Single nucleotide mutation leading to an amino acid substitution in the variant <Emphasis Type="Italic">Tik</Emphasis> soybean Kunitz trypsin inhibitor (SKTI) identified in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &; Zucc.)

The wild soybean (Glycine soja), which is the progenitor of cultivated soybean (Glycine max), is expected to offer more information about genetic variability and more useful mutants for evolutionary research and breeding applications. Here, a total of 1,600 wild soybean samples from China were investigated for genetic variation with regard to the soybean Kunitz trypsin inhibitor (SKTI). A new mutant SKTI, Tik, was identified. It was found to be a Tia-derived codominant allele caused by a transversion point mutation from C to G at nucleotide +171, leading to an alteration of one codon (AAC → AAG) and a corresponding amino acid substitution (Asn → Lys) at the ninth residue. Upon examination of this variant and others previously found in wild soybeans, it became clear that SKTI has undergone high-level evolutionary differentiation. There were more abundant polymorphisms in the wild than in the cultivated soybean.     

Allelic differentiation of Kunitz trypsin inhibitor in wild soybean (Glycine soja)

Soybean Kunitz trypsin inhibitor (SKTI) has several polymorphic types, which are controlled by co-dominant multiple alleles at a single locus. Of these types, Tia and Tib are predominant types, and there are nine differences in amino acids between Tia and Tib. Recently, an intermediate transitional type (Tib ( i5 )) between them was detected. However, other transitional types have not been detected despite surveys of many cultivated and wild soybeans. One of the reasons why other transitional variants have not been found is inferred to be due to the difficulty of the detection of SKTI protein variants by polyacrylamide gel electrophoresis (PAGE). To detect novel variants of SKTI, nucleotide sequence analysis in addition to PAGE was carried out. Four new variants were found from many Japanese wild soybeans. Of these variants, three (designated as Tia ( a1 ), Tia ( a2 ), Tia ( b1 )) were detected through gene sequence analysis on wild soybeans having the same electrophoretic mobility as Tia, and one (Tig) was detected through PAGE. The Tig variant showed a slightly lower electrophoretic mobility than Tic. The nucleotide sequences of Tig were identical to those of Tib except for one T --> C transitional mutation at position +340. The sequences of Tia ( a1 )and Tia ( a2 ) genes were identical to those of Tia with the exception of a G --> A mutation at position +376 and a T --> C mutation at +404, respectively. The sequence of Tia ( b1 ) differed from Tia by three nucleotides: C --> A at position +331, T --> C at +459 and A --> G at +484. Of the three nucleotide changes, two were common to Tia ( b1 ), Tib ( i5 ) and Tib, suggesting that Tia ( b1 ) is an intermediate transitional type between Tia and Tib. Our results suggest that Tib type has been differentiated through a series of mutations from Tia before the domestication of cultivated soybean.     

Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja).

A novel variant of soybean Kunitz trypsin inhibitor (SKTI) was detected in 530 lines of wild soybean (Glycine soja). This variant showed an intermediate electrophoretic mobility between the Tia and Tic types. In isoelectric focusing polyacrylamide gel electrophoresis gels containing urea, this variant had a similar isoelectric point as that of Tia. The genetic analysis of SKTI bands in F2 seeds from crosses of the new variant type with Tia or Tic type showed that this variant type is controlled by a codominant allele at the SKTI locus. We propose the genetic symbol Tif for this novel variant. When the nucleotide sequence of the Tif gene was compared with those of other types of SKTI genes (Tia, Tib, and Tic), the sequence of Tif was identical to that of Tib with the exception of one A-->G transitional mutation occurring at position 676 of Tif. This mutation resulted in an amino acid change from Lys to Glu at the 178 residue. These results suggest that this variant is derived from Tib through a point mutation. In addition, we settled an inconsistency in the number of amino acid differences between Tia and Tib (eight or nine). Analysis of nucleotide and amino acid sequences revealed that Tib was different from Tia by nine amino acids.     

Proteomic characterization of Kunitz trypsin inhibitor variants, Tia and Tib, in soybean [Glycine max (L.) Merrill]

The soybean Kunitz trypsin inhibitor (KTi) has several polymorphic variants. Of these, Tia and Tib, which differ by nine amino acids, are the two main types. In this study, differences in KTi proteome between Tia and Tib were investigated using three soybean cultivars and three mutant lines. Two cultivars, Baekwoon (BW) and Paldal (PD), and one mutant line, SW115-24, were Tia type, whereas one soybean cultivar, Suwon115 (SW115), and two mutant lines, BW-7-2 and PD-5-10, were Tib type. Protein from the six soybean lines was extracted and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), non-denaturing polyacrylamide gel electrophoresis (non-denaturing PAGE), and two-dimensional polyacrylamide gel electrophoresis (2-DE). By SDS-PAGE, there was no difference between soybean cultivars and mutant lines, except for SW115-24. Western blot analysis revealed that, in comparison with Tia, Tib type accumulated relatively low amounts of KTi. By non-denaturing PAGE, the three soybean lines of Tib type were characterized by slower mobility than the three soybean lines of Tia type. Zymography detected eight distinct zones of trypsin inhibitory activity among which Tia and Tib lacked the fifth and sixth zone, respectively. By two-dimensional native polyacrylamide gel electrophoresis (2-DN), the spots related to trypsin inhibitory activity showed different mobilities, whereas only one KTi (21.5?kDa) spot was resolved by 2-DE. By two-dimensional zymography (2-DZ), Tib showed a broader activity zone (pI 4-7) in comparison with Tia (pI 4-5). The results indicate that the genotypes with a different type of KTi present different proteomic profiles and trypsin inhibitory activities.     

Functional analysis of three amino acid residues of purR repressor, Trpl47, Gln-218 and Gln-292 inSalmonella typhimurium

The amber mutation sites of 6 purR(am) mutants were determined by cloning and DNA sequencing. The results showed that the mutations were distributed at three different sites in PurR coding region, G⁷²¹(→A), C⁹³³(→T) and C¹¹⁵⁵(→T), which respectively turn Trp-147,Gln-218 and Gln-292 of PurR into TAG terminal codon. To determine the effect of the three amino acid residues on regulatory function of PurR protein 5 different kinds of tRNA suppressor genes, Su3, Su4, Su6, Su7 and Su9 were used for creating the PurR protein variants with single amino acid substitution. The results indicated that Cys, Glu, Gly, His and Arg which substituted Trp-147 respectively all could not recover the regulation function of PurR. It confirmed that Trp-147 is a critical amino acid for the PurR function. Gln-292 substituted respectively by the same amino acids also could not recover the PurR function, demonstrating that Gln-292 is also an important amino acid residue in PurR.     

1-(<Emphasis Type="Italic">N</Emphasis>-Trifluoroacetylamino)alkylphosphonic acids: synthesis and properties

Summary. The 1-(N-trifluoroacetylamino)alkylphosphonic acids (TFA-AA^P) – sub-products in the synthesis of O,O-dialkyl 1-(N-trifluoroacetylamino)alkylphosphonates and O,O-diethyl 1-aminoalkylphosphonates, were synthesized in two-stage transformations of 1-aminoalkylphosphonic acids including: trifluoroacetylation of 1-aminoalkylphosphonic acids (AA^P) using a trifluoroacetic anhydride/trifluoroacetic acid reagent (AA^P + TFAA/TFA→2) and subsequent hydrolysis of the intermediary compounds 2 into desired TFA-AA^P (2→TFA-AA^P). These intermediates 2 presented mixtures of the type of mixed anhydrides of TFAA and 1-(N-trifluoroacetylamino)alkylphosphonic, pyrophosphonic and polyphosphonic acids, which underwent rapid and quantitative conversion to corresponding TFA-AA^P during treatment with an excess of water. The title acids were isolated by direct evaporation of the corresponding post-reaction mixtures, and their physicochemical proprieties, including deacylation abilities, were determined. TFA-AA^P compounds can be re-converted into the starting amino acids AA^P under respectively mild conditions (AA^P→TFA-AA^P→AA^P).     

Molecular Cloning, Mapping, and Polymorphism of the Porcine SCG2 gene

The secretogranin II (SCG2) gene is associated with the synthesis and secretion of follicle-stimulating hormone and luteinizing hormone. In the present study, we have determined the complete cDNA sequence of pig SCG2, which was submitted to GenBank with accession no. AY870646. Its complete open reading frame of 1,851 nucleotides encodes 616 amino acids. The predicted protein shares 80–87% identity with mouse, human, and bovine SCG2 proteins, and all four species share almost complete identity in the secretoneurin and EM66 domains. Pig SCG2 is a protein of 589 amino acids and 68,132 Da, preceded by a signal peptide of 27 residues. It contains nine pairs of dibasic residues, which are used as potential cleavage sites for generation of physiologically active peptides. Analysis of the SCG2 gene across the INRA-Minnesota porcine radiation hybrid panel indicates close linkage with microsatellite marker SW2608, located on Sus scrofa chromosome 15 (SSC15) q25, which harbors several QTL for ovulation rate and meat quality. Comparative sequencing and EST analysis revealed nine SNPs in porcine SCG2 cDNA, including seven SNPs in the coding region and two SNPs in the 3′ UTR. Four nonsynonymous SNPs (G622A, G1671T, C1718T, and A1790C) resulted in amino acid substitutions of Ala→Thr, Glu→Asp, Pro→Leu, and Asn→Thr, respectively.     

Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various ¹⁴C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [¹⁴C]formate, [2-¹⁴C]glycine and [2-¹⁴C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP → IMP → inosine → hypoxanthine → xanthine and GMP → guanosine → xanthosine → xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.     

Analysis of Chameleon Sequences by Energy Decomposition on a Pairwise Per-residue Basis

The conversion from α-helix to β-strand that has been widely observed in so-called chameleon sequences has received considerable attention since such a structural change may induce many amyloidogenic proteins to self-assemble into fibrils thus causing fatal diseases. Here we report a large scale-analysis of the energetics of secondary structural conversions in a collection of chameleon sequences retrieved from the Protein Data Bank. Major energetic contributions to the secondary structural conversion were analyzed by carrying out energy decomposition on a pairwise per-residue basis, i.e., (i,i), (i,i ± 1), (i,i ± 2), (i,i ± 3), (i,i ± 4) and > (i,i ± 4) intra-/inter-residual interactions. While the overall potential energy differences were subtle, individual residue-based interacting energy differences were observed to vary significantly depending on the specific type of secondary structural conversion. The average energy difference between α-helix and β-strand, <ΔE _α→β>, in the chameleon sequences varied significantly in (i,i), (i,i ± 1) and > (i,i ± 4) interactions. The major energetic factors in secondary structure conversions were electrostatic interactions and the polar term for solvation energy. In addition, residue-based average energy differences in α-helix → β-strand conversions were well-correlated to those in α-helix → random coil → β-strand conversions (R ² = 0.92). Assuming that three secondary structural elements can transform in either direction, this strong correlation indicates that the present energy decomposition method using database structures of chameleon sequences provides a reliable tool for the characterization of secondary structure fluctuations in amino acid sequences.     

Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^–1 and 3.1 × 10⁷ M^–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

A Trypsin Inhibitor from <Emphasis Type="Italic">Sapindus saponaria</Emphasis> L. Seeds: Purification,Characterization, and Activity Towards Pest Insect Digestive Enzyme

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS–PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.     

Structure and function of dnaQ and mutD mutators of Escherichia coli

Summary The nucleotide sequences of the recessivednaQ49 and the dominantmutD5 mutator were determined. ThednaQ49 mutator has a single base substitution in thednaQ gene, thus causing one amino acid change,₉₆Val (GTG)→ Gly (GGG), in the DnaQ protein (ε subunit of DNA polymerase III holoenzyme). ThemutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes,⁷³Leu (TTG)→Trp (TGG) and¹⁶⁴Ala (GCA)→Val (GTA), which were designated themutD52 andmutD51 mutations, respectively. Construction of chimaeric genes carrying one or two of these mutations revealed: (1) eithermutD51 ormutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; (2)mutD51, but notmutD52, exerts the dominant mutator phenotype when present in a low-copy plasmid; (3) the dominantmutD51 mutator activity is suppressed by thednaQ49 mutation when both mutations are present in the same gene. Based on these findings, we devised a model for the action of these mutators.     

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legume crop productivities. Protease inhibitors affect protein degradation and reduce the availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases and the growth and development of A. gemmatalis larvae exposed to soybean Bowman–Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan–Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmatalis was delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage wherein A. gemmatalis fed voraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline information about the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.     

Comparative study on amino acid sequences of Kunitz-type soybean trypsin inhibitors, Tia, Tib, and Tic

The amino acid sequences of three variants of the Kunitz-type trypsin inhibitors, Tia, Tib, and Tic, obtained from some cultivars of soybean were determined by conventional methods. All three inhibitors consisted of 181 amino acid residues. The differences in the amino acid sequences are as follows: Tia E12 G55 Y62 H71 S74 M114 L120 P137 L176; Tib S F N R V I T V; Tic E. The amino acid sequences of Pro(60)-Ser(61) and Asp(154)-Asp(155)-Gly(156)-His(157) of Tia reported previously (Koide & Ikenaka (1973) Eur. J. Biochem. 32, 417-431) were amended to Ser(60)-Pro(61) and His(154)-Asp-Asp-Gly(157), respectively.     

Purification of human seminal acrosin inhibitor and its kinetics

A low molecular mass, naturally occurring acrosin inhibitor has been identified and purified (490-7-fold) from human semen, and kinetic studies have been performed on the association characteristics as well as for the determination of affinity constants (K _i values). The results show thatK _i value (3.34 × 10⁻²) of the inhibitor towards human acrosin is almost three times lower than that of pancreatic trypsin, indicating a much higher specificity and inhibitory property for acrosin. The purified human seminal acrosin inhibitor has a molecular mass of 5.5 kDa and shows a single band using 10–20% gradient SDS PAGE. The work is of great significance for the development of more specific, nontoxic and irreversible inhibitors for human acrosin.     

Structural differences between albumin A and albumin C of the house mouse,Mus musculus

Two albumins, albumin A from C3H mice and albumin C isolated from descendents of the wild mice in which the variant was first uncovered, were found to differ in their electrophoretic properties. Albumin C was shown to bind two more H⁺ ions than albumin A at pH 5.4. Peptide mapping after trypsin digestion revealed that albumin C had three peptides (TP-C1, TP-C2, and TP-C3) which were missing in albumin A. The latter likewise had a peptide (TP-A1) which was not found in albumin C. An amino acid analysis of the variant peptides suggests that TP-A1 had been split into TP-C1 and TP-C2 on digestion with trypsin, because a glutamic acid in TP-A1 was replaced by a lysine. This change would also appropriately alter the electrophoretic properties of albumin C. No obvious counterpart was discovered for TP-C3 of albumin C in albumin A.This work was supported by a grant from the National Research Council of Canada.     

Di-<Emphasis Type="Italic">tert</Emphasis>-butylsilylene-directed α-selective synthesis of <Emphasis Type="Italic">p</Emphasis>-nitrophenyl T-antigen analogues

Seven analogues of p-nitrophenyl T-antigen [Galβ(1→3)GalNAcα(1→O)PNP] have been synthesized as potential substrates for elucidation of the substrate specificity of endo-α-N-acetylgalactosaminidase. These compounds, which are commercially unavailable, include: GlcNAcβ(1→3){GlcNAcβ(1→6)}GalNAcα(1→O)PNP [core 4 type], GalNAcα(1→3)GalNAcα(1→O)PNP [core 5 type], GlcNAcβ(1→6)GalNAcα(1→O)PNP [core 6 type], GalNAcα(1→6)GalNAcα(1→O)PNP [core 7 type], Galα(1→3)GalNAcα(1→O)PNP [core 8 type], Glcβ(1→3)GalNAcα(1→O)PNP and GalNAcβ(1→3)GalNAcα(1→O)PNP. The assembly of these synthetic probes was accomplished efficiently, based on di-tert-butylsilylene(DTBS)-directed α-galactosylation as a key reaction.     

Synergistic effect of DFMO and 2,4-D on regeneration of Tabernaemontana persicariaefolia jacq in vitro

Callus cultures of Tabernaemontana persicariaefolia, (Apocynaceae), an endangered species endemic to the Mascarene Islands, were established from leaf explants on MS medium containing either 5 mg·l⁻¹ 2,4-D and 0.5 mg·l⁻¹ BA or 5 mg·l⁻¹ 2,4-D, 0.5 mg·l⁻¹ BA and 200 mg·l⁻¹ DFMO. Histological studies showed regenerating nodules resembling globular embryos in calli after 4 weeks on the DFMO medium. Green shoot formation was achieved by sequential subculture of the induced calli on media with gradually decreasing 2,4-D concentrations (5→1→0 mg·l⁻¹). Regeneration was greatly stimulated in the presence of DFMO. The first emergence of shoots occured 3 weeks earlier than in untreated callus cultures.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [→ 3)-β-D-Galf-(1 → 5)-β-D-Galf-(1 →] _n → mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [→ 5)-β-D-Galf-(1 →] _n → mannan core. The mannan cores have also been investigated, and are constituted by a (1 → 6)-α-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 → 2) linked α-mannopyranoses. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.