Genome-wide identification, classification and expression analysis of genes encoding putative fasciclin-like arabinogalactan proteins in Chinese cabbage (Brassica rapa L.)

Fasciclin-like arabinogalactan proteins (FLAs), a subclass of arabinogalactan proteins (AGPs), have both predicted AGP-like glycosylated regions and putative fasciclin (FAS) domains, which may function in cell adhesion and communication. Previous studies have identified 21, 27, and 34 FLAs in Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), respectively. In this study, we identified 33 FLAs in the annotated genome of Chinese cabbage (Brassica rapa ssp. pekinensis line Chiifu-401-42). Sequence analysis indicated that FAS domains each contain two highly conserved regions, named H1 and H2, and that 17 FLAs from B. rapa (BrFLAs) possess both of these regions. Prediction of glycosylphosphatidylinositol (GPI) modification sites suggested that 15 BrFLAs were GPI-anchored to the plasma membrane. Additionally, 25 BrFLAs may have been duplicated during the processes that shaped the triplicated genome of the mesopolyploid B. rapa. Expression analyses indicated that BrFLA1, BrFLA11, BrFLA13, BrFLA28 and BrFLA32 were specifically expressed in inflorescence. Meanwhile, BrFLA9 (homologous to AtFLA12) is specifically expressed in stem, and BrFLA6/22 (homologous to AtFLA11) is also highly expressed in stem, suggesting BrFLA6/9/22 may have the same functions as AtFLA11/12 in A. thaliana. Taken together, the identification and bioinformatic analysis of FLAs in B. rapa will open the way for studying their biological functions in plant growth and development as well as evolutionary history of this gene family from A. thaliana to B. rapa.     

The novel inhibitors of serine proteases

Thirty optically active nonprotein α-amino acids and peptides based thereon have been screened for their ability to interact with bovine trypsin and proteinase K from Tritirachium album Limber, which belong to the group of serine proteases. Both structure-based drug design approach and determination of enzyme activity have been used to identify low molecular weight inhibitors of trypsin and proteinase K. Compounds have been selected that according to the docking analysis were able to interact with trypsin and proteinase K. Following the docking analysis measurement of enzymes activity (2R,3S)-β-hydroxyleucine and (2S,3R)-β-hydroxyleucine inhibited both enzymes activity, whereas (S)-α-methyl-β-phenylalanine, (R)-α-methyl-β-phenylalanine, (S)-allylglycine, (R)-allylglycine, (S)-α-allylalanine, (R)-α-allylalanine and allo-O-ethylthreonine inhibited only proteinase K; and N-formyl-(S)-methionyl-(2S,3R)-hydroxyleucine, N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine, N-formyl-(S)-methionyl-(S)-allylglycine and N-formyl-(S)-methionyl-(R)-allylglycine inhibited trypsin. It has been shown that inhibition of trypsin by (2R,3S)-β-hydroxyleucine and N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine is of a competitive mode.     

Cloning,expression and dynamic simulation of TRYP6 from <Emphasis Type="Italic">Leishmania major</Emphasis> (MRHO/IR/75/ER)

Leishmania, a digenetic protozoan parasite causes severe diseases in human and animals. Efficient evasion of toxic microbicidal molecules, such as reactive oxygen species and reactive nitrogen species is crucial for Leishmania to survive and replicate in the host cells. Tryparedoxin peroxidase, a member of peroxiredoxins family, is vital for parasite survival in the presence of antioxidant, hence it is one of the most important molecules in Leishmania viability and then, it may be an appropriate goal for challenging against leishmaniasis. After cloning and sub-cloning of TRYP6 from Leishmania major (MRHO/IR/75/ER), homology modeling of the LmTRYP6 was proposed to predict some functional property of this protein. The refined model showed that the core structure consists of a seven β stranded β-sheet and five α helices which are organized as a central 7-stranded β2-β1-β5-β4-β3-β6-β7 surrounded by 2-stranded β-hairpin, α helices A and D on one side, and α helices B, C and E on the other side. The peroxidatic active site is located in a pocket formed by the residue Pro45, Met46, Thr49, Val51, Cys52, Arg128, Met147 and Pro 148. The catalytic Cys52, located in the first turn of helix αB, is in van der Waals with a Pro45, a Thr49 and an Arg128 that are absolutely conserved in all known Prx sequences. In this study, an attractive molecular target was studied. These results might be used in designing of drugs to fight an important human pathogen.     

β-Carbon stereoselectivity of N-carbamoyl-d-α-amino acid amidohydrolase for α,β-diastereomeric amino acids

N-Carbamoyl-d-α-amino acid amidohydrolase (d-carbamoylase) was found to distinguish stereochemistry not only at the α-carbon but also at the β-carbon of N-carbamoyl-d-α-amino acids. The enzyme selectively acted on one of the four stereoisomers of N-carbamoyl-α,β-diastereomeric amino acids. This simultaneous recognition of two chiral centers by d-carbamoylase was useful for the fine stereoselective synthesis of α,β-diastereomeric amino acids such as threonine, isoleucine, 3,4-methylenedioxyphenylserine and β-methylphenylalanine. The stereoselectivity for the β-carbon was influenced by the pH of the reaction mixture and by the bulk of the substituent at the β-carbon. Received: 18 June 1999 / Received revision: 30 July 1999 / Accepted: 6 August 1999     

Production and structure elucidation of di- and oligosaccharide lipids (biosurfactants) from Tsukamurella sp. nov.

The bacterium Tsukamurella sp. nov., isolated from soil, was found to produce novel glycolipids when grown on sunflower oil as the sole carbon source. The glycolipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The three main components are 2,3-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose, 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose and 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-β-d-galactopyranosyl-(1-6)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranosl which are linked to fatty acids varying in chain length from C₄ to C₁₈. The glycolipids are mainly extracellular but are also found attached to the cell walls. During the cultivation the composition of the glycolipids changed from disaccharide- to tri- and tetrasaccharide lipids. The glycolipids show good surface-active behaviour and have antimicrobial properties. Received: 22 May 1998 / Received revision: 24 August 1998 / Accepted: 26 August 1998     

Origin and evolution of the sulawesi macaques 1. Electrophoretic analysis of hemoglobins

The monkeys on the island of Sulawesi (Celebes), Indonesia, comprise seven species ofMacaca, that isM. maura, M. tonkeana, M. hecki, M. nigrescens, M. nigra, M. ochreata, andM. brunnescens. Hemoglobins from 248 individuals of these seven species were analyzed by isoelectric focusing electrophoresis (IEFE) and by starch gel electrophoresis in the presence of urea (USGE). Eighteen phenotypes consisting of eight molecular types were identified by IEFE analysis. The speciestonkeana inhabiting the central part of the island revealed 11 phenotypes, while peripheral species such asnigrescens andbrunnescens carried only 3 and 2 phenotypes, respectively. On USGE, three α chains and three β chains were identified and named α1, α2, and α6, and β1, β3, and β5, respectively. The α1 chain has the same mobility as the α chains of other macaques, while the α2 chain is less positively charged than α1, and α6 is the least positive among these α chains. The α2 chain is widely distributed in the Sulawesi macaques as the major component. Four species,ochreata, tonkeana, maura, andnigrescens, carried the α1 and α6 chains as minor components. The electrophoretic mobility of β1 was the same as that of other macaques, while β3 and β5 were more positively charged and less positively charged than β1, respectively. All of the Sulawesi species had β3 in high or low gene frequencies and inmaura, tonkeana, andbrunnescens, this type was most abundant. β5 chain existed in the species of the northern peninsula, as the major type. The subordinate type was β3 innigra andnigrescens and β1 inhecki. On the other hand, β1 was most frequently observed inochreata.     

Flavonol glycosides in the leaves ofTrillium apetalon Makino andT. kamtschaticum pallas

Seven flavonol glycosides were isolated from the leaves ofT. apetalon. They were identified chromatographically and spectrally to be: quercetin/kaempferol 3-O-α-arabinopyranosyl-(1→6)-β-galactopyranoside (TQ and TK), quercetin/kaempferol 3-O-[2‴-O-acetyl-α-arabinopyranosyl]-(1→6)-β-galactopyranoside (TAQ and TAK), quercetin 3-O-β-glucoside (ISQ), isorhamnetin 3-O-α-arabinopyranosyl-(1→6)-β-galactopyranoside (TI) and isorhamnetin 3-O-[2‴-O-acetyl-α-arabinopyranosyl]-(1→6)-β-galactopyranoside (TAI). TQ, TAQ, TI and TAI were major constituents. This is the first report on two new isorhamnetin-type glycosides, TI and TAI. The seven flavonol glycosides identical to those ofT. apetalon were isolated and identified in the leaves ofT. kamtschaticum; TQ and TAQ were also major components, but TI and TAI were only minor components. TI and TAI were not detected in the leaves ofT. tschonoskii. These leaf-flavonoid patterns were discussed from a chemosystematic point of view. Part 3 in the series “Studies of the flavonoids of the genusTrillium”. For Part 2 see Yoshitamaet al., (1997) J. Plant Res.110: 379–381.     

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.     

The <Emphasis Type="Italic">Endo-β-Mannanase</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis</Emphasis>, rice,and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4-d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.     

Plant Growth Inhibitory Compounds from Aqueous Leachate of Wheat Straw

When seedlings of lettuce, cress, rice and wheat were incubated with the leachate of wheat straw, the roots growth of lettuce and garden cress were particularly inhibited. The leachate of wheat straw (100 g eq./l) showed 80.5 and 79.4% inhibition for lettuce and cress roots, respectively. The inhibitory activity was stronger as the concentration of wheat straw leachate was greater. This result indicates that allelochemical(s) inhibiting the roots growth of lettuce and cress are leached from the wheat straw into the water. Two potent compounds were isolated from the leachate of the wheat straw and identified as syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan by spectral analyses. Syringoylglycerol 9-O-β-d-glucopyranoside inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 10.0 μM, respectively. On the other hand, l-tryptophan inhibited the roots growth of lettuce and cress at concentrations greater than 0.1 and 1.0 μM, respectively. The content of syringoylglycerol 9-O-β-d-glucopyranoside and l-tryptophan in the leachate of wheat straw (100 g eq./l) was 18.4 ± 0.7 and 6.2 ± 0.6 μM, respectively. Syringoylglycerol 9-O-β-d-glucopyranoside (18.4 μM) showed 21.5 and 13.5% inhibition in the lettuce and cress roots assay, respectively. On the other hand, 6.2 μM of l-tryptophan showed 47.5 and 35.0% inhibition in the lettuce and cress roots assay, respectively. These results suggested that l-tryptophan may be a major contributor to the allelopathy in aqueous leachate of wheat straw and syringoylglycerol 9-O-β-d-glucopyranoside may be a minor contributor.     

Synthesis of p-nitrophenyl sulfated disaccharides with beta-D-(6-sulfo)-GlcNAc units using beta-N-acetylhexosaminidase from Aspergillus oryzae in a transglycosylation reaction

When α-d-GlcNAc-OC₆H₄NO₂ -p and β-d-(6-sulfo)-GlcNAc-OC₆H₄NO₂-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC₆H₄NO₂ -p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC₆H₄NO₂-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC₆H₄NO₂-p (4) was obtained with α-d-Glc-OC₆H₄NO₂ -p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC₆H₄NO₂-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC₆H₄NO₂-p (5) in a yield of 13% based on the amount of 1 added.     

NMR assignment of the Rhodobacter sphaeroides fasciclin-1 domain protein (Fdp)

We report the almost complete assignment of ¹H, ¹³C and ¹⁵N nuclei in the 137-residue his-tagged fasciclin domain protein (Fdp) from Rhodobacter sphaeroides. Fdp is homologous to fasciclin I domains, including Drosophila FAS1 and M. tuberculosis MPB70 and plays a role in cell adhesion.     

甘蓝型油菜BnFLA基因的克隆及表达分析

利用同源克隆法从甘蓝型油菜中获得了1个类成束阿拉伯半乳聚糖蛋白基因(FLA),命名为BnFLA。BnFLA基因开放阅读框长为1 200bp,编码399个氨基酸,分子量为42 885.9Da,等电点为6.37。预测的BnFLA蛋白包含N-端信号肽、2个AGP-like结构域、2个fasciclin-like结构域和C-端GPI-anchor序列。系统进化分析表明BnFLA氨基酸序列与BrFLA17和AtFLA2进化关系较近,一致性分别为98%和87%。qRT-PCR分析表明,BnFLA基因在油菜各组织均有表达,并以下胚轴中表达量最高,其次为子叶,茎秆中表达最少;BnFLA基因的表达受到GA3、BR、IAA、ABA和NaCl的诱导,但受6-BA、蔗糖、低温和PEG抑制。研究认为,油菜中BnFLA基因可能参与激素信号转导途径和非生物胁迫应答。     

A review of acacic acid-type saponins from Leguminosae-Mimosoideae as potent cytotoxic and apoptosis inducing agents

The aim of this review is to highlight updated results on the biologically active saponins from Leguminosae-Mimosoideae. Acacic acid-type saponins (AATS), is a class of very complex glycosides possessing a common aglycon unit of the oleanane-type (acacic acid = 3β, 16α, 21β trihydroxy-olean-12-en-28 oic acid), having various oligosaccharide moieties at C-3 and C-28 and an acyl group at C-21. About sixty molecules of this type have been actively explored in recent years from Leguminosae family, from a chemical point of view and some fifty were reported to possess cancer related activities. These include cytotoxic/antitumor, immunomodulatory, antimutagenic, and apoptosis inducing properties and appear to depend on the acylation and esterification by different moieties at C-21 and C-28 of the acacic acid-type aglycone. One can observe that the (6S) configuration of the outer monoterpenyl moiety (MT) seems more potent in mediating high cytotoxicity than its (6R) isomer. Furthermore, the trisaccharide moiety {β-d-Xylopyranosyl-(1→2)-β-d-Fucopyranosyl-(1→6)- N-Acetamido 2-β-d-Glucopyranosyl-} at C-3, the tetrasaccharide moiety {β-d-Glucopyranosyl-(1→3)-[α-L-Arabinofuranosyl-(1→4)]-α-l-Rhamnopyranosyl-(1→2)-β-d-Glucopyranosyl} at C-28 of the aglycone, and the inner MT hydroxylated at its C-9, having a (6S) configuration can be important substituent patterns for the induction of apoptosis of AATS. Because of their interesting cytotoxic/apoptosis inducing activity, some AATS can be useful in the search for new potential antitumor agents from Fabaceae. Furthermore, the sequence 28-O-{Glc-(1→3)-[Ara_f-(1→4)]-Rha-(1→2)-Glc-Acacic acid}, often encountered in the genera Acacia, Albizia, Archidendron, and Pithecellobium may represent a chemotaxonomic marker of the Mimosoideae subfamily.     

A microcolinearity study at the earliness per se gene Eps-A m 1 region reveals an ancient duplication that preceded the wheat–rice divergence

Wheat flowering is controlled by numerous genes, which respond to environmental signals such as photoperiod and vernalization. Earliness per se (Eps) genes control flowering time independently of these environmental cues and are responsible for the fine tuning of flowering time. We recently mapped the Eps-A ^m 1 gene on the end of Triticum monococcum chromosome arm 1A^mL. As a part of our efforts to clone Eps-A ^m 1 we developed PCR markers flanking this gene within a 2.7 cM interval. We screened more than one thousand gametes with these markers and identified 27 lines with recombination between them. Recombinant lines were used to generate a high-density map and to investigate the microcolinearity between wheat and rice in this region. We mapped ten genes from a 149 kb region located at the distal part of rice chromosome 5 (cdo393 – Ndk3) on a 3.7 cM region on wheat chromosome one. This region is part of an ancient duplication between rice chromosomes 5 and 1. Genes present in both rice chromosomes were less similar to each other than to the closest wheat orthologues, suggesting that this duplication preceded the divergence between wheat and rice. This hypothesis was supported by the presence of 18 loci duplicated both in rice chromosomes 5 and 1 and in the colinear wheat chromosomes from homoeologous groups 1 and 3. Independent gene deletions in wheat and rice lineages explain the alternations of colinearity between rice chromosome 5 and wheat chromosomes 1 and 3. Colinearity between the end of rice chromosome 5 and wheat chromosome 1 was also interrupted by a small inversion, and several non-colinear genes. These results suggest that the distal region of the long arm of wheat chromosome 1 was involved in numerous changes that differentiated wheat and rice genomes. This comparative study provided sufficient markers to saturate the Eps-A ^m 1 gene region and to precisely map this gene within a 0.9 cM interval flanked by the VatpC and Smp loci. Sequences obtained in this study: DQ196178, DQ196179, DQ196180, DQ196181, DQ196182, DQ196183, DQ196184, DQ196185, DQ196186, DQ196187, DQ196488, DQ198537, DQ308530, DQ308531, DQ308532, DQ308533, DQ308534, DQ308535, DQ308536, DQ308537, DQ308538, DQ308539, DQ308540     

Characterization of the <Emphasis Type="Italic">Pragia fontium</Emphasis> Lipopolysaccharides

Lipopolysaccharides (LPSs) of two strains Pragia fontium 97U116 and 27480 were isolated and characterized; they were close to those of other representatives of the family Enterobacteriaceae in fatty acid composition and contained, respectively, 3-hydroxytetradecanoic acid as the predominant component (45.8 and 45.1%), tetradecanoic (23.5 and 28.9%), hexadecanoic (12.6 and 7.9%), hexadecenoic (12.6 and 7.9%), and dodecanoic (4.9 and 4.2%) fatty acids. The O-specific polysaccharides consisted of linear penta- and tetrasaccharide repeating units: →2)-α-D-Galf-(1→3)-α-L-Rhap2Ac-(1→4)-α-D-GlcpNAc-(1→2)-α-L-Rhap-(1→3)-β-D-GlcpNAc-(1→ →4)-β-D-ManpNAc3NAcA-(1→2)-α-L-Rhap-(1→3)-β-L-Rhap-(1→4)-α-D-GlcpNAc-(1→ The LPSs of P. fontium 97U116 and 27480 were serologically active and belonged to different serogroups; they were less toxic than those of strain E. coli O55:B5, but more pyrogenic than the Pyrogenal preparation.     

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997