Inhibition of phosphorylcholine binding to antibodies using synthetic peptides

The amino-acid sequence Phe-Tyr-Met-Glu is unique to phosphorylcholine (PC)-binding antibodies. It occurs in the first complementarity-determining region (CDR1) of the immunoglobulin heavy chains in 89% of all the anti-PC myeloma and hybridoma proteins but is not present in 490 other immunoglobulin heavy chains, 854 light chains or in 2,260 other unrelated proteins. This unique tetrapeptide therefore seems to be involved in PC binding. Here we compare the effectiveness of Phe-Tyr-Met-Glu and other structurally related peptides in inhibiting the binding of PC to PC-binding proteins McPC603 and HOPC8. We also test a surface-simulation peptide that was constructed to mimic the combining site of McPC603. Our data suggest that all these peptides inhibit the binding of PC to PC-binding proteins non-specifically and we show by computer modelling that the surface-simulation peptide does not duplicate the combining site of McPC603.     

Segregation at three loci explains familial and population risk in Hirschsprung disease

Hirschsprung disease (HSCR), the most common hereditary cause of intestinal obstruction, shows considerable variation and complex inheritance. Coding sequence mutations in RET, GDNF, EDNRB, EDN3 and SOX10 lead to long-segment (L-HSCR) and syndromic HSCR but fail to explain the transmission of the much more common short-segment form (S-HSCR). We conducted a genome scan in families with S-HSCR and identified susceptibility loci at 3p21, 10q11 and 19q12 that seem to be necessary and sufficient to explain recurrence risk and population incidence. The gene at 10q11 is probably RET, supporting its crucial role in all forms of HSCR; however, coding sequence mutations are present in only 40% of linked families, suggesting the importance of noncoding variation. Here we show oligogenic inheritance of S-HSCR, the 3p21 and 19q12 loci as RET-dependent modifiers, and a parent-of-origin effect at RET. This study demonstrates by a complete genetic dissection why the inheritance pattern of S-HSCR is nonmendelian.     

Plant pathogens. Mitochondrial control of fungal hybrid virulence

Hybrid species of fungal pathogens that infect wild and cultivated plants are emerging with new virulence and host ranges, posing a threat to agriculture and forestry. Here we show that the virulence of hybrid species of the basidiomycete fungus Heterobasidion annosum (Fr) Bref, a causal agent of root and butt rot in conifers and one of the most economically important forest pathogens, is controlled by their mitochondrial genome. Our results indicate that cooperation between organelles that contain genetic information may influence the phenotype of hybrid phytopathogens.     

Formation of gigantic mitochondria in hypoxic isolated perfused rat hearts

Zusammenfassung Herzen gesunder, erwachsener Ratten wurden mit Krebs-Henseleit-Bikarbonatlösung durchströmt (95% O₂5% CO₂). Bei künstlich herbeigeführter Hypoxie (95% N₂5% CO₂) erschienen die Herzmitochondrien innerhalb von 7 min bedeutend vergrössert und zu 25% miteinander verschmolzen.     

Structural basis for modulation and agonist specificity of HCN pacemaker channels

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.     

Osteoblastic cells regulate the haematopoietic stem cell niche

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.     

Structure of the bacteriophage phi29 DNA packaging motor

Motors generating mechanical force, powered by the hydrolysis of ATP, translocate double-stranded DNA into preformed capsids (proheads) of bacterial viruses and certain animal viruses. Here we describe the motor that packages the double-stranded DNA of the Bacillus subtilis bacteriophage phi29 into a precursor capsid. We determined the structure of the head-tail connector--the central component of the phi29 DNA packaging motor--to 3.2 A resolution by means of X-ray crystallography. We then fitted the connector into the electron densities of the prohead and of the partially packaged prohead as determined using cryo-electron microscopy and image reconstruction analysis. Our results suggest that the prohead plus dodecameric connector, prohead RNA, viral ATPase and DNA comprise a rotary motor with the head-prohead RNA-ATPase complex acting as a stator, the DNA acting as a spindle, and the connector as a ball-race. The helical nature of the DNA converts the rotary action of the connector into translation of the DNA.