Inferring meaningful pathways in weighted metabolic networks

An approach is presented for computing meaningful pathways in the network of small molecule metabolism comprising the chemical reactions characterized in all organisms. The metabolic network is described as a weighted graph in which all the compounds are included, but each compound is assigned a weight equal to the number of reactions in which it participates. Path finding is performed in this graph by searching for one or more paths with lowest weight. Performance is evaluated systematically by computing paths between the first and last reactions in annotated metabolic pathways, and comparing the intermediate reactions in the computed pathways to those in the annotated ones. For the sake of comparison, paths are computed also in the un-weighted raw (all compounds and reactions) and filtered (highly connected pool metabolites removed) metabolic graphs, respectively. The correspondence between the computed and annotated pathways is very poor (<30%) in the raw graph; increasing to approximately 65% in the filtered graph; reaching approximately 85% in the weighted graph. Considering the best-matching path among the five lightest paths increases the correspondence to 92%, on average. We then show that the average distance between pairs of metabolites is significantly larger in the weighted graph than in the raw unfiltered graph, suggesting that the small-world properties previously reported for metabolic networks probably result from irrelevant shortcuts through pool metabolites. In addition, we provide evidence that the length of the shortest path in the weighted graph represents a valid measure of the "metabolic distance" between enzymes. We suggest that the success of our simplistic approach is rooted in the high degree of specificity of the reactions in metabolic pathways, presumably reflecting thermodynamic constraints operating in these pathways. We expect our approach to find useful applications in inferring metabolic pathways in newly sequenced genomes.     

Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning

The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.     

Biochemical fates of alpha hemoglobin bound to alpha hemoglobin-stabilizing protein AHSP

Alpha hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds free alpha hemoglobin (alphaHb) to maintain its structure and limit its pro-oxidant activity. Prior studies have defined two different alphaHb.AHSP complexes. Binding of AHSP to Fe(II) alphaHb induces an unusual configuration in which the F helix of the globin becomes disordered and the heme ring becomes solvent-exposed. Over time, this intermediate oxidizes to form a stable hemichrome in which the proximal (F8) and distal (E7) histidines are coordinated to the heme iron atom. The addition of betaHb to either Fe(II) or Fe(III) alphaHb.AHSP displaces AHSP to generate tetrameric (alpha(2)beta(2)) HbA species. The biochemical properties and in vivo significance of the two alphaHb.AHSP complexes are poorly understood. Here we show that Fe(III) alphaHb.AHSP forms from auto-oxidation of oxygenated alphaHb bound to AHSP and that this process is greatly accelerated at physiologic temperature and oxygen pressures. In contrast to free Fe(III) alphaHb hemichromes, AHSP-bound Fe(III) alphaHb does not precipitate and can be recycled into functional HbA. This requires enzymatic reduction of AHSP-bound alphaHb, either prior to or after extraction by beta subunits. In contrast, reaction of Fe(II) alphaHb-AHSP with betaHb generates functional HbA directly. Our findings support a model in which AHSP can either stabilize alphaHb transiently en route to HbA formation during normal erythropoiesis or convert excessive free alphaHb into a more chemically inert state from which recovery of alphaHb is possible by redox cycling.     

Is the ApoE polymorphism associated with dilated cardiomyopathy?

Apolipoprotein E (ApoE) is 34 kDa protein involved in the modulation of cholesterol transport and homeostasis. Polymorphism of the ApoE gene has been implicated in many chronic cardiovascular and neuronal diseases. ApoE epsilon4 allele has been reported to be associated with increased risk of cardiovascular diseases such as myocardial infarction, hypertension, coronary heart disease, etc. Fifty patients with the end-stage dilated cardiomyopathy (DCM) and advanced congestive heart failure were examined in our study. For evaluation of ApoE polymorphism, novel approach of fast screening of ApoE gene polymorphism by combination of PCR and blotting (CVD StripAssay) was used. Individual genotypes were correlated with basic cardiologic clinical parameters. The reported frequency of this allele in Caucasian population is 14.7 %. Our results showed that in patients with DCM frequency of the ApoE epsilon4 allele is 40 %. Frequency of the genotype epsilon2/4 was 58 % and epsilon3/4 was 22 %. Comparison with control Caucasian groups monitored by others clearly revealed that frequency of epsilon4 alelle is increased in patients with advanced stages of DCM. This observation suggests association of ApoE polymorphism with severe form of DCM. Physiological consequences of this observation remain to be clarified.     

Synthesis and characterization of HE-24.8: a polymeric contrast agent for magnetic resonance angiography

The physical and biological properties of a water-soluble polymeric contrast agent based on a complex of N-(2-hydroxypropyl)methacrylamide copolymer with gadolinium (HE-24.8) were investigated, and its potential for experimental magnetic resonance (MR) angiography was assessed. Relaxivities of Gd-DTPA-BMA, Gd-DTPA-HSA (human serum albumin), and HE-24.8 were determined at 1.5 T. Thermic stability and biocompatibility of HE-24.8 were assessed in vitro and by analyzing kinetics and organ distribution in rats for up to 2 weeks. For comparison, HE-24.8- and Gd-DTPA-HSA-enhanced micro-MR angiographies of brain, chest, and subcutaneous tumors in rats were performed. T1 relaxivity of HE-24.8 (21.3 +/- 1.1 mM(-1) s(-1)) was 5-fold higher than that of Gd-DTPA-BMA (4.1 +/- 0.1 mM(-1) s(-1)) and twice as high as that of Gd-DTPA-HSA (12.4 +/- 0.2 mM(-1) s(-1)). Varying the molecular weight of the polymer (15-46 kDa) did not significantly change the T1 relaxivity. In rats, 20 and 10% of the injected dose of HE-24.8 was detected at 24 and 168 h postinjection, respectively. Upon a relatively rapid initial renal clearance, no specific retention in any organ was noted, with some exception for the reticulo-endothelial system. No measurable release of gadolinium from the polymer-Gd complex or cell toxicity was observed during its incubation in aqueous environment. Excellent display of rat and tumor vascularization was achieved with Gd-DTPA-HSA and HE-24.8; however, contrast of vessels was higher in HE-24.8-enhanced scans. HE-24.8 is considered a macromolecular contrast agent highly suited for experimental MR studies.     

Adaptive radiation and hybridization in Wallace's Dreamponds: evidence from sailfin silversides in the Malili Lakes of Sulawesi

Adaptive radiations are extremely useful to understand factors driving speciation. A challenge in speciation research is to distinguish forces creating novelties and those relevant to divergence and adaptation. Recently, hybridization has regained major interest as a potential force leading to functional novelty and to the genesis of new species. Here, we show that introgressive hybridization is a prominent phenomenon in the radiation of sailfin silversides (Teleostei: Atheriniformes: Telmatherinidae) inhabiting the ancient Malili Lakes of Sulawesi, correlating conspicuously with patterns of increased diversity. We found the most diverse lacustrine species-group of the radiation to be heavily introgressed by genotypes originating from streams of the lake system, an effect that has masked the primary phylogenetic pattern of the flock. We conclude that hybridization could have acted as a key factor in the generation of the flock's spectacular diversity. To our knowledge, this is the first empirical evidence for massive reticulate evolution within a complex animal radiation.     

Grey, a novel mutation in the murine Lyst gene, causes the beige phenotype by skipping of exon 25

The murine beige mutant phenotype and the human Chediak-Higashi syndrome are caused by mutations in the murine Lyst (lysosomal trafficking regulator) gene and the human CHS gene, respectively. In this report we have analyzed a novel murine mutant Lyst allele, called Lyst(bg-grey), that had been found in an ENU mutation screen and named grey because of the grey coat color of affected mice. The phenotype caused by the Lyst(bg-grey) mutation was inherited in a recessive fashion. Melanosomes of melanocytes associated with hair follicles and the choroid layer of the eye, as well as melanosomes in the neural tube-derived pigment epithelium of the retina, were larger and irregularly shaped in homozygous mutants compared with those of wild-type controls. Secretory vesicles in dermal mast cells of the mutant skin were enlarged as well. Test crosses with beige homozygous mutant mice (Lyst(bg)) showed that double heterozygotes (Lyst(bg)/Lyst(bg-grey)) were phenotypically indistinguishable from either homozygous parent, demonstrating that the ENU mutation was an allele of the murine Lyst gene. RT-PCR analyses revealed the skipping of exon 25 in Lyst(bg-grey) mutants, which is predicted to cause a missense D2399E mutation and the loss of the following 77 amino acids encoded by exon 25 but leave the C-terminal end of the protein intact. Analysis of the genomic Lyst locus around exon 25 showed that the splice donor at the end of exon 25 showed a T-to-C transition point mutation. Western blot analysis suggests that the Lyst(bg-grey) mutation causes instability of the LYST protein. Because the phenotype of Lyst(bg) and Lyst(bg-grey) mutants is indistinguishable, at least with respect to melanosomes and secretory granules in mast cells, the Lyst(bg-grey) mutation defines a critical region for the stability of the murine LYST protein.     

Morphologic and molecular characterization of two novel Krt71 (Krt2-6g) mutations: Krt71 rco12 and Krt71 rco13

We have analyzed two novel mouse mutant strains, Rco12 and Rco13, displaying a wavy pelage and curly vibrissae that have been identified in an ENU screen for dominant mutations affecting the pelage. The mutations were mapped to mouse Chromosome 15 and identified as missense point mutations in the first exon of the Krt71 (formerly called Krt2-6g) gene causing alterations of amino acid residue 143 from alanine to glycine (Rco12) and residue 146 from isoleucine to phenylalanine. The morphologic analyses demonstrated that both mutations cause identical phenotypes leading to the formation of filamentous aggregates in Henle’s and Huxley’s layers of the inner root sheath (IRS) of the hair follicle that leads to the bending of the hair shaft. Both novel mutations are located in the immediate vicinity of previously identified mutations in murine Krt71 that cause similar phenotypes and alter the helix initiation motif of the keratin. The characterization of these mutants demonstrates the importance of this Krt71 domain for the formation of linear IRS intermediate filaments.     

The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis

Mammalian Staufen2 (Stau2) is a member of the double-stranded RNA-binding protein family. Its expression is largely restricted to the brain. It is thought to play a role in the delivery of RNA to dendrites of polarized neurons. To investigate the function of Stau2 in mature neurons, we interfered with Stau2 expression by RNA interference (RNAi). Mature neurons lacking Stau2 displayed a significant reduction in the number of dendritic spines and an increase in filopodia-like structures. The number of PSD95-positive synapses and miniature excitatory postsynaptic currents were markedly reduced in Stau2 down-regulated neurons. Akin effects were caused by overexpression of dominant-negative Stau2. The observed phenotype could be rescued by overexpression of two RNAi cleavage-resistant Stau2 isoforms. In situ hybridization revealed reduced expression levels of beta-actin mRNA and fewer dendritic beta-actin mRNPs in Stau2 down-regulated neurons. Thus, our data suggest an important role for Stau2 in the formation and maintenance of dendritic spines of hippocampal neurons.