Enhancement of dissolution and bioavailability of flurbiprofen by low molecular weight chitosans.

The dissolution behavior and absorption of flurbiprofen (FP) following oral administration from:three types of chitosans (LM chitosans), with different molecular weights and degree of acetylation, have been studied in comparison with those of the drug alone. The solubility of FP increased with concentrations of LM chitosan, especially in the case of C-III, with the highest degree of deacetylation degree among the three chitosans. This indicates that amino groups of LM chitosan play an important role in its interaction with FP. Moreover, spectroscopic studies, including NMR data, indicate that the binding involves interactions between the carboxyl group of FP and the amino group of the chitosans. The dissolution rates of FP for a C-III kneaded mixture were enhanced with increasing amounts of C-III. The oral absorption of FP from a C-III kneaded mixture was improved to a significant extent, compared to FP alone. These results suggest that FP from LM chitosan kneaded mixture increases the dissolution rate and improves the bioavailability of the drug by the formation of a water-soluble complex.

Purification, molecular cloning and ethylene-inducible expression of a soluble-type epoxide hydrolase from soybean (Glycine max [L.] Merr.).

A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23-mer oligonucleotides with sequences corresponding to an internal region of eight amino-acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase-like precursor consisting of 341 amino-acid residues, suggesting that 25 amino-acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide-diol converting activity using styrene oxide as substrate. The Vmax and Km values for styrene oxide are 1.36 micromol x min-1 x mg-1 and 1500 microM, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp fragment from this clone was found to cover the entire coding region together with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. The coding region of the gene, interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self-defense systems of plant.

Molecular cloning and ethylene-inducible expression of Chib1 chitinase from soybean (Glycine max (L.) Merr.).

A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.

An Isoelectric Separation of Soybean ß-Amylase Isoforms and Their Enzymic Characteristics.

Authentic soybean ß-amylase preparation, purified to homogeneity as judged by SDS-PAGE by using an affinity purification step, was composed of four pI-differing isoforms. By chromatofocusing, these isoforms were separated into three fractions, designated as fractions 1-3 in the order of elution. Fraction 1 contained two isoforms having the same molecular mass (55,989 Da), as measured by mass spectrometric analysis, with different pIs, 5.32 (Isoform I) and 5.22 (Isoform II). Fraction 2 showed a single isoform having a molecular mass of 55,994 Da and having a pI of 5.09. This component, named Isoform III, existed rather in excess in a mixture of the authentic enzyme isoforms. The remainder (fraction 3) also contained a single component (Isoform IV) which has a molecular mass of 56,310 Da with a pI of 4.97. Chemical analyses indicated that the N-termini and the C-terminal tripeptides of four pI-separated isoforms mentioned are similar to one another, and are blocked and are NH2-Val-Asp-Gly-COOH, respectively. Moreover, enzymic properties involving specific activity and the value of kcat/Km for the above three fractions are almost the same, and also agreed completely with those of an unfractionated authentic ß-amylase preparation.

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

A class III acidic endochitinase is specifically expressed in the developing seeds of soybean (Glycine max [L.] Merr.).

A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase specific activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was purified from mature dry seeds. Based upon the selected part (the residue positions 10-17) of the determined N-terminal 38 amino acid sequence, a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The resulting 1340 bp cDNA was comprised of a 5'-untranslated region of 39 bases, a coding region corresponding to a 25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34,269, calculated pI 4.7), and a 235 nucleotide 3'-terminal untranslated region including 24 bases of the poly(A) tail. By comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than 50% identical to every class III acidic chitinase, but has no significant similarity to other families of chitinases. The comparison also showed that the C-terminal region of this chitinase is markedly extended, by at least 31 residues. Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean. Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed defense, not only in development but also in dormancy of soybean seed. This is the first reported isolation and cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose that this enzyme would be classified into a new class of chitinase PR-8 family, together with a Sesbania homologue.

A rapid purification method for soybean Bowman-Birk protease inhibitor using hydrophobic-interaction chromatography.

A protein fraction was isolated from defatted soybean flour by extraction at acid pH, 40% ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration chromatography. SDS-PAGE, under reducing conditions, confirmed it as a homogeneous preparation. This conclusion was consistent with N-terminal amino acid sequence data (20 cycles) which showed a major sequence identical to those reported for soybean Bowman-Birk-type protease inhibitor (BBI), and also indicated a minimum 95% purity based on recoveries of PTH-amino acid residues. The purified fraction inhibited both trypsin and chymotrypsin with average specific activities of 350 and 672 units mg(-1), respectively. Compared with classical BBI purification, this procedure is very rapid requiring only 72-96 h to achieve a yield of 37 mg purified BBI per 200 g starting material.

Phosphonothrixin, a novel herbicidal antibiotic produced by Saccharothrix sp. ST-888. I. Taxonomy, fermentation, isolation and biological properties.

A novel herbicidal compound, phosphonothrixin, was found in the fermentation broth of Saccharothrix sp. ST-888 cultured on a vegetable juice medium. The compound exhibiting acidic and hydrophilic properties was obtained when the fermentation broth of ST-888 was subjected to ion exchange chromatography, gel filtration chromatography and ion-pair chromatography. Phosphonothrixin significantly inhibited germination of gramineous and broadleaf weeds. Foliar application of this antibiotic gave rise to chlorosis in all of the plants tested.

Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site.

By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH2-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze -Asn248-Asn249- but not -Asn249-Val250-, in the heptapeptide Gly-Asn248-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.

cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors.

A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.