Low sequence similarity and gene content of symbiotic clusters of Bradyrhizobium sp. WM9 (Lupinus) indicate early divergence of “lupin” lineage in the genus Bradyrhizobium

Two sequenced nodulation regions of lupin Bradyrhizobium sp. WM9 carried the majority of genes involved in the Nod factor production. The nod region I harbored: nolA, nodD, nodA, nodB, nodC, nodS, nodI, nodJ, nolO, nodZ, fixR, nifA, fixA, nodM, nolK and noeL. This gene arrangement resembled that found in the nodulation region of Bradyrhizobium japonicum USDA110, however strain WM9 harbored only one nodD gene copy, while the nodM, nolK and noeL genes had no counterparts in the 410 kb symbiotic region of strain USDA110. Region II harbored nolL and nodW, but lacked an nodV gene. Both regions carried ORFs that lacked similarity to the published USDA110 sequences, though they had homologues in symbiotic regions of Rhizobium etli, Sinorhizobium sp. NGR234 and Mesorhizobium loti. These differences in gene content, as well as a low average sequence identity (70%) of symbiotic genes with respect to B. japonicum USDA110 were in contrast with the phylogenetic relationship of USDA110 and WM9 revealed by the analysis of 16S rDNA and dnaK sequences. This most likely reflected an early divergence of symbiotic loci, and possible co-speciation with distinct legumes. During this process the loss of a noeI gene and the acquisition of a nolL gene could be regarded as an adaptation towards these legumes that responded to Nod factors carrying 4-O-acetylfucose rather than 2-O-methylfucose. This explained various responses of lupins and serradella plants to infection by mutants in nodZ and nolL genes, knowing that serradella is a stringent legume while lupins are more promiscuous legumes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Analysis of microsatellite instability and loss of heterozygosity in breast cancer with the use of a well characterized multiplex system

Analysis of microsatellite instability (MI) and loss of heterozygosity (LOH) is recommended for screening patients with sporadic and hereditary malignancies. This study shows an application of a fluorescent hexaplex PCR system for microsatellite typing on A.L.F. DNA Sequencer (Pharmacia Biotech). This technique detects changes in microsatellites providing a time-efficient, reliable and accurate method for MI and LOH analyses. The Fragment Manager software was used for automated size calculation and quantitation of DNA fragments, enabling rapid and precise measurement of allelic ratios. We examined 70 breast cancer and 70 control DNA specimens, classified all the patterns of microsatellite alterations, and set up MI and LOH assessment criteria for the automated multiplex fluorescent method.     

Amide proton temperature coefficients as hydrogen bond indicators in proteins

Correlations between amide proton temperature coefficients (_HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show _HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 3₁₀ helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures.     

Synthesis and X-ray structures of sulfate esters of fructose and its isopropylidene derivatives. Part 1: 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose 1-sulfate and 4,5-O-isopropylidene-beta-D-fructopyranose 1-sulfate

2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose 1-sulfate have been synthesized by treatment of 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose with pyridine-sulfur trioxide complex. Direct hydrolysis of the isopropylidene group at C-4, C-5 gave 2,3-O-isopropylidene-beta-D-fructopyranose 1-sulfate. The crystal and molecular structures of ammonium (1a) and potassium (1b) salts of diisopropylidene derivative and ammonium (2) salt of monoisopropylidene derivative were determined by X-ray crystallography. Data for 1a and 1b were collected in 120 K and in 150 K for 2. All salts crystallized in P2(1)2(1)2(1) space group. There are three independent anions in asymmetric unit in 1b. Pyranose rings in the diisopropylidene derivative salts studied adopt 2S(0) twist boat conformation, whereas in the monoisopropylidene exists in a slightly distorted chair conformation (4C(1)). A staggered conformation is preferred by the sulfate group as indicated by values of C-(ester)-S-O(terminal) torsion angles: -173.2(4) degrees in 1a, 175.1(6) degrees in anion A of 1b, 170.8(6) degrees in anion C of 1b and 177.9(2) degrees in 2. However, strong interactions such as potassium-oxygen and H-bonds may affect the geometry: in anion B of 1b the value of the torsion angle is 139.4(6) degrees.     

NMR structures of two variants of bovine pancreatic trypsin inhibitor (BPTI) reveal unexpected influence of mutations on protein structure and stability

Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues.     

Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate

Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants.     

Measuring protein conformational changes by FRET/LRET

Fluorescence resonance energy transfer (FRET) provides a unique means of measuring interatomic distances in biological molecules in real time. Recent advances have been made in the application of this technique to studies of conformational changes in proteins. New ways of introducing fluorescence probes into proteins, newly developed fluorescence probes, and progress in the technologies for fluorescence signal detection have greatly expanded the range of applications of FRET. In particular, studies of conformational changes in proteins at a single molecule level and in the native in vivo context of a living cell are now possible.     

Evaluation of mouse preimplantation embryos exposed to oxidative stress cultured with insulin-like growth factor I and II, epidermal growth factor, insulin, transferrin and selenium

Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress.