Genetic complementation of rhizobial nod mutants with Frankia DNA: artifact or reality?

Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciaenodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine Nod^? R. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA ^? , nodB ^? , nodC ^? , nodD ^? , nodF? ^? , nodL ^? , nodH ^? ) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciaenodC::Tn5 mutant. The Nod⁺ rhizobial transconjugants were isolated and analysed. The Nod⁺ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur.     

The crystal and molecular structure of VH amylose by electron diffraction analysis

The crystal and molecular structures of VH amylose were determined by a constrained linked-atom least-squares refinement, utilizing intensities measured from electron diffraction patterns and stereochemical restraints. Hexagonal platelet single crystals were grown from dilute aqueous ethanol solution and their electron diffraction diagrams analysed. These data indicated that the amylose chains were crystallized in a hexagonal lattice with a = b = 13.65 A, c (chain axis) = 8.05 A and space group P6(5)22. The best model obtained using the base plane data coupled with a stereochemical refinement yielded R = 0.24 (R' = 0.25). It corresponded to a system of left-handed 6-fold helices packed on an hexagonal net but with statistically random up/down chain disorder. A column of six water molecules was present within each helical repeat. Additionally, the gap between each pair of adjacent helices was bridged by two water molecules positioned so as to allow hydrogen bonding with chains of either sense. This proposed crystal structure differs somewhat from previous reports which invoked orthorhombic lattices and requires a regularly alternating arrangement of up and down chains to account for the intensity. Suggestions are made to account for these differences.     

Embedding softened herbarium material in Spurr's resin for histological studies.

Plant organs, including stems, rhizomes, leaves, roots, petals, sporangia and flower pedicels obtained from dried herbarium specimens of a variety of plant species have been softened with Aerosol OT and subsequently dehydrated in a graded series of acetones and embedded in Spurr's resin. Although the quality of preservation varied, sections of a variety of materials showed excellent cellular preservation. Sections stained through the resin with toluidine blue O and examined with either bright field microscopy or with crossed polarizers showed good cell detail. Histochemical tests for callose, polysaccharides, and cellulosic walls, using sections from which the resin had been removed by sodium methoxide and then viewed with an epifluorescence microscope, gave excellent results.     

Transition metal carbonyl labeling of proteins. A novel approach to a solid-phase two-site immunoassay using Fourier transform infrared spectroscopy.

Labeling of bovine serum albumin (BSA) and anti-human thyroid stimulating hormone (hTSH) monoclonal antibodies (mAbs) was performed using (N-succinimidyl 4-pentynoate)hexacarbonyldicobalt (NSCo2(CO)6). Conditions of coupling were different depending on the protein to be labeled, denaturation of the mAbs occuring with high percentages of organic solvent in the reaction mixture. The influence of reaction time and initial concentration of NSCo2(CO)6 was examined. They were both shown to affect the final coupling rate of the metal carbonyl probe. Preservation of the immunoreactivity toward 125I-hTSH was observed for five conjugates having different NSCo2(CO)6: mAb molar ratios when compared to unmodified and peroxidase-labeled mAbs. Finally, a preliminary study of the quantitative detection of the metal carbonyl mAbs on microtiter wells was achieved using Fourier transform infrared spectroscopy.     

Trait anxiety, submaximal physical exercise and blood androgens

This study evaluates the relationship between trait anxiety and both androgen and gonadotrophic hormone levels at rest and during severe physical exercise. Twelve volunteers were selected among 160 untrained male collegial students and classified as anxious (N = 6) or non-anxious (N = 6) subjects according to their scores on three trait-anxiety tests (STAI, IPAT, 16 PF). Serum delta 4-androgen (testosterone and delta 4-androstenedione), delta 5-androgen (DHEA and DHEA-SO4) and gonadotrophin (LH and FSH) concentrations were measured by radioimmunoassay before, during and after 20 minutes of intensive bicycle exercise (80% of maximal heart rate). Results indicate significantly lower serum delta 4-androgens in anxious subjects before exercise. However, for each subject and irrespective of his anxiety level, all measured serum androgen concentrations increased significantly during exercise, although delta 4-androstene-dione remained lower in anxious subjects than in non-anxious ones. Serum LH concentrations (but not FSH) were significantly higher in anxious subjects throughout the observation periods. However, exercise induced in each subject a significant decrease in the serum level of both gonadotrophic hormones. The results suggest that trait anxiety level may constitute an important factor that affects both pre-exercise and exercise serum androgen concentrations in untrained subjects.     

XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5’ cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.     

Thermodynamics and dynamics of amyloid peptide oligomerization are sequence dependent

Aggregation of the full‐length amyloid‐β (Aβ) and β2‐microglobulin (β2m) proteins is associated with Alzheimer's disease and dialysis‐related amyloidosis, respectively. This assembly process is not restricted to full‐length proteins, however, many short peptides also assemble into amyloid fibrils in vitro. Remarkably, the kinetics of amyloid‐fibril formation of all these molecules is generally described by a nucleation‐polymerization process characterized by a lag phase associated with the formation of a nucleus, after which fibril elongation occurs rapidly. In this study, we report using long molecular dynamics simulations with the OPEP coarse‐grained force field, the thermodynamics and dynamics of the octamerization for two amyloid 7‐residue peptides: the β2m83‐89 NHVTLSQ and Aβ16‐22 KLVFFAE fragments. Based on multiple trajectories run at 310 K, totaling 2.2 μs (β2m83‐89) and 4.8 μs (Aβ16‐22) and starting from random configurations and orientations of the chains, we find that the two peptides not only share common but also very different aggregation properties. Notably, an increase in the hydrophobic character of the peptide, as observed in Aβ16‐22 with respect to β2m83‐89 impacts the thermodynamics by reducing the population of bilayer β‐sheet assemblies. Higher hydrophobicity is also found to slow down the dynamics of β‐sheet formation by enhancing the averaged lifetime of all configuration types (CT) and by reducing the complexity of the CT transition probability matrix. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mixed lymphocyte culture suppressor cells can be induced by non-HLA differences in man

Lymphocytes (A) sensitized in vitro by cells from a HLA-identical sibling (B) for 8 days showed inhibiting effects when added to fresh mixed lymphocyte cultures (MLC) where A responders were stimulated by cells from other family members in a ratio of 1:1:1. In 23 of 31 such pairs tested in 15 families, proliferative activities in these 6-day second-step MLC were inhibited by 54 +/- 18% in the presence of A'B sensitized cells as compared to control cultures with modulating A' cells similarly preincubated but in the absence of B stimulators. In addition, A'B could also suppress MLC responses of B in 12 of the 17 pairs in which this was tested. Inhibition was not due to cytotoxic elimination of stimulators and it was radiation sensitive. Suppression appeared to be specific but it did not seem to be restricted by HLA-A, -B, or -DR determinants. Hence, these results indicate that suppressor cells generated after priming by HLA-identical cells can regulate allogeneic proliferative responses even when they are directed to HLA differences.