Analysis of hydroxyproline isomers and hydroxylysine by reversed-phase HPLC and mass spectrometry

Collagens, the most abundant mammalian proteins, contain a high content of hydroxylated amino acids, such as, 3- and 4-cis-/trans-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl). Whereas the global content of 4-Hyp was studied by amino acid analysis, no technique to determine all five hydroxyamino acids simultaneously in collagens has been reported. Here, we report the separation of all five hydroxyamino acids as well as two Hyp epimers from all other proteinogenic amino acids after derivatization with N(2)-(5-fluoro-2,4-dinitrophenyl)-l-valine amide (l-FDVA) by RPC-UV-ESI-MS. The general applicability of this method is shown for three Hyp-containing peptides as well as collagen type I.     

Lipopolysaccharide transport to the cell surface: periplasmic transport and assembly into the outer membrane

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.     

Endophytic colonization and biocontrol performance of Pseudomonas fluorescens PICF7 in olive (Olea europaea L.) are determined neither by pyoverdine production nor swimming motility

Pseudomonas fluorescens PICF7 is an indigenous inhabitant of olive (Olea europaea L.) rhizosphere, able to display endophytic lifestyle in roots, to induce a wide range of defence responses upon colonization of this organ and to exert effective biological control against Verticillium wilt of olive (VWO) (Verticillium dahliae). We aimed to evaluate the involvement of specific PICF7 phenotypes in olive root colonization and VWO biocontrol effectiveness by generating mutants impaired in swimming motility (fliI) or siderophore pyoverdine production (pvdI). Besides, the performance of mutants with diminished in vitro growth in potato dextrose agar medium (gltA) and cysteine (Cys) auxotrophy was also assessed. Results showed that olive root colonization and VWO biocontrol ability of the fliI, pvdI and gltA mutants did not significantly differ from that displayed by the parental strain PICF7. Consequently, altered in vitro growth, swimming motility and pyoverdine production contribute neither to PICF7 VWO suppressive effect nor to its colonization ability. In contrast, the Cys auxotroph mutant showed reduced olive root colonization capacity and lost full biocontrol efficacy. Moreover, confocal laser scanning microscopy revealed that all mutants tested were able to endophytically colonize root tissue to the same extent as wild‐type PICF7, discarding these traits as relevant for its endophytic lifestyle.     

Bite force and body mass of the fossil rodent Telicomys giganteus (Caviomorpha,Dinomyidae)

An exceptionally well-preserved skull of the Pliocene rodent Telicomys giganteus allowed the first estimation of body mass and analysis of the bite mechanics of this species of South American giant rodent. In this study, we reconstructed the main anatomical features of the skull of this Pliocene rodent and related them to the bite force at the incisors. The average of an estimation body mass gives 100 kg. We also estimated the bite force using three different techniques. Two methods suggest that bite forces at the incisors have a range of 500–1000 N. However, the incisors seem to be stronger than expected for this bite force, implying that the bite forces may have been greater than 2000 N. We consider the hypothesis of defense against predators or other agonistic behavior to explain our results.     

Genetic structure of La Cabrera, Spain, from surnames and migration matrices

The genetic structure of La Cabrera (province of Léon, Spain), a highly isolated and inbred population (alpha3 = 0.00482), is analyzed by applying multivariate methods (nonmetric multidimensional scaling, Mantel test, Monmonier's algorithm) to different biodemographic data sets. Isonymy, parent-offspring migration (total, males, females), and marital migration matrices were obtained from 5,714 marriages recorded in 37 parishes (clustered in 4 municipalities) between 1880 and 1989. The aim of the study is to investigate the relationships between the genetic and geographic structures of the area. Endemicity values (diagonal of parent-offspring migration matrices), calculated for both sexes at two hierarchical levels (parishes and municipalities), show that female mobility follows the virilocal migration model at the higher (municipalities) level and the uxorilocal model at the parish level. Analysis of isonymy and parent-offspring migration matrices shows high correspondence between the genetic structure and geographic location of the parishes. In fact, the main reproductive barriers, constructed using Monmonier's algorithm, generally coincide with geographic barriers, highlighting increasing isolation patterns from northwest to southeast. Moreover, the analysis of isonymous relationships, which are influenced by earlier population movements, identifies three parishes whose outlier positions are explained by historical-cultural or geographic reasons. The positive and highly significant values (0.32 < or = r < or = 0.51;p < or = 0.001) given by the Mantel tests underline the dependence of the genetic structure on geographic distance. In confirmation of the endemicity results, the lowest correlation value (r = 0.32) is given by the female migration matrix. When the outlier parishes are omitted from the analysis, the correlation between isonymy and geographic distance increases from 0.35 to 0.46, and the values from the other migration matrices remain unchanged. In conclusion, the combination of different data sets and methods allows a wider and more coherent reconstruction of the historical changes in the genetic structure of the La Cabrera population.     

Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.     

The C-terminal loop of the homing endonuclease I-CreI is essential for site recognition, DNA binding and cleavage

Meganucleases are sequence-specific endonucleases with large cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These enzymes open novel perspectives for genome engineering in a wide range of fields, including gene therapy. A new crystal structure of the I-CreI dimer without DNA has allowed the comparison with the DNA-bound protein. The C-terminal loop displays a different conformation, which suggests its implication in DNA binding. A site-directed mutagenesis study in this region demonstrates that whereas the C-terminal helix is negligible for DNA binding, the final C-terminal loop is essential in DNA binding and cleavage. We have identified two regions that comprise the Ser138–Lys139 and Lys142–Thr143 pairs whose double mutation affect DNA binding in vitro and abolish cleavage in vivo. However, the mutation of only one residue in these sites allows DNA binding in vitro and cleavage in vivo. These findings demonstrate that the C-terminal loop of I-CreI endonuclease plays a fundamental role in its catalytic mechanism and suggest this novel site as a region to take into account for engineering new endonucleases with tailored specificity.     

Exploitation of the shrimp trawl fishery in the period 1991-1999 at the Gulf of Nicoya, Costa Rica

In Costa Rica, the Gulf of Nicoya shrimp fishery originated in 1952 and represented one of the most important economic activities in the region. Nevertheless, overfishery reduced the captured volumes to levels that prevent this commercial activity. I analyzed official fishery statistics between 1991 and 1999. These species involved are: two species of white shrimp, (Litopenaeus occidentalis and L. stylirostris), the "titi" shrimp (Xiphopenaeus riveti), the brown shrimp (Farfantepenaeus californiensis), the "pinki" shrimp (F. brevirostris) and the "fidel" shrimp (Solenocera agassizi). All the species reached the Maximum Sustainable Yield in the decades of 1970 and 1980 and are now found at over-exploitation levels. I recommend that this shrimp trawl fishery be completely closed down.     

Triatoma mexicana (Hemiptera: Reduviidae) in Guanajuato, Mexico: house infestation and seasonal variation

Triatoma mexicana was described by Herrich-Schaeffer in 1848. In 1940, a male specimen was found in Hidalgo. In 1970, this species was recorded in the state of Queretaro. Later, it was registered in Guanajuato and San Luis Potosi. In the present paper we performed an investigation in 545 dwellings from three counties in the state of Guanajuato, Mexico, from March 2003 to May 2004. The search and capture of triatomines were seasonally performed indoors and outdoors. Entomological indexes were calculated. The risk and no risk relations between triatomine presence and housing construction materials were analyzed. Fourteen triatomines were collected indoors and 151 outdoors. The vectors were collected in houses built with either risky and non-risky materials. Adults go indoors but do not settle there, hence, no relationship was found between the building materials and infestation of houses. Conventional interventions like house improvement or insecticide spraying are not efficient for the control of T. mexicana, because its developmental cycle is accomplished outdoors in the area surrounding the houses.