Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather than long‐distance dispersal origin for the disjunct distribution of the Chilean endemic Dioscorea biloba (Dioscoreaceae) around the Atacama Desert

Aim The Chilean endemic Dioscorea biloba (Dioscoreaceae) is a dioecious geophyte that shows a remarkable 600 km north–south disjunction in the peripheral arid area of the Atacama Desert. Its restricted present‐day distribution and probable Neogene origin indicate that its populations have a history linked to that of the Atacama Desert, making this an ideal model species with which to investigate the biogeography of the region. Location Chile, Atacama Desert and peripheral arid area. Methods Two hundred and seventy‐five individuals from nine populations were genotyped for seven nuclear microsatellite loci, and plastid trnL–F and trnT–L sequences were obtained for a representative subset of these. Analyses included the estimation of genetic diversity and population structure through clustering, Bayesian and analysis of molecular variance analyses, and statistical parsimony networks of chloroplast haplotypes. Isolation by distance was tested against alternative dispersal hypotheses. Results Microsatellite markers revealed moderate to high levels of genetic diversity within populations, with those from the southern Limarí Valley showing the highest values and northern populations showing less exclusive alleles. Bayesian analysis of microsatellite data identified three genetic groups that corresponded to geographical ranges. Chloroplast phylogeography revealed no haplotypes shared between northern and southern ranges, and little haplotype sharing between the two neighbouring southern valleys. Dispersal models suggested the presence of extinct hypothetical populations between the southern and northern ranges. Main conclusions Our results are consistent with prolonged isolation of the northern and southern groups, mediated by the life‐history traits of the species. Significant isolation was revealed at both large and moderate distances as gene flow was not evident even between neighbouring valleys. Bayesian analyses of microsatellite and chloroplast haplotype diversity identified the southern area of Limarí as the probable area of origin of the species. Our data do not support recent dispersal of D. biloba from the southern range into Antofagasta, but indicate the fragmentation of an earlier wider range, concomitant with the Pliocene–Pleistocene climatic oscillations, with subsequent extinctions of the Atacama Desert populations and the divergence of the peripheral ones as a consequence of genetic drift.     

Structural Basis for Silicic Acid Uptake by Higher Plants

Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.     

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459–464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context.Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.Abbreviations BSA Bovine srum albumin - mab Monoclonal antibody - PBS Phosphate buffered saline - PMSF Phenylmethyl sulfonyl fluoride     

Absolute configuration and conformation of the pure opioid antagonist (+)-2,9 alpha-dimethyl-5-(m-hydroxyphenyl)morphan.

(+)-2,9 alpha-Dimethyl-5-(m-hydroxyphenyl)morphan is the only phenylmorphan analog whose affinity for opioid kappa-receptors is greater than its affinity for opioid mu-receptors. Pharmacologically, the compound is a pure opioid antagonist devoid of agonist activity in in vivo assays of antinociception. The absolute configuration of the compound has been determined to be (1R,5S,9R) from an X-ray crystallographic study of the chloride salt. Thus, the absolute configuration corresponds to that of the atypical opioid agonist (-)-phenylmorphan while the weak atypical agonist (-)-2,9 alpha-dimethyl-5-(m- hydroxyphenyl)morphan corresponds to the potent morphine-like (+)-phenylmorphan. The preferred orientations of the phenyl ring for the two stereoisomers were determined using the molecular mechanics program MM2-87 and found to vary from that of the two parent compounds. The atypical properties of the two 9 alpha-methyl analogs is consistent with an opioid ligand model which proposes that morphine-like properties require a particular range of phenyl orientations. There was good agreement between the structure obtained from X-ray crystallography and computed with the MM2-87 program.     

Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Socially isolated? How parents and neighbourhood adults influence youth behaviour in disadvantaged communities

William Julius Wilson’s model of adult joblessness, community disorganization and their effects on youth problem behaviour de-emphasizes the range in children’s outcomes across socially disorganized communities, and says little about the factors that influence this variation. It also does not address the processes by which family structure and relationships affect the well-being of African-American and poor youth. My work is part of a larger research agenda that has begun to address these issues by focusing on the differential rates of sexual activity among youth living in disadvantaged environments, and developing models to explain this variation. This work suggests that units of socially cohesive, stable adults exist among the social networks of successful children and families in poor neighbourhoods. It also points to the existence and functioning of alternative two-parent family structures and offers hypotheses for how family environment interacts with neighbourhood context to influence youth behaviour.