The first step of aminoacylation at the atomic level in histidyl-tRNA synthetase

The crystal structure of an enzyme–substrate complex with histidyl-tRNA synthetase from Escherichia coli, ATP, and the amino acid analog histidinol is described and compared with the previously obtained enzyme–product complex with histidyl-adenylate. An active site arginine, Arg-259, unique to all histidyl-tRNA synthetases, plays the role of the catalytic magnesium ion seen in seryl-tRNA synthetase. When Arg-259 is substituted with histidine, the apparent second order rate constant (kcat/Km) for the pyrophosphate exchange reaction and the aminoacylation reaction decreases 1,000-fold and 500-fold, respectively. Crystals soaked with MnCl2 reveal the existence of two metal binding sites between β- and γ-phosphates; these sites appear to stabilize the conformation of the pyrophosphate. The use of both conserved metal ions and arginine in phosphoryl transfer provides evidence of significant early functional divergence of class II aminoacyl-tRNA synthetases.

Effectiveness of home care programmes for patients with incurable cancer on their quality of life and time spent in hospital: systematic review

Objective: To investigate whether for patients with incurable cancer comprehensive home care programmes are more effective than standard care in maintaining the patients’ quality of life and reducing their “readmission time” (percentage of days spent in hospital from start of care till death).

Purification of the Trehalase GMTRE1 from Soybean Nodules and Cloning of Its cDNA. GMTRE1 Is Expressed at a Low Level in Multiple Tissues1

Trehalose (α-d-glucopyranosyl-1,1-α-d-glucopyranoside), a disaccharide widespread among microbes and lower invertebrates, is generally believed to be nonexistent in higher plants. However, the recent discovery of Arabidopsis genes whose products are involved in trehalose synthesis has renewed interest in the possibility of a function of trehalose in higher plants. We previously showed that trehalase, the enzyme that degrades trehalose, is present in nodules of soybean (Glycine max [L.] Merr.), and we characterized the enzyme as an apoplastic glycoprotein. Here we describe the purification of this trehalase to homogeneity and the cloning of a full-length cDNA encoding this enzyme, named GMTRE1 (G. max trehalase 1). The amino acid sequence derived from the open reading frame of GMTRE1 shows strong homology to known trehalases from bacteria, fungi, and animals. GMTRE1 is a single-copy gene and is expressed at a low but constant level in many tissues.

Heat-Shock-Induced Changes in Intracellular Ca2+ Level in Tobacco Seedlings in Relation to Thermotolerance1

Exposure of plants to elevated temperatures results in a complex set of changes in gene expression that induce thermotolerance and improve cellular survival to subsequent stress. Pretreatment of young tobacco (Nicotiana plumbaginifolia) seedlings with Ca2+ or ethylene glycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid enhanced or diminished subsequent thermotolerance, respectively, compared with untreated seedlings, suggesting a possible involvement of cytosolic Ca2+ in heat-shock (HS) signal transduction. Using tobacco seedlings transformed with the Ca2+-sensitive, luminescent protein aequorin, we observed that HS temperatures induced prolonged but transient increases in cytoplasmic but not chloroplastic Ca2+. A single HS initiated a refractory period in which additional HS signals failed to increase cytosolic Ca2+. However, throughout this refractory period, seedlings responded to mechanical stimulation or cold shock with cytosolic Ca2+ increases similar to untreated controls. These observations suggest that there may be specific pools of cytosolic Ca2+ mobilized by heat treatments or that the refractory period results from a temporary block in HS perception or transduction. Use of inhibitors suggests that HS mobilizes cytosolic Ca2+ from both intracellular and extracellular sources.

Histone H1 overexpressed to high level in tobacco affects certain developmental programs but has limited effect on basal cellular functions.

Histone H1, a major structural component of chromatin fiber, is believed to act as a general repressor of transcription. To investigate in vivo the role of this protein in transcription regulation during development of a multicellular organism, we made transgenic tobacco plants that overexpress the gene for Arabidopsis histone H1. In all plants that overexpressed H1 the total H1-to-DNA ratio in chromatin increased 2.3-2.8 times compared with the physiological level. This was accompanied by 50-100% decrease of native tobacco H1. The phenotypic changes in H1-overexpressing plants ranged from mild to severe perturbations in morphological appearance and flowering. No correlation was observed between the extent of phenotypic change and the variation in the amount of overexpressed H1 or the presence or absence of the native tobacco H1. However, the severe phenotypic changes were correlated with early occurrence during plant growth of cells with abnormally heterochromatinized nuclei. Such cells occurred considerably later in plants with milder changes. Surprisingly, the ability of cells with highly heterochromatinized nuclei to fulfill basic physiological functions, including differentiation, was not markedly hampered. The results support the suggestion that chromatin structural changes dependent on H1 stoichiometry and on the profile of major H1 variants have limited regulatory effect on the activity of genes that control basal cellular functions. However, the H1-mediated chromatin changes can be of much greater importance for the regulation of genes involved in control of specific developmental programs.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

Dynamic mapping at the laminar level of odor-elicited responses in rat olfactory bulb by functional MRI

We have applied functional MRI (fMRI) based on blood oxygenation level-dependent (BOLD) image-contrast to map odor-elicited olfactory responses at the laminar level in the rat olfactory bulb (OB) elicited by iso-amyl acetate (10−2 dilution of saturated vapor) with spatial and temporal resolutions of 220×220×1,000 μm and 36 s. The laminar structure of the OB was clearly depicted by high-resolution in vivo anatomical MRI with spatial resolution of 110×110×1,000 μm. In repeated BOLD fMRI measurements, highly significant (P < 0.001) foci were located in the outer layers of both OBs. The occurrence of focal OB activity within a domain at the level of individual glomeruli or groups of glomeruli was corroborated on an intra- and inter-animal basis under anesthetized conditions with this noninvasive method. The dynamic studies demonstrated that the odor-elicited BOLD activations were highly reproducible on a time scale of minutes, whereas over tens of minutes the activations sometimes varied slowly. We found large BOLD signal (ΔS/S = 10–30%) arising from the olfactory nerve layer, which is devoid of synapses and composed of unmyelinated fibers and glial cells. Our results support previous studies with other methods showing that odors elicit activity within glomerular layer domains in the mammalian OB, and extend the analysis to shorter time periods at the level of individual glomeruli or groups of glomeruli. With further improvement, BOLD fMRI should be ideal for systematic analysis of the functional significance of individual glomeruli in olfactory information encoding and of spatiotemporal processing within the olfactory system.

Perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting

To investigate the nature of plasticity in the adult visual system, perceptual learning was measured in a peripheral orientation discrimination task with systematically varying amounts of external (environmental) noise. The signal contrasts required to achieve threshold were reduced by a factor or two or more after training at all levels of external noise. The strong quantitative regularities revealed by this novel paradigm ruled out changes in multiplicative internal noise, changes in transducer nonlinearites, and simple attentional tradeoffs. Instead, the regularities specify the mechanisms of perceptual learning at the behavioral level as a combination of external noise exclusion and stimulus enhancement via additive internal noise reduction. The findings also constrain the neural architecture of perceptual learning. Plasticity in the weights between basic visual channels and decision is sufficient to account for perceptual learning without requiring the retuning of visual mechanisms.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.