Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

N-acetylcysteine and captopril protect DNA and cells against radiolysis by fast neutrons

N-Acetylcysteine and captopril, respectively mucolytic and antihypertensive drugs, contain free sulfhydryl groups. Since in general thiols have well-established radioprotective abilities, we sought putative radioprotective effects of these drugs against therapeutic fast neutrons. We show that pBR322 plasmid DNA is indeed protected against radiolytic strand breakage by both drugs. The oxygen independent protection is consistent with a hydroxyl radical scavenging mechanism. A clonogenicity assay reveals an increase of the survival of SCL-1 cultured keratinocytes irradiated in the presence of the drugs compared with cells irradiated without drugs. Our results suggest possible interferences between treatment with drugs bearing-SH groups and radiotherapy.     

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles⁵. The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction^*force interaction, ANOVA, P<0.05) and MF (contraction^*force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.     

The genome of the non-cultured,bacterial-like organism associated with citrus greening disease contains thenusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase

We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the noncultured, bacterial-like organism (BLO) associated with citrus greening disease. Nucleotide sequence determination has shown that fragment In-2.6 is part of therplKAJL-rpoBC gene cluster, a well-known operon in eubacteria. The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be thenusG gene. InEscherichia coli, nusG is also immediately upstream ofrplKAJL-rpoBC. Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase. Fragment In-0.6 could not be identified.When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined. At lower stringencies, In-2.6 was able to detect also the African strain. The implications of these reults in the taxonomical position of the greening BLO are discussed.     

Trade-offs and synergies between ecosystem productivity and stability in temperate grasslands

Aim

It is crucial to monitor how the productivity of grasslands varies with its temporal stability for management of these ecosystems. However, identifying the direction of the productivity–stability relationship remains challenging because ecological stability has multiple components that can display neutral, positive or negative covariations. Furthermore, evidence suggests that the direction of the productivity–stability relationship depends on the biotic interactions and abiotic conditions that underlie ecosystem productivity and stability. We decipher the relationships between grassland productivity and two components of its stability in four habitat types with contrasting environments and flora.

Location

France.

Time period

2000–2020.

Major taxa

Grassland plant species.

Methods

We used c. 20,000 vegetation plots spread across French permanent grasslands and remotely sensed vegetation indices to quantify grassland productivity and temporal stability. We decomposed stability into constancy (i.e., temporal invariability) and resistance (i.e., maximum deviation from average) and deciphered the direct and indirect effects of abiotic (namely growing season length and nitrogen input) and biotic (namely plant taxonomic diversity, trait diversity and community-weighted mean traits) factors on productivity–stability relationships using structural equation models.

Results

We found a positive relationship between productivity and constancy and a negative relationship between productivity and resistance in all habitats. Abiotic factors had stronger effects on productivity and stability compared with biotic factors. A longer growing season enhanced grassland productivity and constancy. Nitrogen input had positive and negative effects on grassland productivity and resistance, respectively. Trait values affected the constancy and resistance of grassland more than taxonomic and trait diversity, with effects varying from one habitat to another. Productivity was not related to any biotic factor.

Main conclusions

Our findings reveal how vital it is to consider both the multiple components of stability and the interaction between environment and biodiversity to gain an understanding of the relationships between productivity and stability in real-world ecosystems, which is a crucial step for sustainable grassland management.     

Modeling of protein loops by simulated annealing.

A method is presented to model loops of protein to be used in homology modeling of proteins. This method employs the ESAP program of Higo et al. (Higo, J., Collura, V., & Garnier, J., 1992, Biopolymers 32, 33-43) and is based on a fast Monte Carlo simulation and a simulated annealing algorithm. The method is tested on different loops or peptide segments from immunoglobulin, bovine pancreatic trypsin inhibitor, and bovine trypsin. The predicted structure is obtained from the ensemble average of the coordinates of the Monte Carlo simulation at 300 K, which exhibits the lowest internal energy. The starting conformation of the loop prior to modeling is chosen to be completely extended, and a closing harmonic potential is applied to N, CA, C, and O atoms of the terminal residues. A rigid geometry potential of Robson and Platt (1986, J. Mol. Biol. 188, 259-281) with a united atom representation is used. This we demonstrate to yield a loop structure with good hydrogen bonding and torsion angles in the allowed regions of the Ramachandran map. The average accuracy of the modeling evaluated on the eight modeled loops is 1 A root mean square deviation (rmsd) for the backbone atoms and 2.3 A rmsd for all heavy atoms.     

Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression

In Pseudomonas aeruginosa, several exoproteins synthesized with a signal sequence (elastase, lipase, phospholipases, alkaline phosphatase and exotoxin A) are secreted by a two-step mechanism. They first cross the inner membrane in a signal sequence-dependent way, and are further translocated across the outer membrane in a second step requiring secretion functions encoded by several xcp genes. Ten xcp genes have already been characterized (Bally et al., 1992a). In this study, two additional xcp genes, xcpP and xcpQ, are described. They are located in the 40 min region of the chromosome where they probably define an operon, divergent from the xcpR–Z operon previously characterized in this region. These two genes encode two proteins, XcpP and XcpQ, similar to PulC and PulD of the pul system of Klebsiella oxytoca. Moreover, the two divergent operons share a common regulation which is growth-phase dependent.     

Spatial components of foraging behavior in an African Ponerine ant,Paltothyreus tarsatus

Colonies of the African stink ant Paltothyreus tarsatuslocated in the forest have nests with shorter horizontal galleries and a smaller total foraging surface than colonies located in open areas. Each solitary worker specializes on the same central or peripheral hunting zone but she does not specialize on a particular sector during group-retrieving. The search for prey is characterized by a wandering walk with spatial parameters varying in two ways. Capture of a termite releases a path characterized by sinuosity and a decrease in speed of movement. In contrast, a failure in the course of an attempted capture releases an increase in both sinuosity and speed of movement corresponding to a socalled reserve behavior. Each worker shortens her retrieving trip in comparison with her search trip and the straightness of the homing paths depends on the size and shape of the prey. Our data show that behavioral flexibility at the individual level in P. tarsatusis important in determining spatial foraging strategy at the colony level.     

Identification and characterization of adhesive factors of Clostridium difficile involved in adhesion to human colonic enterocyte-like Caco-2 and mucus-secreting HT29 cells in culture

Experiments reported in this communication showed that the highly toxinogenic Cd 79685, Cd 4784, and Wilkins Clostridium difficile strains and the moderately toxinogenic FD strain grown in the presence of blood adhere to polarized monolayers of two cultured human intestinal cell lines: the human colonic epithelial Caco-2 cells and the human mucus-secreting HT29-MTX cells. Scanning electron microscopy revealed that the bacteria interacted with well-defined apical microvilli of differentiated Caco-2 cells and that the bacteria strongly bind to the mucus layer that entirely covers the surface of the HT29-MTX cells. The binding of C. difficile to Caco-2 cells developed in parallel with the differentiation features of the Caco-2 cells, suggesting that the protein(s) which constitute C. difficile-binding sites are differentiation-related brush border protein(s). To better define this interaction, we tentatively characterized the mechanism(s) of adhesion of C. difficile with adherence assays. It was shown that heating of C. difficile grown in the presence of blood enhanced the bacterial interaction with the brush border of the enterocyte-like Caco-2 cells and the human mucus-secreting HT29-MTX cells. A labile surface-associated component was involved in C. difficile adhesion since washes of C. difficile grown in the presence of blood without heat shock decreased adhesion. After heating, washes of C. difficile grown in the presence of blood did not modify adhesion. Analysis of surface-associated proteins of C. difficile subjected to different culture conditions was con-ducted. After growth of C. difficile Cd 79685, Cd 4784, FD and Wilkins strains in the presence of blood and heating, two predominant SDS-extractable proteins with molecular masses of 12 and 27 kDa were observed and two other proteins with masses of 48 and 31 kDa disappeared. Direct involvement of the 12 and 27 kDa surface-associated proteins in the adhe-sion of C. difficile strains was demonstrated by using rat polycolonal antibodies pAb 12 and pAb 27 directed against the 12 and 27kDa proteins. Indeed, adhesion to Caco-2 cell monoiayers of C. difficiie strains grown in the presence of blood, without or with heat-shock, was blocked. Taken together, our results suggest that C. difficiie may utilize blood components as adhesins to adhere to human intestinal cultured cells.