Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.     

Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by Alcanivorax borkumensis SK2

A novel mutant of the marine oil-degrading bacterium Alcanivorax borkumensis SK2, containing a mini-Tn5 transposon disrupting a "tesB-like" acyl-coenzyme A (CoA) thioesterase gene, was found to hyperproduce polyhydroxyalkanoates (PHA), resulting in the extracellular deposition of this biotechnologically important polymer when grown on alkanes. The tesB-like gene encodes a distinct novel enzyme activity, which acts exclusively on hydroxylated acyl-CoAs and thus represents a hydroxyacyl-CoA-specific thioesterase. Inactivation of this enzyme results in the rechanneling of CoA-activated hydroxylated fatty acids, the cellular intermediates of alkane degradation, towards PHA production. These findings may open up new avenues for the development of simplified biotechnological processes for the production of PHA as a raw material for the production of bioplastics.     

Mouse Emi2 is required to enter meiosis II by reestablishing cyclin B1 during interkinesis

During interkinesis, a metaphase II (MetII) spindle is built immediately after the completion of meiosis I. Oocytes then remain MetII arrested until fertilization. In mouse, we find that early mitotic inhibitor 2 (Emi2), which is an anaphase-promoting complex inhibitor, is involved in both the establishment and the maintenance of MetII arrest. In MetII oocytes, Emi2 needs to be degraded for oocytes to exit meiosis, and such degradation, as visualized by fluorescent protein tagging, occurred tens of minutes ahead of cyclin B1. Emi2 antisense morpholino knockdown during oocyte maturation did not affect polar body (PB) extrusion. However, in interkinesis the central spindle microtubules from meiosis I persisted for a short time, and a MetII spindle failed to assemble. The chromatin in the oocyte quickly decondensed and a nucleus formed. All of these effects were caused by the essential role of Emi2 in stabilizing cyclin B1 after the first PB extrusion because in Emi2 knockdown oocytes a MetII spindle was recovered by Emi2 rescue or by expression of nondegradable cyclin B1 after meiosis I.     

Fragrep： An Efficient Search Tool for Fragmented Patterns in Genomic Sequences

Many classes of non-coding RNAs （ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum） can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.     

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.     

Racial differences in genome-wide methylation profiling and gene expression in breast tissues from healthy women

Breast cancer is more common in European Americans (EAs) than in African Americans (AAs) but mortality from breast cancer is higher among AAs. While there are racial differences in DNA methylation and gene expression in breast tumors, little is known whether such racial differences exist in breast tissues of healthy women. Genome-wide DNA methylation and gene expression profiling was performed in histologically normal breast tissues of healthy women. Linear regression models were used to identify differentially-methylated CpG sites (CpGs) between EAs (n = 61) and AAs (n = 22). Correlations for methylation and expression were assessed. Biological functions of the differentially-methylated genes were assigned using the Ingenuity Pathway Analysis. Among 485 differentially-methylated CpGs by race, 203 were hypermethylated in EAs, and 282 were hypermethylated in AAs. Promoter-related differentially-methylated CpGs were more frequently hypermethylated in EAs (52%) than AAs (27%) while gene body and intergenic CpGs were more frequently hypermethylated in AAs. The differentially-methylated CpGs were enriched for cancer-associated genes with roles in cell death and survival, cellular development, and cell-to-cell signaling. In a separate analysis for correlation in EAs and AAs, different patterns of correlation were found between EAs and AAs. The correlated genes showed different biological networks between EAs and AAs; networks were connected by Ubiquitin C. To our knowledge, this is the first comprehensive genome-wide study to identify differences in methylation and gene expression between EAs and AAs in breast tissues from healthy women. These findings may provide further insights regarding the contribution of epigenetic differences to racial disparities in breast cancer.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Relationship between assemblages of mycorrhizal fungi and bacteria on grass roots

Soils support an enormous microbial diversity, but the ecological drivers of this diversity are poorly understood. Interactions between the roots of individual grass species and the arbuscular mycorrhizal (AM) fungi and bacteria in their rhizoplane were studied in a grazed, unimproved upland pasture. Individual root fragments were isolated from soil cores, DNA extracted and used to identify plant species and assess rhizoplane bacterial and AM fungal assemblages, by amplifying part of the small-subunit ribosomal RNA gene, followed by terminal restriction fragment length polymorphism analysis. For the first time we showed that AM fungal and bacterial assemblages are related in situ and that this relationship occurred at the community level. Principal coordinate analyses of the data show that the AM fungi were a major factor determining the bacterial assemblage on grass roots. We also report a strong influence of the composition of the plant community on AM fungal assemblage. The bacterial assemblage was also influenced by soil pH and was spatially structured, whereas AM fungi were influenced neither by the bacteria nor by soil pH. Our study shows that linkages between plant roots and their microbial communities exist in a complex web of interactions that act at individual and at community levels, with AM fungi influencing the bacterial assemblage, but not the other way round.     

Employment of 16S rDNA gene sequencing techniques for improved identification of difficult-to-identify bacterial veterinary pathogens

To employ 16S rDNA PCR and automated sequencing techniques to identify a collection of bacterial veterinary pathogens from avian, equine, canine and ovine sources, that have proven difficult to identify, employing conventional cultural techniques. Universal or “broad-range” eubacterial PCR was performed on a collection of 46 difficult-to-identify bacterial isolates originating from clinical veterinary specimens. 16S rDNA PCR was performed using two sets of universal primers to successfully generate a composite amplicon of 1,068 bp, which was sequenced to obtain each isolate’s identity. Sequence analysis was able to identify all isolates examined with relative ease. Where the use of molecular identification methods is justified, such as in outbreak control or bioterrorism in animal health, employment of partial 16S rDNA PCR and sequencing employing universal or “broad-range” 16S rDNA, provides a valuable and reliable method of identification of such pathogens.