Chemotaxonomy of Portuguese Ulex: quinolizidine alkaloids as taxonomical markers

Six species of Portuguese Ulex L. in a total of nineteen populations were studied by GC-EIMS as to their content in quinolizidine alkaloids. Sparteine, beta-isosparteine, jussiaeiine A, N-methylcytisine, cytisine, 5,6-dehydrolupanine, rhombifoline, lupanine, jussiaeiine B, N-formylcytisine, N-acetylcytisine, anagyrine, jussiaeiine C, jussiaeiine D, pohakuline, baptifoline, and epibaptifoline were detected. Analysis of the chromatograms showed that the chemical profile of all species was mainly composed of N-methylcytisine, cytisine, anagyrine, and jussiaeiines A, B, C and D. Therefore a quantification study of these alkaloids in all the populations studied was done by GC. These data were then submitted to cluster analysis and principal component analysis, which allowed the definition of five chemotypes and the recognition of hybrids. N-methylcytisine, cytisine, and jussiaeiines A, C and D are recognized as markers of this genus in Portugal.     

Retention of D genome chromosomes in pentaploid wheat crosses

The transfer of genes between Triticum aestivum (hexaploid bread wheat) and T. turgidum (tetraploid durum wheat) holds considerable potential for genetic improvement of both these closely related species. Five different T. aestivum/T. turgidum ssp. durum crosses were investigated using Diversity Arrays Technology (DArT) markers to determine the inheritance of parental A, B and D genome material in subsequent generations derived from these crosses. The proportions of A, B and D chromosomal segments inherited from the hexaploid parent were found to vary significantly among individual crosses. F(2) populations retained widely varying quantities of D genome material, ranging from 99% to none. The relative inheritance of bread wheat and durum alleles in the A and B genomes of derived lines also varied among the crosses. Within any one cross, progeny without D chromosomes in general had significantly more A and B genome durum alleles than lines retaining D chromosomes. The ability to select for and manipulate this non-random segregation in bread wheat/durum crosses will assist in efficient backcrossing of selected characters into the recurrent durum or hexaploid genotype of choice. This study illustrates the utility of DArT markers in the study of inter-specific crosses to commercial crop species.     

Norditerpenoid alkaloids from Delphinium species

From the aerial parts of four Delphinium species 11 known and 3 new norditerpenoid alkaloids have been isolated: from D. dissectum Huth: delavaine A/B, deoxylycoctonine, methyllycaconitine; new: 10-hydroxymethyllycaconitine; from D. excelsum Reichenb.: delcaroline, delectinine, delterine, methyllycaconitine; new: 10-hydroxymethyllycaconitine, 18-O-methyldelterine and 10-hydroxynudicaulidine; from D. grandiflorum L.: delcosine, deltatsine, grandiflorine, methyllycaconitine; from D. triste Fisch.: delcosine, macrocentridine, 14-dehydrodelcosine. The structures of the new alkaloids were established on the basis of MS, 1H, 13C, DEPT, homonuclear COSY, HMQC and HMBC NMR spectroscopic techniques.     

Asn124 of Cel5A from Hypocrea jecorina not only provides the N-glycosylation site but is also essential in maintaining enzymatic activity

To investigate the function of N-glycosylation of Cel5A (endoglucanase II) from Hypocrea jecorina, two N-glycosylation site deletion Cel5A mutants (rN124D and rN124H) were expressed in Saccharomyces cerevisiae. The weights of these recombinant mutants were 54 kDa, which were lower than that of rCel5A. This result was expected to be attributed to deglycosylation. The enzyme activity of rN124H was greatly reduced to 60.6% compared with rCel5A, whereas rN124D showed slightly lower activity (10%) than that of rCel5A. rN124D and rN124H showed different thermal stabilities compared with the glycosylated rCel5A, especially at lower pH value. Thermal stabilities were reduced and improved for rN124D and rN124H, respectively. Circular dichroism spectroscopy showed that the modification of secondary structure by mutation may be the reason for the change in enzymatic activity and thermal stability. [BMB Reports 2014; 47(5): 256-261]     

A hybrid model to study pathological mutations of the human ADP/ATP carriers

The adenine nucleotide carrier (Ancp) plays an essential role in the metabolism of cellular energy by catalyzing the transport of ADP and ATP across the inner mitochondrial membrane. Previous reports have indicated that mutations in the HANC1 gene, encoding the muscle isoform of human Ancp (HAnc1p), are directly involved in several diseases, including autosomal dominant progressive external ophthalmoplegia and cardiomyopathies. In this work, we studied three pathogenic HANC1 mutations at the biochemical level. To do so, we expressed the DdANCA gene, encoding the unique Ancp carrier of Dictyostelium discoideum (DdAncAp), in a yeast strain lacking all endogenous ANC genes. Our results indicate that DdAncAp is a good model for the human protein. It allows the carrier to be studied in yeast, and provides information on how the HANC1 mutations impair ADP/ATP transport in humans. A94D, A126D and V291M mutations, corresponding to A90D, A123D and V289M in HAnc1p, respectively, did not affect levels of DdAncAp in yeast mitochondria. However, while the wild-type DdAncAp fully restored growth of the ANC-null yeast strain on a non-fermentable carbon source, the carriers encompassing either the A94D or the A126D mutation failed to complement the null strain. The effect of the V291M mutation was not as pronounced, but led to impairment mainly of the nucleotide translocation process per se. These findings provide new insights into the mechanisms responsible for the diseases induced by HAnc1p mutations.     

Polyketides from Penicillium sp. JP-1, an endophytic fungus associated with the mangrove plant Aegiceras corniculatum

Four polyketides, leptosphaerone C (1), penicillenone (2), arugosin I (3) and 9-demethyl FR-901235 (4), as well as five known compounds, bacillosporin A (5), bacillosporin C (6), sequoiamonascin D (7), sequoiatone A (8), and sequoiatone B (9) were isolated from the Penicillium sp. JP-1, an endophytic fungus isolated from Aegiceras corniculatum. Their structures were determined by spectroscopic methods, mainly by 2D NMR spectroscopic analyses. Compound 1 showed cytotoxicity against A-549 cells with an IC50 value of 1.45 microM, while compound 2 showed cytotoxicity against P388 cells with an IC50 value of 1.38 microM.     

A new ecdysteroid with unique 9beta-OH and four other ecdysteroids from Silene italica ssp. nemoralis

A new natural ecdysteroid, 9beta,20-dihydroxyecdysone (1) and four related compounds 5alpha-20-hydroxyecdysone (2), 5alpha-2-deoxy-integristerone A (3), integristerone A (4) and 22-deoxy-integristerone A (5) were isolated from the herb of Silene italica ssp. nemoralis. Compound 1 is the C-9 epimer of the known 9alpha,20-dihydroxyecdysone (6) and represents a peculiar steroid skeleton. The structures of the compounds were elucidated by 1D and 2D NMR, IR and MS spectroscopy.     

Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability

Lipase L1 from Geobacillus stearothermophilus L1 contains an unusual extra domain, making a tight intramolecular interaction with the main catalytic domain through a Zn2+-binding coordination. To elucidate the role of the Zn2+, we disrupted the Zn2+-binding site by mutating the zinc-ligand residues (H87A, D61A/H87A, and D61A/H81A/H87A/D238A). The activity vs. temperature profiles of the mutant enzymes showed that the disruption of the Zn2+-binding site resulted in a notable decrease in the optimal temperature for maximal activity from 60 to 45-50 degrees C. The mutations also abolished the Zn2+-induced thermal stabilization. The wild-type enzyme revealed a 34.6-fold increase in stabilization with the addition of Zn2+ at 60 degrees C, whereas the mutant enzymes exhibited no response to Zn2+. Additional circular dichroism spectroscopy studies also confirmed the structural stabilizing role of Zn2+ on lipase L1 at elevated temperatures.     

Cross-reactivity between sera from dogs experimentally infected with Dirofilaria immitis and crude extract of Toxocara canis

This study was performed to investigate whethere there is cross-reactivity between Dirofilaria immitis and three intestinal nematodes of dogs. In ELISA, D. immitis-infected dog sera obtained at the 4th molting stage (9-11 weeks) and microfilaremic stage (25-30 weeks) were shown to be highly reactive with crude extract of T. canis. In immunoblotting, some antigenic fractions, 44, 57, 88, 100 kDa of crude extract of T. canis, were found to be positive reaction with sera of dogs infected with D. immitis. However, little or no cross-reaction were observed between sera of D. immitis-infected dogs and crude extract antigen of T. vulpis or A. caninum. These result suggest that there are partial cross reaction between sera of D. immitis-infected dogs and the antigen of T. canis.     

Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis.

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.     

Tyrolobibenzyls E and F from Scorzonera humilis and distribution of caffeic acid derivatives,lignans and tyrolobibenzyls in European taxa of the subtribe Scorzonerinae (Lactuceae,Asteraceae)

A chemosystematic study of the subtribe Scorzonerinae, a subtribe of the Lactuceae tribe of the Asteraceae family was performed, using the recently discovered tyrolobibenzyls as well as lignans and caffeic acid derivatives as diagnostic characters. In addition to the known compounds two new tyrolobibenzyls (E and F) were isolated and their structures were established by mass spectrometry and 1D and 2D NMR spectroscopy. Twenty four samples from rootstocks of seventeen different Scorzonerinae taxa, comprising members of three genera (Podospermum, Scorzonera, and Tragopogon), were analyzed. Tyrolobibenzyls A (1), B (2), C (5), D (3), E (6), and F (4) were identified in crude extracts by means of HPLC retention times, on-line UV spectra and on-line MS/MS spectra. Quantification of these compounds was performed by HPLC, using 2,2-bis-(4-hydroxyphenyl)-propane as an internal standard. Tyrolobibenzyls A-F were only detected in samples from Scorzonera humilis, while chlorogenic acid and 3,5-dicaffeoylquinic acid were detected in all samples investigated. In contrast, caffeoyl tartaric acid and cichoric acid were not detectable in any member of the subtribe Scorzonerinae.     

The tornabeatins, four tetrahydro-2-furanone derivatives from the lichenized ascomycete Tornabea scutellifera (With.) J.R. Laundon

Tornabeatins A, B, C and D, have been isolated as new natural products from the lichenized ascomycete Tornabea scutellifera, and their structures elucidated using UV, IR, MS, 1D and 2D NMR spectral data and chemical degradation.     

Nucleotide Substitution Patterning within the Meloidogyne rDNA D3 Region and Its Evolutionary Implications

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among acollection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified from genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in an isolate.     

Seasonal Fluctuations of Soil and Tissue Populations of Ditylenchus dipsaci and Aphelenchoides ritzemabosi in Alfalfa

Population dynamics of A. ritzemabosi and D. dipsaci were studied in two alfalfa fields in Wyoming. Symptomatic stem-bud tissue and root-zone soil from alfalfa plants exhibiting symptoms of D. dipsaci infection were collected at intervals of 3 to 4 weeks. Both nematodes were extracted from stem tissue with the Baermann funnel method and from soil with the sieving and Baermann funnel method. Soil moisture and soil temperature at 5 cm accounted for 64.8% and 61.0%, respectively, of the variability in numbers of both nematodes in soil at the Big Horn field. Also at the Big Horn field, A. ritzemabosi was found in soil on only three of the 14 collection dates, whereas D. dipsaci was found in soil on 12 dates. Aphelenchoides ritzemabosi was found in stem tissue samples on 9 of the 14 sampling dates whereas D. dipsaci was found on all dates. Populations of both nematodes in stem tissue peaked in October, and soil populations of both peaked in January, when soil moisture was greatest. Numbers of D. dipsaci in stem tissue were related to mean air temperature 3 weeks prior to tissue collection, while none of the climatic factors measured were associated with numbers of A. ritzemabosi. At the Dayton field, soil moisture plus soil temperature at 5 cm accounted for 98.2% and 91.4% of the variability in the soil populations of A. ritzemabosi and D. dipsaci, respectively. Aphelenchoides ritzemabosi was extracted from soil at two of the five collection dates, compared to extraction of D. dipsaci at three dates. Aphelenchoides ritzemabosi was collected from stem tissue at six of the seven sampling dates while D. dipsaci was found at all sampling dates. The only environmental factor that was associated with an increase in the numbers of both nematodes in alfalfa stem tissue was total precipitation 1 week prior to sampling, and this occurred only at the Dayton field. Numbers of A. ritzemabosi in stem tissue appeared to be not affected by any of the environmental factors studied, while numbers of D. dipsaci in stem tissue were associated with cumulative monthly precipitation, snow cover at time of sampling, and the mean weekly temperature 3 weeks prior to sampling. Harvesting alfalfa reduced the numbers of A. ritzemabosi at the Big Horn field and both nematodes at the Dayton field.     

Antifungal diterpenes from Hypoestes serpens (Acanthaceae)

Two new diterpenes, fusicoserpenol A and dolabeserpenoic acid A, with antifungal activity, were isolated from leaves of Hypoestes serpens (Acanthaceae). Their structures were elucidated by means of spectrometric methods including 1D and 2D NMR experiments and MS analysis. X-ray crystallographic analysis confirmed the structure of fusicoserpenol A and established the relative configuration.     

P450 enzymes from the bacterium Novosphingobium aromaticivorans

Twelve of the fifteen potential P450 enzymes from the bacterium Novosphingobium aromaticivorans, which is known to degrade a wide range of aromatic hydrocarbons, have been produced via heterologous expression in Escherichia coli. The enzymes were tested for their ability to bind a range of substrates including polyaromatic hydrocarbons. While two of the enzymes were found to bind aromatic compounds (CYP108D1 and CYP203A2), the others show binding with a variety of compounds including linear alkanes (CYP153C1) and mono- and sesqui-terpenoid compounds (CYP101B1, CYP101C1, CYP101D1, CYP101D2, CYP111A1, and CYP219A1). A 2Fe-2S ferredoxin (Arx-A), which is associated with CYP101D2, was also produced. The activity of five of the P450 enzymes (CYP101B1, CYP101C1, CYP101D1, CYP101D2, and CYP111A2) was reconstituted with Arx-A and putidaredoxin reductase (of the P450cam system from Pseudomonas putida) in a Class I type electron transfer system. Preliminary characterisation of the majority of the substrate oxidation products is reported.     

Fine structure of body wall cuticle of females of Meloidodera charis,Atalodera lonicerae,and Sarisodera hydrophila (Heteroderidae)

The body wall cuticle of adult females of Meloidodera charis, Atolodera lonicerae, and Sarisodera hydrophila is examined by transmission electron and light microscopy for comparison with Heterodera schachtii and previous observations of additional species of Heterodera, Globodera, and Punctodera. The cuticle of M. charis is least complex, consisting of layers A, B, C (with A outermost), and varies in overall thickness from 3 to 8 μm. As in other species, the cuticle is thickest in mature specimens. The cuticle of A. lonicerae is 6-9 μm thick; unlike M. charis it has an innermost layer, D, in addition to A, B, and C. The cuticle of S. hydrophila varies from 14 to 30 μm thick and includes a D layer similar to A. lonicerae; layer C is subdivided into additional zones relative to other heteroderids, and the external portion of the cuticle is infused with an electron-dense material. The presence of a D layer in A. lonicerae and S. hydrophila is a character state which is shared with Globodera spp. and Punctodera sp. The electron-dense material in the outer layers of S. hydrophila also occurs in Globodera spp. and Punctodera sp. On the other hand, H. schachtii resembles other Heterodera spp. as well as M. charis by the absence of a D layer and lack of electron-dense material in the outer layers. The pattern of occurrence of shared character states, including those of the cuticle, may be useful for phylogenetic analysis of Heteroderidae.     

Steroidal saponins from the roots of Asparagus racemosus

Five steroidal saponins, shatavarins VI-X, together with five known saponins, shatavarin I (or asparoside B), shatavarin IV (or asparinin B), shatavarin V, immunoside and schidigerasaponin D5 (or asparanin A), have been isolated from the roots of Asparagus racemosus by RP-HPLC and characterized by spectroscopic (1D and 2D NMR experiments) and spectrometric (LCMS) methods.     

Glucosinolates in Diplotaxis and Eruca leaves: diversity, taxonomic relations and applied aspects

Leaf glucosinolates of 42 Diplotaxis and 21 Eruca accessions were studied. Total content ranged from 0.25 to more than 70 g kg(-1) dry wt. The 13 clusters, defined on the basis of glucosinolate composition, belonged to two glucosinolate-rich groups, characterised by the prevalence of a single component, and one low-glucosinolate group, with a profile not dominated by any individual component. A sinigrin-rich cluster (D. ibicensis, D. berthautii, D. ilorcitana, D. siettiana, D. tenuisiliqua, D. brevisiliqua, and D. virgata) and a gluconapin-rich cluster (D. catholica, D.siifolia, D. virgata, and D. ollivieri) included all the species previously classified in the nigra phylogenetic lineage. D. virgata was confirmed to be a critical taxon, with one accession slightly diverging from the others. D. siifolia subsp. vicentina was separated from the others in a glucobrassicin-rich cluster. D. harra, a rather isolated representative of sub-genus Hesperidium, clustered together D. assurgens in a sinalbin-rich cluster. Another well defined cluster was represented by D. brachycarpa (gluconasturtin). The two sub-species of D. erucoides were well differentiated by their glucosinolate profile. The low glucosinolate species: D. tenuifolia, D. viminea, D. cretacea, D. muralis (subgenus Diplotaxis), and E. vesicaria, all previously included in the rapa/oleracea lineage, belonged to seven less defined clusters, mainly differing on the presence/absence or the relative abundance of some components (glucoraphanin, glucolepidin, 4-hydroxy-glucobrassicin, 4-phenylbutyl gls, glucoerucin and neoglucobrassicin). The data support previous taxonomic works. Glucosinolate-rich taxa, with well characterised profiles may be suitable for industrial uses, whereas the variability of edible D. tenuifolia and E. vesicaria may represent a basis for breeding horticultural types.     

Cardiobutanolide,a styryllactone from Goniothalamus cardiopetalus

A styryllactone namely cardiobutanolide was isolated from the stem bark of Goniothalamus cardiopetalus together with four known styryllactones goniothalamin, goniodiol, goniofufurone, goniofupyrone and known acetogenins squamocin and an epimeric mixture of goniodonin and 34-epi-goniodonin. The structure of the new compound was elucidated on the basis of 1D and 2D NMR experiments and mass spectroscopic techniques.