Hepoxilin A3 (HXA3) synthase deficiency is causative of a novel ichthyosis form

Non-bullous congenital ichthyosis erythroderma (NCIE) and lamellar ichthyosis (LI) are characterized by mutations in 12R-lipoxygenase (12R-LOX) and/or epidermal lipoxygenase 3 (eLOX3) enzymes. The eLOX3 lacks oxygenase activity, but is capable of forming hepoxilin-type products from arachidonic acid-derived hydroperoxide from 12R-LOX, termed 12R-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12R-HpETE). Mutations in either of two enzymes lead to NCIE or LI. Moreover, 12R-LOX-deficient mice exhibit severe phenotypic water barrier dysfunctions. Here, we demonstrate that 12R-HpETE can also be transformed to 8R-HXA(3) by hepoxilin A(3) (HXA(3)) synthase (12-lipoxygenase), which exhibits oxygenase activity. We also presented a novel form of ichthyosis in a patient, termed hepoxilin A(3) synthase-linked ichthyosis (HXALI), whose scales expressed high levels of 12R-LOX, but were deficient of HXA(3) synthase.     

Effects of geographical heterogeneity in species interactions on the evolution of venom genes

Geographical heterogeneity in the composition of biotic interactions can create a mosaic of selection regimes that may drive the differentiation of phenotypes that operate at the interface of these interactions. Nonetheless, little is known about effects of these geographical mosaics on the evolution of genes encoding traits associated with species interactions. Predatory marine snails of the family Conidae use venom, a cocktail of conotoxins, to capture prey. We characterized patterns of geographical variation at five conotoxin genes of a vermivorous species, Conus ebraeus, at Hawaii, Guam and American Samoa, and evaluated how these patterns of variation are associated with geographical heterogeneity in prey utilization. All populations show distinct patterns of prey utilization. Three ‘highly polymorphic’ conotoxin genes showed significant geographical differences in allelic frequency, and appear to be affected by different modes of selection among populations. Two genes exhibited low levels of diversity and a general lack of differentiation among populations. Levels of diversity of ‘highly polymorphic’ genes exhibit a positive relationship with dietary breadth. The different patterns of evolution exhibited by conotoxin genes suggest that these genes play different roles in prey capture, and that some genes are more greatly affected by differences in predator–prey interactions than others. Moreover, differences in dietary breadth appear to have a greater influence on the differentiation of venoms than differences in the species of prey.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

A Survey of Intestinal Protozoan Infections among Gastroenteritis Patients during a 3-Year Period (2004-2006) in Gyeonggi-do (Province), South Korea

The incidence and etiology of parasite-associated gastroenteritis during 2004-2006 in Gyeonggi-do (province), South Korea was determined by means of antigen detection ELISA on 6,071 stool specimens collected from 6 general hospitals. At least 1 parasitic agent was detected in 3.4% (208/6,071) of the stool samples. Among these, Giardia lamblia was the most numerous (152 cases; 2.5%), followed by Entamoeba histolytica (25 cases; 0.4%), Cryptosporidium parvum (23 cases; 0.4%), and mixed infections (8 cases; 0.1%). Patients aged 1-5 years had the largest proportion (69.2%; 144/208) of parasite-positive stool specimens. Parasite-mediated gastroenteritis was most common from June to September. The detection rate gradually increased from 2004 to 2006. This study shows that parasite-mediated gastroenteritis may be significant among children in Korea and that parasite infection surveillance should be constantly performed.     

A revision of the Chinese Trigonalyidae (Hymenoptera,Trigonalyoidea)

The Chinese fauna of the family Trigonalyidae Cresson, 1887, is revised, keyed and fully illustrated for the first time. Eight genera of this family (Bakeronymus Rohwer, 1922, Bareogonalos Schulz, 1907, Jezonogonalos Tsuneki, 1991, re-instated, Lycogaster Shuckard, 1841, Orthogonalys Schulz, 1905, Pseudogonalos Schulz, 1906, Taeniogonalos Schulz, 1906 and Teranishia Tsuneki, 1991) are recorded from China. The genus Ischnogonalos Schulz, 1907, is synonymized with Taeniogonalos Schulz, 1906. In total 40 valid species are recognized. Twenty species are new for science: Jezonogonalos elliptifera sp. n., J. jiangliae sp. n., J. luteata sp. n., J. nigrata sp. n., Lycogaster angustula sp. n., L. flavonigrata sp. n., L. nigralva sp. n., Orthogonalys cheni sp. n., O. clypeata sp. n., O. robusta sp. n., Pseudogonalos angusta sp. n., Taeniogonalos bucarinata sp. n., T. cordata sp. n., T. geminata sp. n., T. sculpturata sp. n., T. triangulata sp. n., T. tricolorisoma sp. n., T. uncifera sp. n., Teranishia crenulata sp. n. and T. glabrata sp. n. Two species are reported new for China: Orthogonalys elongata Teranishi, 1929 and Nanogonalos flavocincta Teranishi, 1929 (renamed to Taeniogonalos subtruncata nom. n.). Seven new synonyms are proposed: Poecilogonalos yuasai Teranishi, 1938, and P. maga taiwana Tsuneki, 1991, of Taeniogonalos taihorina (Bischoff, 1914); Taiwanogonalos minima Tsuneki, 1991, and T. similis Tsuneki, 1991, of Taeniogonalos alticola (Tsuneki, 1991); P. intermedia Chen, 1949, and P. unifasciata Chen, 1949, of Taeniogonalos formosana (Bischoff, 1913). Six taxa are recognised as valid species: Bakeronymus seidakka Yamane & Terayama, 1983, Jezonogonalos laeviceps (Tsuneki, 1991), J. satoi (Tsuneki, 1991), Taeniogonalos alticola (Tsuneki, 1991), T. flavoscutellata (Chen, 1949) and T. gestroi (Schulz, 1908). Five new combinations are made: Jezonogonalos laeviceps (Tsuneki, 1991), comb. n., J. satoi (Tsuneki, 1991), comb. n., Taeniogonalos flavoscutellata (Chen, 1949), comb. n., T. gestroi (Schulz, 1908), comb. n. and T. lachrymosa (Westwood, 1874), comb. n. Lectotypes are designated for Lycogaster violaceipennis Chen, 1949, Poecilogonalos flavoscutellata Chen, 1949, P. rufofasciata Chen, 1949, and P. tricolor Chen, 1949.     

A revision of the Chinese Stephanidae (Hymenoptera, Stephanoidea)

Stephanidae Leach, 1815 (Hymenoptera: Stephanoidea) from China are revised. Five genera are reported from China: Foenatopus Smith, 1861; Megischus Brullé, 1846; Parastephanellus Enderlein, 1906; Schlettererius Ashmead, 1900; and Stephanus Jurine (in Panzer), 1801, and the genera are keyed. All the Chinese species are described and illustrated and new synonyms are established. Keys to species of the five genera occurring in China and adjacent regions are provided.SIX SPECIES ARE NEW TO SCIENCE: Foenatopus brevimaculatussp. n., Foenatopus maculiferussp. n., Foenatopus yangisp. n., Parastephanellus angulatussp. n., Parastephanellus brevicoxalissp. n. and Parastephanellus zhejiangensissp. n. One species, Parastephanellus matsumotoi van Achterberg, 2006, is newly recorded from China.The following 9 new synonyms are proposed: Foenatopus aratifrons Enderlein, 1913 and Foenatopus yunnanensis Chao, 1964, new synonymys for Foenatopus annulitarsus Enderlein, 1913; Foenatopus cerviculatus (Chao, 1964) and Foenatopus chaoi Belokobylskij, 1995 for Foenatopus chinensis (Elliott, 1919); Foenatopus formosanus Enderlein, 1913 for Foenatopus cinctus (Matsumura, 1912); Foenatopus simillimus (Elliott, 1920) and Foenatopus trilineatus (Elliott, 1920) for Foenatopus flavidentatus (Enderlein, 1913); Foenatopus trilobatus (Elliott, 1920) for Foenatopus ruficollis (Enderlein, 1913); Parastephanellus austrochinensis Belokobylskij, 1995 for Parastephanellus brevistigma Enderlein, 1913. A lectotype is designated for Diastephanus trilineatus Elliott, 1920.     

Synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone from D-glucose

We have described the synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone by the reduction of a keto-conduritol derivative, the latter being prepared in five steps from (-)-(2S,3R,4S,5S)-2,3,4-tribenzyloxy-5-hydroxycyclohexanone, which is in turn readily synthesized from D-glucose.     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

NH(4)(+)-stimulated low-K(+) uptake is associated with the induction of H(+) extrusion by the plasma membrane H(+)-ATPase in sorghum roots under K(+) deficiency

The effect of external inorganic nitrogen and K⁺ content on K⁺ uptake from low-K⁺ solutions and plasma membrane (PM) H⁺-ATPase activity of sorghum roots was studied. Plants were grown for 15 days in full-nutrient solutions containing 0.2 or 1.4 mM K⁺ and inorganic nitrogen as NO₃^-, NO₃^-/NH₄⁺ or NH₄⁺ and then starved of K⁺ for 24, 48 and 72 h. NH₄⁺ in full nutrient solution significantly affected the uptake efficiency and accumulation of K⁺, and this effect was less pronounced at the high K⁺ concentration. In contrast, the translocation rate of K⁺ to the shoot was not altered. Depletion assays showed that plants grown with NH₄⁺ more efficiently depleted the external K⁺ and reached higher initial rates of low-K⁺ uptake than plants grown with NO₃^-. One possible influence of K⁺ content of shoot, but not of roots, on K⁺ uptake was evidenced. Enhanced K⁺-uptake capacity was correlated with the induction of H⁺ extrusion by PM H⁺-ATPase. In plants grown in high K⁺ solutions, the increase in the active H⁺ gradient was associated with an increase of the PM H⁺-ATPase protein concentration. In contrast, in plants grown in solutions containing 0.2 mM K⁺, only the initial rate of H⁺-pumping and ATP hydrolysis were affected. Under these conditions, two specific isoforms of PM H⁺-ATPase were detected, independent of the nitrogen source and deficiency period. No change in enzyme activity was observed in NO₃^--grown plants. The results suggest that K⁺ homeostasis in NH₄⁺-grown sorghum plants may be regulated by a high capacity for K⁺ uptake, which is dependent upon the H⁺-pumping activity of PM H⁺-ATPase.     

Evolution of unusual morphologies in Lentibulariaceae (bladderworts and allies) and Podostemaceae (river-weeds): a pictorial report at the interface of developmental biology and morphological diversification

Background Various groups of flowering plants reveal profound (‘saltational’) changes of their bauplans (architectural rules) as compared with related taxa. These plants are known as morphological misfits that appear as rather large morphological deviations from the norm. Some of them emerged as morphological key innovations (perhaps ‘hopeful monsters’) that gave rise to new evolutionary lines of organisms, based on (major) genetic changes.Scope This pictorial report places emphasis on released bauplans as typical for bladderworts (Utricularia, approx. 230 secies, Lentibulariaceae) and river-weeds (Podostemaceae, three subfamilies, approx. 54 genera, approx. 310 species). Bladderworts (Utricularia) are carnivorous, possessing sucking traps. They live as submerged aquatics (except for their flowers), as humid terrestrials or as epiphytes. Most Podostemaceae are restricted to rocks in tropical river-rapids and waterfalls. They survive as submerged haptophytes in these extreme habitats during the rainy season, emerging with their flowers afterwards. The recent scientific progress in developmental biology and evolutionary history of both Lentibulariaceae and Podostemaceae is summarized.Conclusions Lentibulariaceae and Podostemaceae follow structural rules that are different from but related to those of more typical flowering plants. The roots, stems and leaves – as still distinguishable in related flowering plants – are blurred (‘fuzzy’). However, both families have stable floral bauplans. The developmental switches to unusual vegetative morphologies facilitated rather than prevented the evolution of species diversity in both families. The lack of one-to-one correspondence between structural categories and gene expression may have arisen from the re-use of existing genetic resources in novel contexts. Understanding what developmental patterns are followed in Lentibulariaceae and Podostemaceae is a necessary prerequisite to discover the genetic alterations that led to the evolution of these atypical plants. Future molecular genetic work on morphological misfits such as bladderworts and river-weeds will provide insight into developmental and evolutionary aspects of more typical vascular plants.     

Evolutionary history of the Afro-Madagascan Ixora species (Rubiaceae): species diversification and distribution of key morphological traits inferred from dated molecular phylogenetic trees

Background and Aims

Previous work on the pantropical genus Ixora has revealed an Afro-Madagascan clade, but as yet no study has focused in detail on the evolutionary history and morphological trends in this group. Here the evolutionary history of Afro-Madagascan Ixora spp. (a clade of approx. 80 taxa) is investigated and the phylogenetic trees compared with several key morphological traits in taxa occurring in Madagascar.

Methods

Phylogenetic relationships of Afro-Madagascan Ixora are assessed using sequence data from four plastid regions (petD, rps16, rpoB-trnC and trnL-trnF) and nuclear ribosomal external transcribed spacer (ETS) and internal transcribed spacer (ITS) regions. The phylogenetic distribution of key morphological characters is assessed. Bayesian inference (implemented in BEAST) is used to estimate the temporal origin of Ixora based on fossil evidence.

Key Results

Two separate lineages of Madagascan taxa are recovered, one of which is nested in a group of East African taxa. Divergence in Ixora is estimated to have commenced during the mid Miocene, with extensive cladogenesis occurring in the Afro-Madagascan clade during the Pliocene onwards.

Conclusions

Both lineages of Madagascan Ixora exhibit morphological innovations that are rare throughout the rest of the genus, including a trend towards pauciflorous inflorescences and a trend towards extreme corolla tube length, suggesting that the same ecological and selective pressures are acting upon taxa from both Madagascan lineages. Novel ecological opportunities resulting from climate-induced habitat fragmentation and corolla tube length diversification are likely to have facilitated species radiation on Madagascar.     

AtCYP20-2 is an auxiliary protein of the chloroplast NAD(P)H dehydrogenase complex

AtCYP20-2 is one of 16 immunophilins in thylakoid lumen. The presence of the isomerase domain in AtCYP20-2, an enrichment of AtCYP20-2 in the stroma membranes and it’s co-migration with NAD(P)H dehydrogenase (NDH) in native gels provide evidence that AtCYP20-2 is an auxiliary protein of NDH. When different NDH mutants were studied, AtCYP20-2 was found to be strongly reduced especially in mutants deficient in the membrane domain of NDH, thus suggesting a role in the assembly of NDH hydrophobic domain. Lack of AtCYP20-2, however, did not lead to severe malfunction of NDH, indicating redundancy in the function of lumenal immunophilins.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

A model of Taura syndrome virus (TSV) epidemics in Litopenaeus vannamei

Taura syndrome virus (TSV) is a highly virulent pathogen of Litopenaeus vannamei, has affected shrimp aquaculture throughout the world, and threatens wild populations. Despite its importance, little work has been done on the pathogen's formal epidemiology. Therefore we developed a compartment model for epidemics of TSV in closed populations of L. vannamei. The model includes five compartments, uninfected susceptible, prepatently infected, acutely infected, chronically infected, and dead infected shrimp. The transmission coefficients, patency coefficient, virulence coefficients, and removal coefficient (disappearance of dead infected shrimp) control the dynamics of the model. We estimated the coefficients in laboratory studies and inserted the estimates in the model to characterize TSV epidemics and to estimate the basic reproduction ratio R(0) and threshold density for TSV epidemics in L. vannamei. Further we examined through computer simulation the effect of varying the coefficients on R(0). Decreases in transmission decrease R(0), decreases in virulence increase R(0), increases in patency do not affect R(0), and increases in recovery most likely increase R(0) but under some conditions might decrease it.     

Multiple origins of the determinate growth habit in domesticated common bean (Phaseolus vulgaris)

Background and Aims

The actual number of domestications of a crop is one of the key questions in domestication studies. Answers to this question have generally been based on relationships between wild progenitors and domesticated descendants determined with anonymous molecular markers. In this study, this question was investigated by determining the number of instances a domestication phenotype had been selected in a crop species. One of the traits that appeared during domestication of common bean (Phaseolus vulgaris) is determinacy, in which stems end with a terminal inflorescence. It has been shown earlier that a homologue of the arabidopsis TFL1 gene – PvTFL1y – controls determinacy in a naturally occurring variation of common bean.

Methods

Sequence variation was analysed for PvTFL1y in a sample of 46 wild and domesticated accessions that included determinate and indeterminate accessions.

Key Results

Indeterminate types – wild and domesticated – showed only synonymous nucleotide substitutions. Determinate types – observed only among domesticated accessions – showed, in addition to synonymous substitutions, non-synonymous substitutions, indels, a putative intron-splicing failure, a retrotransposon insertion and a deletion of the entire locus. The retrotransposon insertion was observed in 70 % of determinate cultivars, in the Americas and elsewhere. Other determinate mutants had a more restricted distribution in the Americas only, either in the Andean or in the Mesoamerican gene pool of common bean.

Conclusions

Although each of the determinacy haplotypes probably does not represent distinct domestication events, they are consistent with the multiple (seven) domestication pattern in the genus Phaseolus. The predominance of determinacy in the Andean gene pool may reflect domestication of common bean prior to maize introduction in the Andes.     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

Cytogenetic and molecular evidence suggest multiple origins and geographical parthenogenesis in Nothoscordum gracile (Alliaceae)

Background and Aims

Nothoscordum gracile is an apomitic tetraploid widely distributed throughout the Americas and naturalized in many temperate regions of other continents. It has been suggested to form a species complex with sexual and apomictic N. nudicaule and N. macrostemon. Tetraploids of these species also share a structurally heterozygous chromosome complement 2n = 19 (13M + 6A). In this work, the origin of N. gracile and its relationships with its related species was investigated based on cytological and molecular data.

Methods

Cytogenetic analyses were based on meiotic behaviour, CMA bands, localization of 5S and 45S rDNA sites, and genomic in situ hybridization (GISH). Nuclear ITS and plastidial trnL-trnF sequences were also obtained for most individuals.

Key Results

Proximal CMA bands were observed in the long arms of all acrocentrics of 2x and 4x N. macrostemon but not in diploid and some tetraploid cytotypes of N. nudicaule. Samples of N. gracile showed a variable number of CMA bands in the long arms of acrocentrics. Analysis of ITS sequences, dot-blot, GISH, and 5S and 45S rDNA sites, revealed no differentiation among the three species. The trnL-trnF cpDNA fragment showed variation with a trend to geographical structuring irrespective of morphospecies and fully congruent with karyotype variation.

Conclusions

The 2n = 19 karyotype was probably formed by a centric fusion event occurring in N. nudicaule and later transmitted to tetraploid cytotypes of N. macrostemon. Diploids of N. nudicaule and N. macrostemon appeared as consistent recently diverged species, whereas tetraploid apomicts seem to constitute an assemblage of polyploid hybrids originating from multiple independent hybridization events between them, part of which are morphologically recognizable as N. gracile.     

Stereoselective reactions of (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene derivatives form 27-nor-3,20,23,26-tetrahydroxy-cholesten-22-ones and related bromo ketones

The previously reported analog of pregnenolone having a 3,4-dihydro-2H-pyran attached via a Cz.sbnd;C bond to the C-20 position (1), stereoselectively reacts with m-chloroperoxybenzoic acid in methanol at -5 degrees C. Acid-catalyzed hydrolysis of the isolated intermediates gives good yields of mostly a new 27-norcholesterol analog: (20R,23R)-3,20,23,26-tetrahydroxy-27-norcholest-5-en-22-one-3-acetate (2a, and a smaller amount of its 23S enantiomer 2b). Three different conditions of epoxidation and methanolysis followed by acid-catalyzed hydrolysis typically produce approximately 2:1 ratios of the 23R:23S diastereoisomers with a C-23 hydroxy group at the new asymmetric center. Bromine also reacts stereoselectively with (20R)-3,20-dihydroxy-(3',4'-dihydro-2'H-pyranyl)-5-pregnene (4) giving mostly (20R,23R)-23-bromo-3,20,26-trihydroxy-27-norcholest-5-en-22-one (7a). Thus both major steroidal products 2a and 7a have the same C-23R configuration. Assignment of molecular structures and the absolute configurations to 1 and 2a were based on elemental analysis, mass spectra, nuclear magnetic resonance, FTIR infrared spectroscopic analysis and X-ray crystallography. Mechanisms are discussed for stereochemical selectivity during epoxidation and bromination of the 3,4-dihydro-2H-pyranyl ring in 1 and 4.