Production of a COX-2 inhibitor, 2,4,5-trimethoxybenzaldehyde, with submerged cultured Antrodia camphorata.

AIMS: To investigate the active ingredient in fruiting bodies and to produce it with cultured mycelium in Antrodia camphorata (BCRC 35398). METHODS AND RESULTS: The volatile components from the fruiting bodies, the liquid cultured broth of A. camphorata and Cinnamomum kanehirae wood were separately isolated by steam distillation-solvent extraction and identified by gas chromatography-mass spectrometry. In the fruiting bodies, a COX-2 inhibitor 2,4,5-trimethoxybenzaldehyde (TMBA) was found to be the most abundant constituent, but was totally absent in its cultured broth and its natural host, C. kanehirae wood. On feeding with the acid-digested sawdust of C. kanehirae wood or vanillin to the broth for culture, TMBA was produced in both cultured broths. CONCLUSION: The TMBA identified in fruiting bodies was an active ingredient whose functions consisted with the reported experiences of this mushroom. Feeding vanillin to culture broth could produce TMBA containing mycelium product like its fruiting bodies did. SIGNIFICANCE AND IMPACT OF THE STUDY: This study found an active ingredient in fruiting bodies of A. camphorata and elucidated this compound derived from digested sawdust of C. kanehirae wood. A feasible method was also developed to produce TMBA containing mycelium by feeding vanillin.

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Analysis of minimal sequences on JC virus VP1 required for capsid assembly.

Human JC virus (JCV) belongs to the family of Polyomaviridae. The viral capsid is composed of 72 capsomeres. Five VP1 molecules make up a capsomere structure. To investigate the minimal sequences on JCV VP1 polypeptide required for capsid assembly, the first 12 (Delta N12) and 19 (Delta N19) amino acids at the N-terminus and the last 16 (Delta C16), 17 (Delta C17), and 31 (Delta C31) amino acids at the C-terminus of VP1 were truncated and expressed in E. coli. The VP1 proteins of Delta N12 and Delta C16 were able to self-assemble into a virus-like particle similar to that of wild-type (WT) VP1. However, the mutant proteins of Delta N19, Delta C17, and Delta C31 formed a pentameric capsomere structure as demonstrated by a 10-50% sucrose gradient centrifugation and electron microscopy. These results suggest that the 12 amino-terminal and 16 carboxy-terminal amino acids of VP1 are dispensable for the formation of virus-like particles, and further truncation at either end of VP1 leads to the loss of this property.

Identification of a DNA encapsidation sequence for human polyomavirus pseudovirion formation.

Human polyomavirus is a naked capsid virus containing a closed circular double-stranded DNA genome. The mechanism of DNA encapsidation for the viral progeny formation is not fully understood. In this study, DNA encapsidation domain of the major capsid protein, VP1, of the human polyomavirus JCV was investigated. When the first 12 amino acids were deleted, the E. coli expressed VP1 (Delta N12VP1) failed to encapsidate the host DNA although the integrity of the capsid-like structure was maintained. In addition, capsid-like particles of Delta N12VP1 did not package exogenous DNA in vitro, which is in contrast to that of the full-length VP1 protein. These findings suggest that the N-terminal of the first 12 amino acids of VP1 were responsible for DNA encapsidation. The importance of amino acids in the DNA encapsidation domain was determined further using site-directed mutagenesis. All of the positively charged amino acids at the N-terminal region of VP1 were essential for DNA encapsidation. The results indicate that the N-terminal region of the human polyomavirus major capsid protein VP1 may be involved in viral genome encapsidation during progeny maturation.

Interstitial colocalization of two cervid satellite DNAs involved in the genesis of the Indian muntjac karyotype.

A number of repetitive DNA clones were generated from PCR amplifications of Indian muntjac genomic DNA using primer sequences derived from a white tailed deer satellite II DNA sequence. One clone (Mmv-0.7) was characterized and shown to be a cervid satellite II DNA clone. Multiple colored FISH studies with cervid satellite I (C5) and this satellite II clone (Mmv-0.7) to Chinese muntjac metaphase chromosomes localized both satellite DNAs at the pericentromeric regions of all chromosomes except for chromosome 3 and the Y chromosome, whereas chromosome 3 exhibited pericentromeric satellite II DNA only. Where distinguishable, the pericentromeric satellite II signals appeared terminally oriented with respect to satellite I. Six pairs of Chinese muntjac autosomes had interstitial satellite I sites with four of these autosomal pairs (chromosomes 1, 2 and two other smaller autosomal pairs) also exhibiting interstitial satellite II signals. An interstitial site on the X chromosome was found to have satellite II signals. For the Indian muntjac chromosomes, FISH studies revealed a pericentromeric hybridization for satellites I and II as well as 27 distinct interstitial hybridization sites, each having at least one of the satellite DNAs. These data were used to more precisely define the chromosome fusion-associated breakpoints that presumably led to the formation of the present-day Indian muntjac karyotype. It further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a 2n = 70 ancestral karyotype rather than from an intermediate 2n = 46 Chinese muntjac-like karyotype.

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families.

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.

The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells.

The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli. VP1 protein expressed in E. coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells. Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations. The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope. The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction. DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro. Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells. These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3. This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses. Furthermore, capsid-like particles of JC virus VP1 generated in E. coli potentially could be used as a human gene transfer vector.

Structure and genomic organization of porcine RACK1 gene.

The cDNA encoding porcine RACK1 protein was isolated from porcine spleen cDNA library. The deduced protein sequence of porcine RACK1 cDNA shows that it contains 317 amino acid residues, and shares nearly 100% identity with its vertebrate counterparts. Noticeably, the RACK1 protein was differentially expressed in various porcine tissues. High expression of RACK1 protein was observed in the tissues including thymus, pituitary, spleen and liver, whereas there was no detectable expression in muscle. The genomic DNA of porcine RACK1 with approximate 7.5 kb was constructed by both polymerase chain reaction amplification and genomic library screening. It consists of eight exons intervened by seven introns, and most of the intron/exon splice sites conform to the GT/AG rule. The promoter region contains functional serum response element, YY1-like binding site and AP1 site, which is supported by the finding that the expression of RACK1 gene in cultured porcine ST cells has a serum response as well as a TPA response.

Description of Saccharomonospora xinjiangensis sp. nov. based on chemical and molecular classification.

Comparative studies of morphology, physiology, biochemistry and chemical composition of cells, and phylogenetic analysis based on 16S rRNA gene sequences were carried out with strains XJ-54T and XJ-58 of the genus Saccharomonospora and type strains of related genera. The results indicated that the two strains are different from known members of the genus Saccharomonospora. A new species with the name Saccharomonospora xinjiangensis sp. nov. is proposed. The new species is characterized by the presence of longitudinal pairs of spores on both the aerial and the vegetative hyphae and contains phosphatidyl ethanolamine, phosphatidyl choline and unknown glucosamine-containing phospholipids, and the major menaquinones MK-9(H2), MK-9(H4) and MK-7(H4).

Analysis of the regulatory region of the lysC gene of Escherichia coli.

A lysC-lac'Z fusion plasmid was constructed to study the regulatory region of the lysC gene. Analysis by deletion mutations confirmed the existence of an alternative promoter, P2, located upstream of the previously identified promoter, P1. The transcription start site of promoter P2 was located 85 base pairs upstream the transcription start site of promoter P1. Both promoters are regulated by lysine.

Genomic cloning and sequence analysis of Taiwan-3 human polyomavirus JC virus.

Four different-strains of human polyomavirus JC virus (JCV), CY, Taiwan-1, Taiwan-2, and Taiwan-3, have been found in pregnant women and autoimmune disease patients in Taiwan. In this study, we report the cloning and sequencing of the Taiwan-3 JCV, virus isolated from the urine of an immunosuppressed patient with rheumatoid arthritis. The viral genome was amplified by polymerase chain reaction and then cloned into a prokaryotic replicative plasmid, pGEM-7Zf(-). The genomic DNA was sequenced and found to comprise 5,111 base pairs. The enhancer-promoter region of the viral genome lacks a copy of pentanucleotide-A (GGGAA) and pentanucleotide-B (AAAGC) compared to the CY archetypal JCV. There are 108 nucleotides altered in the total genome, excluding the variable part of the enhancer-promoter region, between Mad-1 (the prototype JC virus) and Taiwan-3. The enhancer-promoter region has approximately 25% of the altered nucleotides, resulting in amino acid changes in the open reading frames for I.T. capsid proteins (VP1, VP2, and VP3), and the agno protein. The cloned Taiwan-3, genome will provide an source for physiologic and pathologic investigation of the JCV virus in the future.

A target-specific chimeric toxin composed of epidermal growth factor and Pseudomonas exotoxin A with a deletion in its toxin-binding domain.

We have fused the epidermal growth factor (EGF) to the amino terminus of Pseudomonas exotoxin A (PE) to create a cytotoxic agent, designated EGF-PE, which preferentially kills EGF-receptor-bearing cells. In this study, we analyzed the effect of the Ia domain, the binding domain of PE on the cytotoxicity of EGF-PE towards EGF-receptor-bearing cells and tried to develop a more potent EGF-receptor-targeting toxin. EGF-PE molecules with sequential deletions at the amino terminus of PE were constructed and expressed in E. coli strain BL21(DE3). The cytotoxicity of these chimeric toxins was then examined. Our results show that the amino-terminal and carboxy-terminal regions of the Ia domain of PE are important for the cytotoxicity of a PE-based targeting toxin. To design a more potent PE-based EGF-receptor-targeting toxin, a chimeric toxin, named EGF-PE(delta 34-220), which had most of the Ia domain deleted but retained amino acid residues 1-33 and 221-252 of this domain, was constructed. EGF-PE(delta 34-220) has EGF-receptor-binding activity but does not show PE-receptor-binding activity and is mildly cytotoxic to EGF-receptor-deficient NR6 cells. As expected, EGF-PE(delta 34-220) is a more potent cytotoxic agent towards EGF-receptor-bearing cells than EGF-PE(delta 1-252), where the entire Ia domain of PE was deleted. In addition, EGF-PE(delta 34-220) was shown to be extremely cytotoxic to EGF-receptor-bearing cancer cells, such as A431, CE81T/VGH, and KB-3-1 cells. We also found that EGF-PE(delta 34-220) was highly expressed in BL21(DE3) and could be easily purified by urea extraction. Thus, EGF-PE(delta 34-220) can be a useful cytotoxic agent towards EGF-receptor-bearing cells.

Molecular cloning and sequence analysis of the cellobiohydrolase I gene from Trichoderma koningii G-39.

The cellobiohydrolase I gene, cbh1, has been cloned from an enhanced cellulase-producing strain, Trichoderma koningii G-39. Sequence comparisons show that T. koningii cbh1 is identical to that of T. reesei with the exception of 6 bp--two causing silent substitutions in the coding region, three differing in one of the introns, and one in 5'-noncoding region. Thus, it should encode an identical CBHI to that of T. reesei despite the differences in morphological characters of the two species. Analysis of approximately 1.4 kb of the 5' flanking region shows a number of surprisingly interesting putative regulatory features. There are no unusual features within about 600 bp upstream of the translation start ATG. However, prior to the 600-bp region, there are seven CAAT sequences, a number of direct and inverted repeats, and two C/T-rich regions. Also, there are five consensus 5'-(G/C)PyGGGG-3' sequences that have been identified to be carbon catabolite repressor binding sites of Aspergillus nidulans CREA and Saccharomyces cerevisiae MIG1 repressors. The structural organization around these consensus sequence regions is similar to those of A. nidulas alcR and alcA promoters. While the production of large amounts of CBHI by T. koningii upon induction apparently correlates with the large number of CAAT boxes in the 5' upstream untranslated region of cbh1, the presence of five CREA/MIG1 repressor-binding consensus sequences in the region suggests the wide-domain carbon catabolite repression regulatory system that controls the A. nidulans ethanol regulon, and yeast GAL genes transcription might also be operative and responsible for regulation of T. koningii cbh1 transcription.

Effects of lysine-sensitive aspartokinase III on lysine biosynthesis in Escherichia coli K-12.

A promoterless lysC gene, coding for Escherichia coli aspartokinase III (AKase III), has been cloned by phenotypic complementation using plasmid pUC19 as the vector. The hybrid plasmid obtained, pUC19AK3, preserved the ribosome binding site and transcriptional termination signal of the gene but with a lac promoter. E. coli strains containing the recombinant plasmid had high levels of AKase III activity. AKase III activity from expressing strains was inhibited by lysine, leucine, and S-(2-aminoethyl)-L-cysteine (AEC) but not by threonine and methionine. The overexpressed AKase III enzyme had a molecular weight of about 50 kD from SDS-polyacrylamide gel electrophoresis. N-terminal amino acid sequence analysis confirmed that the product from the hybrid plasmid was identical to native AKase III rather than a fusion protein. Moreover, overexpression of AKase III significantly increased lysine excretion in the plasmid-harboring E. coli strain DH1. This increase in the level of AKase III activity also affected other metabolites than lysine. Addition of aspartate to the medium brought about significant increases in lysine excretion. A maximum increase (about 8-fold) in lysine accumulation was observed 45 minutes after incubation in minimal medium containing 0.2% aspartate as compared to aspartate-free medium.

Purification and characterization of an endoxylanase from Trichoderma koningii G-39.

Trichoderma koningii G-39 produced xylanases in submerged culture using oat spelt xylan or crystalline cellulose, Avicel, as the sole carbon source. A low-Mr xylanase was purified from the culture filtrate by ion-exchange chromatography on SP-Trisacryl-M and gel filtration on Fractogel TSK HW-50F. It was homogeneous on SDS/PAGE and isoelectric focusing. A typical procedure provided about 11-fold purification with 4.5% protein yield and 50% activity recovery. The purified enzyme has an Mr value of about 21,500 and a pI of 8.9. Its specific activity was 6100 units/mg of protein, with optimal activity towards 0.5% xylan at about pH 5.5 and 60 degrees C. The purified enzyme had no activity against CM-cellulose with a degree of substitution of 0.63. It also showed no beta-xylosidase activity. The Km and Vmax. values, as determined with the soluble fraction of oat spelt xylan as substrate, were 0.70 mg/ml and 1.85 x 10(6) mumol/min per mg of enzyme respectively. Hg2+ (1 mM) and SDS (10 mM) completely inhibited xylanase activity, whereas Ca2+ showed no significant effect on the enzyme activity at 1 mM, but gave 80% inhibition at 10 mM. The enzyme contained about 4.4% carbohydrate and showed an immunological relationship to a cellobiohydrolase from the same fungal strain.

Specificity of the acid protease from Monascus kaoliang towards the B-chain of oxidized insulin.

The proteolytic specificity of the acid protease from Monascus kaoliang has been investigated using the B-chain of performic acid-oxidized insulin as peptide substrate. Six splittings were detected after 1 h digestion and 12 splittings were found after 12 h incubation at 37 degrees C, pH 4.8. The bonds most susceptible to the acton of M. kaoliang acid protease were Phe(24)-Phe(25), Leu(15)-Tyr(16) and Tyr(16)-Leu(17). Among the acid proteases compared, the specificity of M. kaoliang acid protease on the B-chain of oxidized insulin is more closely related to that of penicillopepsin with which it has ten cleavage sites in common. N-Acetyl-L-phenylalanyl-L-3,5-diiodotyrosine, a synthetic substrate for pepsin, was resistant to the hydrolysis of M. kaoliang acid protease.

Laser Raman studies on the conformation of Pro-Leu-Gly-NH2.

Laser Raman spectra of Pro-Leu-Gly-NH2, the factor that inhibits release of pituitary melanotropin, have been obtained in the solid state, in dimethylsuloxide and in aqueous solution. The amide I frequencies were observed at 1650 and 1687 cm-1 in the solid state and at 1669 cm-1 in dimethylsulfoxide. The conformation of this tripeptide has been proposed by 1H-NMR studies in [2H]-dimethylsulfoxide and revealed by X-ray analysis to be type II beta-turn. These observed amide I frequencies thus are characteristic of type II beta-turn conformation. The relatively lower amide I frequency, 1645 cm-1, observed in 2H2O indicates that the conformation of the peptide in aqueous solution could be different from those in solid state and in dimethylsulfoxide. pH changes hae no significant effect on aqueous solution spectra, except for the shape of the amide III band. The maximum of the amide III band shifted from 1259 cm-1 at pH 2.0 to 1242 cm-1 at pH 11.7. The amide III peaks in the solid state were at 1234 and 1268 cm-1.

Laser Raman studies on cobrotoxin.

Laser Raman spectra of cobrotoxin under various conditions have been obtained. Comparison of the spectra of native cobrotoxin in lyophilized form and in aqueous solution indicates that the secondary structures of cobrotoxin are not significantly affected by the removal of the aqueous solvent. On going from the native to the partially reduced and the completely reduced, carboxy-methylated forms, characteristic peaks of the C-S-S-C and tyrosine ring in the region of 500--900 cm-1 showed definite changes in structure. The partially reduced form gave two peaks at 502 and 524 cm-1, suggesting difference in the conformation of the remaining disulfide bonds. As indicated by the present work, the conformation of the main chain of cobrotoxin in the native unperturbed state, in the partially reduced and in the completely reduced forms are the coexistence of beta-pleated sheet with random-coil structure, predominantly random coil, and predominantly random coil with the existence of an alpha-helix type structure, respectively. The effect of pH on the conformation of cobrotoxin in solution appeared to give rise to the change of the local structure of two aromatic residues common to all snake neurotoxins.

Purification and characterization of an acid protease from Monascus kaoliang.

An acid protease from Monascus kaoliang was purified by consecutive applications of fractional acetone precipitation, batchwise CM-cellulose method and DEAE-cellulose column chromatography. The preparation was homogeneous on disc polyacrylamide gel electrophoresis at pH 4.5 and 7.5. The yield was about 30% with overall increase in specific activity of about 6-fold. The molecular weight as determined by SDS gel electrophoresis was about 34,000. The enzyme was a glycoprotease as indicated by specific carbohydrate staining on gels. It possessed the nature of an acid protease with a pH optimum at 3.0 toward heat-denatured casein and was stable over the range of pH 3.0 to 6.0. Reducing agents and thiol poisons had no effect on this enzyme, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inactivate this protease, indicating the probable absence of serine residue in the active site. The enzyme was inactivated by reaction with the carboxy-group specific reagent, 1,2-epoxy-3-(p-nitrophenoxy) propane (EPNP). Pepstatin, a specific inhibitor for pepsin, was shown to inhibit this enzyme strongly. However, biacetyl (2,3-butadione) had little effect on this protease, although it inactivated pepsin to an 85% activity loss. Also, p-bromophenacyl bromide, another specific inhibitor of pepsin, failed to inactivate this acid protease.