3 showed essentially the same substrate specificity and inhibitor sensitivity to that of purified liver DD3. To analyze the role of cysteines (145, 154, 188, 193 and 206) in the enzymatic activity of DD3, site-directed mutagenesis of DD3 using the polymerase chain reaction method was performed. Mutants (C145S, C154S, C188S, C193S and C206S) were analyzed for substrate specificity, cofactor binding and inactivation by disulfide (dithio-bis(2-nitrobenzoic acid), alkylating reagent (N-ethylmaleimide) and oxidants (naphthoquinone and H(2)O(2)) Results indicated that these five cysteines of rDD3 may not be directly involved in substrate or cofactor binding. Mutant C193S showed strong resistance to SH-reagents unlike wild-type DD3 (WT) or other mutants. Both the WT and the other mutants showed essentially the same sensitivity to SH-reagents. Cofactor (NADP(+)) protected mutants C145S, C188S and C206S from inactivation as well as WT, while NAD(+) was not protective. Our present results indicate that Cys193, which is located close to the NADP(+)-binding site, may be involved in the alteration of enzymatic activity.     

Lipopolysaccharide-induced IL-18 secretion from murine Kupffer cells independently of myeloid differentiation factor 88 that is critically involved in induction of production of IL-12 and IL-1beta

IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection.     

Cyclophilin A-independent replication of a human immunodeficiency virus type 1 isolate carrying a small portion of the simian immunodeficiency virus SIV(MAC) gag capsid region

Hybrid viruses between human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus strain mac (SIV(MAC)) are invaluable to various fields of HIV-1 research. To date, however, no replication-competent HIV-1 strain containing the gag capsid (CA) region of SIV(MAC) has been reported. To obtain the viable gag gene chimeric virus in an HIV-1 background, seven HIV-1 strains carrying a part of SIV(MAC) CA or a small deletion in the CA region were constructed and examined for their biological and biochemical characteristics. While all the recombinants and mutants were found to express Gag and to produce progeny virions on transfection, only one chimeric virus, which has 18 bp of SIV gag CA sequence in place of the region encoding the HIV-1 CA cyclophilin A (CyPA)-binding loop, was infectious for human cell lines. Although this chimeric virus was unable to grow in monkey lymphocytic cells like wild-type (wt) HIV-1 did, it grew much better than wt virus in the presence of cyclosporin A in a human cell line which supports HIV-1 replication in a CyPA-dependent manner. These results indicate that the transfer of a small portion of the SIV(MAC) CA region to HIV-1 could confer the CyPA-independent replication potential of SIV(MAC) on the virus.     

Thermal preconditioning protects rat cardiac muscle cells from doxorubicin-induced apoptosis

Doxorubicin (DOX=adriamycine), an effective chemotherapeutic agents for cancers, has severe cardiotoxicity. In the paresent study, we examined the protective effect of thermal preconditioning (TP) against apoptosis of rat cardiac muscle cells induced by DOX. Treatment with DOX (10 microM) for 24 hrs resulted in apoptosis of cardiac muscle cells, which was evaluated by examining "DNA ladder" formation and TUNEL staining. The number of TUNEL-positive cells was significantly decreased in cells subjected to TP by incubation at 42 degrees C for 30 min, 24 hrs prior to DOX-treatment. Antisense oligonucleotides of the heat shock protein (HSP) 70 blunted this effect. These results indicate that DOX-induced apoptosis in cardiac muscle cells is prevented by TP, at least in part, via a HSP70-mediated mechanism.     

Synthesis of alkyl fucosides through β-glucosidase-catalyzed condensation of fucose and 1-alcohols

-Glucosidase from almond catalyzed condensations of fucose and 1-alcohols with carbon numbers of 6 to 8 produce the corresponding 1-alkyl -d-fucosides. The enzyme also catalyzed the condensation of galactose and 1-hexanol. The conversion for the synthesis of hexyl fucoside was higher than those for the syntheses of hexyl glucoside and galactoside. The effect of initial saccharide concentrations on the conversions to alkyl glycosides was examined. The conversions at day 8 were almost the same at the low initial concentrations of the saccharides. However, the conversion significantly decreased as the concentrations increased. The surfactant properties of the prepared alkyl glycosides were measured. The critical micelle concentrations of the alkyl fucosides were lower than those of the glucosides and galactosides with the same alkyl chains.     

Soybean glycinin A1aB1b subunit has a molecular chaperone-like function to assist folding of the other subunit having low folding ability

Soybean (Glycine max L.) glycinin is composed of five subunits which are classified into two groups (group I: A1aB1b, A1bB2, and A2B1a; group II: A3B4 and A5A4B3). All the common soybean cultivars contain both group I and II subunits (Maruyama, N. et al., Phytochemistry, 64, 701-708 (2003)). The biosynthesis of group I starts earlier compared with that of the A3B4 subunit during seed development (Meinke, D.W. et al., Planta, 153, 130-139 (1981)). We have revealed that group I A1aB1b was mostly expressed as a soluble protein, but that A3B4 was expressed mainly as an insoluble protein in Escherichia coli under the same expression conditions; namely, A1aB1b had higher folding ability than A3B4. We therefore assumed that A1aB1b assists folding of group II subunits like a molecular chaperone does. In order to ascertain this, A1aB1b and A3B4 were co-expressed in E. coli. All of the expressed proteins of A3B4 were recovered in a soluble fraction. To confirm this result, we also co-expressed A1aB1b with modified A3B4 versions having extremely low folding ability. All expressed modified A3B4 versions were soluble. These results clearly suggest that A1aB1b has a molecular chaperone-like function in their folding.     

Accumulation and degradation of thiamin-binding protein and level of thiamin in wheat seeds during seed maturation and germination

Changes in the levels of thiamin-binding globulin and thiamin in wheat seeds during maturation and germination were studied. The thiamin-binding activity of the seed proteins increased with seed development after flowering. The thiamin content of the seeds also increased with development. Thiamin-binding activity decreased during seed germination. On the other hand, immunological analysis using an antibody directed against the thiamin-binding protein isolated from wheat seeds showed that the thiamin-binding globulin accumulated in the aleurone layer of the seeds during maturation, and then the protein was degraded and disappeared during seed germination. These results suggested that the thiamin-binding globulin of wheat seeds was synthesized and accumulated in the aleurone layer of the seeds with seed development, similar to the thiamin-binding albumin in sesame seeds, and that thiamin bound to the thiamin-binding globulin in the dormant wheat seeds for germ growth during germination.     

Improved bile acid-binding ability of soybean glycinin A1a polypeptide by the introduction of a bile acid-binding peptide (VAWWMY)

We have previously identified a potential bile acid-binding peptide sequence (VAWWMY) in acidic polypeptide A1a of the soybean glycinin A1aB1b subunit (Choi, S. K., et al., Biosci. Biotechnol. Biochem., 66, 2395-2401 (2002)). In this study, we introduced the nucleotide sequence encoding this peptide in the coding DNA which corresponds to amino acids between 251 and 256, and 282 and 287 into the A1a polypeptide by replacement to respectively give modified versions A1aM1 and A1aM2. A fluorescence analysis demonstrates that their bile acid-binding ability was improved compared to A1a. Moreover, modified proglycinin A1aB1b with the VAWWMY sequence at the same sites as those of A1aM1 and A1aM2 was judged to assume the correct conformation. These results suggest the possibility of developing transgenic crops to accumulate the modified glycinin.     

The solubilization and biological activities of Aspergillus beta-(1 --> 3)-D-glucan

We have recently demonstrated that the cell wall beta-glucan of Candida albicans could be solubilized by sodium hypochlorite, followed by dimethylsulfoxide-extraction (NaClO-DMSO method). In this study, applying this method to Aspergillus spp., we prepared mycelial cell wall beta-glucan and examined its physical properties and immunotoxicological activity. The acetone-dried mycelia of Aspergillus spp. were oxidized by the NaClO-DMSO method. An analysis of (13)C NMR spectra revealed the preparations to be composed of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan. Also, the proportion of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan varied. Furthermore, a solubilized Aspergillus beta-glucan (ASBG) was prepared from OX-Asp by urea-autoclave treatment. ASBG showed limulus activity similar to Candida solubilized beta-glucan (CSBG), and there was little difference in the activity of ASBG between various Aspergillus spp. ASBG affected the production of IL-8 by human peripheral blood mononuclear cells (PBMC). ASBG should be useful for analyzing the clinical role of beta-glucan.