Cryptic species and systematics of the hynobiid salamanders of the Liua–Pseudohynobius complex: Molecular and phylogenetic perspectives

Using mitochondrial DNA sequencing and allozyme electrophoresis, we examined 18 populations of the Liua–Pseudohynobius complex, endemic to China. Based on their phylogenetic affiliation and exhibited fixed allelic differences, the complex comprises at least six species, two of which are previously unknown cryptic species. The complex is clearly divided into two groups, genus Liua including Liua shihi and Liua tsinpaensis, and genus Pseudohynobius including Pseudohynobius flavomaculatus, Pseudohynobius shuichengensis and the two new species. The previously often used genus name Ranodon is inappropriate, because the type species of the genus, Ranodon sibricus, is distantly related to this complex. The species diversity among Chinese hynobiid salamanders are far from being recognized and further effort should be directed at extensive field collection in central and western China.     

Phylogeny of the Rhizobium–Allorhizobium–Agrobacterium clade supports the delineation of Neorhizobium gen. nov.

The genera Agrobacterium, Allorhizobium, and Rhizobium belong to the family Rhizobiaceae. However, the placement of a phytopathogenic group of bacteria, the genus Agrobacterium, among the nitrogen-fixing bacteria and the unclear position of Rhizobium galegae have caused controversy in previous taxonomic studies. To resolve uncertainties in the taxonomy and nomenclature within this family, the phylogenetic relationships of generic members of Rhizobiaceae were studied, but with particular emphasis on the taxa included in Agrobacterium and the “R. galegae complex” (R. galegae and related taxa), using multilocus sequence analysis (MLSA) of six protein-coding housekeeping genes among 114 rhizobial and agrobacterial taxa. The results showed that R. galegae, R. vignae, R. huautlense, and R. alkalisoli formed a separate clade that clearly represented a new genus, for which the name Neorhizobium is proposed. Agrobacterium was shown to represent a separate cluster of mainly pathogenic taxa of the family Rhizobiaceae. A. vitis grouped with Allorhizobium, distinct from Agrobacterium, and should be reclassified as Allorhizobium vitis, whereas Rhizobium rhizogenes was considered to be the proper name for former Agrobacterium rhizogenes. This phylogenetic study further indicated that the taxonomic status of several taxa could be resolved by the creation of more novel genera.     

The light-harvesting chlorophyll a/b · protein complex of the green alga Acetabularia mediterranea isolation and characterization of two subunits

In the green alga Acetabularia mediterranea a light-harvesting chlorophyll a/b · protein complex of 67 000 daltons has been found which contains two polypeptide chains of 21 500 and 23 000 daltons. These two polypeptides were isolated on a preparative scale and were further characterized by several different methods. Both polypeptides proved to be very similar. While their amino acid and sugar compositions as well as their immunochemical properties were almost identical the tryptic peptides and the cyanogen bromide fragments of the two polypeptides revealed minor but significant differences. The 67 000-dalton chlorophyll a/b · protein complex and its two polypeptide components were compared to the light-harvesting chlorophyll a/b · protein of higher plants.     

Visual learning and memory of Drosophila melanogaster wild type CS and the mutants dunce1, amnesiac, turnip and rutabaga

Dunce¹, amnesiac, turnip and rutabaga, mutants of Drosophila melanogaster deficient in olfactory learning and/or memory, were tested for visual learning ability and memory. All these mutants are able to learn conditioned visual information, but not as well as the corresponding wildtype CS. Memory of all four mutants is reduced during the first 30 min after training, but indistinguishable from that of the wildtype two hours after conditioning.     

Identification of the first deletion–insertion involving the complete structure of GAA gene and part of CCDC40 gene mediated by an Alu element

Pompe disease is an uncommon autosomal recessive glycogen storage disorder caused by deficiency of acid α-glucosidase. Classic infantile form triggers severe cardiomyopathy, hypotonia, and respiratory failure, leading to death within the first two years of life. The majority of patients with Pompe disease have been reported to have point mutations in the GAA gene. We report the first complex deletion–insertion encompassing the complete structure of GAA gene and a large fragment of the gene CCDC40 in a patient with very severe form of Pompe disease. Sequencing analysis of breakpoints allowed us to determine the potential implication of an Alu repeat in the pathogenic mechanism. We suggest that molecular strategy of Pompe disease should include systematic analysis of large rearrangements.     

Hormonal aspects of oögenesis in the flies Phormia regina and Sarcophaga bullata

The corpora allata (CA) and median neurosecretory cells (MNC) of Phormia regina and Sarcophaga bullata become active with increasing age of the fly, on a diet of sugar alone. To prevent or retard oögenesis the CA or MNCs must be removed shortly after emergence, with subsequent protein meals. Topical JH application partially compensates for CA or MNC removal. This shows that the MNC activate the CA, and not vice versa. The trauma of either operation slightly depresses egg development.Injection of ecdysone into both species in the stage of initial yolk deposition causes the primary oöcytes to degenerate. This leads to development of the penultimate oöcytes. Older and younger egg stages are not sensitive to ecdysone. In P. regina the application of JH to females with developing primary oöcytes stimulates yolk deposition in the penultimate oöcytes.     

Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization,and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens

The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis and Streptomyces vinaceus, respectively, that shared the same 16S rRNA gene sequences with the clade, were subjected to multilocus sequence analysis (MLSA), DNA–DNA hybridization (DDH) and phenotypic characterization for a comprehensive reevaluation. The 13 strains still formed a distinct, albeit loosely related, clade in the phylogenetic tree based on concatenated sequences of aptD, gyrB, recA, rpoB and trpB genes, supported by a high bootstrap value and different tree-making algorithms, with MLSA evolutionary distances ranging from 0 to 0.003. DDH values among these strains were well above the 70% cut-off point for species delineation. Based on the genotypic data of MLSA and DDH, combined with key phenotypic properties in common, it is proposed that the 10 species and subspecies of the S. albidoflavus clade, namely S. albidoflavus, S. canescens, S. champavatii, S. coelicolor, S. felleus, S. globisporus subsp. caucasicus, S. griseus subsp. solvifaciens, S. limosus, S. odorifer and S. sampsonii, should be merged into a single genomic species, for which the name S. albidoflavus is retained, and that the three strains S. galilaeus CGMCC 4.1320, S. sioyaensis CGMCC 4.1306 and S. vinaceus CGMCC 4.1305 should be assigned to S. albidoflavus as well. The results also indicated that MLSA could be the procedure of choice for distinguishing between species within Streptomyces 16S rRNA gene clades.     

Determination of the CCR5∆32 frequency in Emiratis and Tunisians and the screening of the CCR5 gene for novel alleles in Emiratis

Background

The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented that variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5?32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5?32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians).

Methodology

The prevalence of CCR5 gene variants including CCR5?32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5?32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing.

Results

Among Emiratis, the allele frequency of the CCR5?32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002. Moreover, the prevalence of the CCR5?32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans.

Conclusion

We conclude that the allele frequency of the most critical CCR5 polymorphism (?32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.     

A study of the pollen grains of Amaranthus spinosus Linné and A. Dubius Mart ex Thellung and their hybrids

A pollen-morphology study of Amaranthus spinosus, A. dubius, and their hybrids has been carried out. Three pollen types have been observed, namely (1) Type A: micrograins; (2) Type B: grains with smaller pores; and (3) Type C: grains with larger pores. Type B is characteristic of A. spinosus, Type C of A. dubius, and the micrograins of the hybrids. Pollen size range, and frequency of the various morphotypes serve to throw light on the biosystematics of the plants studied.     

Study of the inclusion of the (R)- and (S)-camphor enantiomers in α-cyclodextrin by X-ray crystallography and molecular dynamics

The inclusion of (R)- and (S)-camphor compounds in α-cyclodextrin has been studied by X-ray crystallography. The crystal structures of the complexes reveal that one guest molecule is accommodated inside the cavity formed by a head-to-head cyclodextrin dimer. In the crystal lattice, the dimers form layers which are successively shifted by half a dimer. In both (R)- and (S)-cases, the camphor molecule exhibits disorder and occupies three major sites with orientations that can be described as either ‘polar’ or ‘equatorial’. Molecular dynamics simulations performed for the observed complexes indicate that although the carbonyl oxygen of both (R)- and (S)-camphor switches between different hydrogen bonding partners, it maintains the observed mode of ‘polar’ or ‘equatorial’ alignment.     

Phospholipase C and sphingomyelinase activities of the Clostridium perfringens α-toxin

α-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. α-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of α-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of α-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Genetic and phytochemical analysis of the in vitro regenerated Pilosocereus robinii by ISSR,SDS–PAGE and HPLC

Pilosocereus robinii is a rare species which is experiencing sudden population collapse. Identifying and developing effective conservation and management strategies to halt the forestall extinction of this species is crucial. The present study was conducted to assess the best conditions for in vitro propagation of this plant in regard to its morphogenic, genetic as well as the chemical potentials. A successful in vitro propagation system of P. robinii has been developed. MS hormone-free medium induced the best root morphogenic potential. The plants were acclimatized in the greenhouse at 100% survival rate. Besides, the somaclonal variations between the in vitro raised plants were analyzed using PCR-ISSR markers and SDS–PAGE protein, where the regenerated explants on MS medium supplemented with TDZ were the highest in inducing new specific marker bands. Sh6 ISSR primer showed the highest polymorphism value, 81.8% with 33 total amplified fragments, while Sh3 ISSR primer showed the lowest value with polymorphic percentage of 14.3%. Furthermore, SDS–PAGE protein analysis showed no variation in protein pattern of the studied treatments. On the other side, HPLC analysis of the in vitro plantlets extracts has shown that 2iP based treatments were the highest in organic acids accumulation, while the phenolic constituents' accumulation was found to reach its peak in the BA based treatments.     

Synthesis and characterisation of κ-P and κ-P,N palladium(II) complexes of the open cage water soluble aminophosphine PTN

New palladium(II) complexes containing the water soluble aminophosphine PTN ligand (PTN = 7-phospha-3,7-dimethyl-1,3,5-triazabicyclo[3.3.1]nonane) in 1:1 and 1:2 ratio Pd/PTN ligand, respectively, were prepared and fully characterised by mono and bidimensional ³¹P, ¹H and ¹³C NMR techniques showing that PTN can adopt both κ¹-P and κ²-P,N coordination modes. The complexes with Pd/PTN ratio 1:2 are highly soluble in water at room temperature. Suitable crystals for X-ray structure determination were obtained for the neutral complex κ²-P,N-Pd(PTN)(OAc)₂ (1) and for the monocationic complex [Pd(κ²-P,N-PTN)(κ¹-P-PTN)Cl][PF₆] (5).     

En route to a genome-based classification of Archaea and Bacteria?

Given the considerable promise whole-genome sequencing offers for phylogeny and classification, it is surprising that microbial systematics and genomics have not yet been reconciled. This might be due to the intrinsic difficulties in inferring reasonable phylogenies from genomic sequences, particularly in the light of the significant amount of lateral gene transfer in prokaryotic genomes. However, recent studies indicate that the species tree and the hierarchical classification based on it are still meaningful concepts, and that state-of-the-art phylogenetic inference methods are able to provide reliable estimates of the species tree to the benefit of taxonomy. Conversely, we suspect that the current lack of completely sequenced genomes for many of the major lineages of prokaryotes and for most type strains is a major obstacle in progress towards a genome-based classification of microorganisms. We conclude that phylogeny-driven microbial genome sequencing projects such as the Genomic Encyclopaedia of Archaea and Bacteria (GEBA) project are likely to rectify this situation.     

n-Alkanes and ω-hydroxyalkanoic acids from the needles of twenty-eight Picea species

The needle wax of twenty-eight species of Picea has been investigated. The quantitative patterns of the n-alkanes and ω-hydroxyalkanoic acids isolated from these waxes support the view that the genus should not be divided into sections.     

Isolation and synthesis of trans- and cis-(−)-clovamides and their deoxy analogues from the bark of Dalbergia melanoxylon

N-(3′,4′-Dihydroxy-trans-cinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine [(?)-clovamide], the major phenolic metabolite (0.1%) in the bark of Dalbergia melanoxylon, is associated with minor proportions of its cis-isomer, and similar pairs of geometrical isomers of their deoxy analogues N-(4′-hydroxycinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine and N-(4′-hydroxycinnamoyl)-3-(4-hydroxyphenyl)-L-alanine. (?)-Trans-clovamide is synthesized by direct condensation of the acid chloride of caffeic acid with L-DOPA. Diagnostic CD spectra of these compounds and ¹³C spectra of (?)-trans- and (?)-cis-clovamides are recorded.     

Importance of spores and δ-endotoxin protein crystals of Bacillus thuringiensis in Galleria mellonella

Larvae of Galleria mellonella were fed on a honey-rich artificial food containing live spores or toxic crystals of Bacillus thuringiensis serotype V or various combinations of both. In this food; 1:1 combinations were 10 times more potent than live spores alone and about 10⁴ times more potent than crystals alone. Reduction in the proportion of spores, but not in that of crystals, decreased the slope of probit lines from 3.4 to 0.6. One or more factors in the spore are at least partly responsible for the potency of serotype V in G. mellonella. The results suggest than an observed gross loss of potency of this serotype in beehives is more likely to be due to death of spores than to deterioration of crystals. The reaction of G. mellonella to serotype V is nearest to that of a type 3 host species. Spores of serotype I are almost inactive in this host.