Triploidy in callus culture ofVicia faba L. investigated by the Giemsa C-banding technique

Summary Callus tissue ofVicia faba has been grown from explants of immature embryos. The karyological analysis by the Giemsa C-banding technique has shown triploid and tri-aneuploid mitoses, all with an extra chromosome and with structurally altered karyotypes. Estimates of the amounts of interphase heterochromatin (chromocentres) indicate endopolyploidy up to 12 n.This work was financially supported by the Research Council of the S.R. Croatia — SIZIV. The authors wish to express their gratitude to the Industroprojekt Zagreb for the use of the image analyser, to Mr M. Denih for the area determinations of drawings, and to Dr D. Schweizer, Vienna, for critical comments.     

Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2

TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs.     

A high-intensity highly coherent soft X-ray femtosecond laser seeded by a high harmonic beam

Synchrotrons have for decades provided invaluable sources of soft X-rays, the application of which has led to significant progress in many areas of science and technology. But future applications of soft X-rays--in structural biology, for example--anticipate the need for pulses with much shorter duration (femtoseconds) and much higher energy (millijoules) than those delivered by synchrotrons. Soft X-ray free-electron lasers should fulfil these requirements but will be limited in number; the pressure on beamtime is therefore likely to be considerable. Laser-driven soft X-ray sources offer a comparatively inexpensive and widely available alternative, but have encountered practical bottlenecks in the quest for high intensities. Here we establish and characterize a soft X-ray laser chain that shows how these bottlenecks can in principle be overcome. By combining the high optical quality available from high-harmonic laser sources (as a seed beam) with a highly energetic soft X-ray laser plasma amplifier, we produce a tabletop soft X-ray femtosecond laser operating at 10 Hz and exhibiting full saturation, high energy, high coherence and full polarization. This technique should be readily applicable on all existing laser-driven soft X-ray facilities.     

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.     

Noradrenaline and serotonin selectively modulate thalamic burst firing by enhancing a hyperpolarization-activated cation current

Neurons in many regions of the mammalian nervous system generate action potentials in two distinct modes: rhythmic oscillations in which spikes cluster together in a cyclical manner, and single spike firing in which action potentials occur relatively independently of one another. Which mode of action potential generation a neuron displays often varies with the behavioural state of the animal. For example, the shift from slow-wave sleep to waking and attentiveness is associated with a change in thalamic neurons from rhythmic burst firing to repetitive single spike activity, and a greatly increased responsiveness to excitatory synaptic inputs. This marked change in firing pattern and excitability is controlled in part by ascending noradrenergic and serotonergic inputs from the brainstem, although the cellular mechanisms of this effect have remained largely unknown. Here we report that noradrenaline and serotonin enhance a mixed Na+/K+ current which is activated by hyperpolarization (Ih) and that this enhancement may be mediated by increases in intracellular concentration of cyclic AMP. This novel action of noradrenaline and serotonin reduces the ability of thalamic neurons to generate rhythmic burst firing and promotes a state of excitability that is conducive to the thalamocortical synaptic processing associated with cognition.     

Acetylcholine inhibits identified interneurons in the cat lateral geniculate nucleus

The transmission of visual information from retina to cortex through the dorsal lateral geniculate nucleus (LGNd) is controlled by non-retinal inputs. Enhanced visually evoked responses in cat LGNd relay cells during periods of increased alertness have been attributed in large part to increased rate of acetylcholine (ACh) release by fibres ascending from the brainstem reticular formation. ACh can modulate geniculate visual responses in vivo, but comparatively little is known about the underlying ionic mechanisms of these cholinergic actions. Although direct excitation of LGNd relay neurons has been shown in vitro, the situation is complicated because cholinergic axons form numerous and complex synapses not only with relay cells, but also with inhibitory interneurons, and electrical activation of the brainstem cholinergic neurons reduces inhibitory postsynaptic potentials in the LGNd. We report here that morphologically characterized interneurons in the cat LGNd possess distinctive electrophysiological properties in comparison with those of relay cells and are inhibited by ACh through a muscarinic receptor-mediated increase in potassium conductance. Together the direct excitation of relay cells and inhibition of intrageniculate interneurons allow the ascending cholinergic system to exert a powerful facilitatory influence over the transfer of visual information to the cerebral cortex.     

Inhibitory influence of acid citrate dextrose on the activity of dehydrogenase in erythrocytes

Zusammenfassung Der viel verwendete gerinnungshemmende Zusatz ACD (acid citric dextrose) vermindert die Aktivität der Glucose-6-Phosphat-Dehydrogenase und anderer Dehydrogenasen der Erythrozyten. Diese Wirkung kommt durch pH-Verschiebung zustande.