Mitochondrial targeting sequences may form amphiphilic helices.

Twenty three mitochondrial targeting sequences have been analysed with regard to their potential for forming amphiphilic helices. It is shown that most if not all of these sequences can be expected to form helices with high hydrophobic moments in a suitable environment. In the few cases studied so far, the segments of maximal hydrophobic moment coincide closely with 'critical' regions defined by deletions and point mutations.     

Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering

In N-glycosylated glycoproteins, carbohydrate is attached to Asn in the sequence Asn-X-Ser/Thr, where X denotes any amino acid. However, the presence of this consensus peptide does not always lead to glycosylation. We have compiled an extensive collection of glycosylated and non-glycosylated Asn-X-Thr/Ser sites and present a statistical study based on this data set. Our results indicate that non-glycosylated sites tend to be found more frequently towards the C termini of glycoproteins, and that proline residues in positions X and Y in the consensus Asn-X-Thr/Ser-Y strongly reduce the likelihood of N-linked glycosylation. Beyond this, there are no obvious local sequence features that seem to correlate with the absence or presence of N-linked glycosylation. These findings are discussed in terms of the prediction and engineering of glycosylation sites in secretory proteins.     

Molecular characterization of three Mhc class II B haplotypes in the ring-necked pheasant

We investigated the class II B genes in free-ranging population of the ring-necked pheasant Phasianus colchicus by a combination of restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), and DNA sequencing. Special attention was paid to the variation in the second exon, which encodes the peptide-binding ₁-domain. The population was introduced, but it still exhibited major histocompatibility complex polymorphism with at least three segregating class II B haplotypes and consequently six genotypes. We found two class II B genes associated with each haplotype. The class II B genes of birds had until then only been molecularly characterized in the domestic chicken. the pheasant genes were highly variable, although one of the amplified sequences was found in two different haplotypes. Taken together, the most polymorphic positions (residues 37 and 38) were not identical in any of the predicted protein sequences, but all except one of the motifs had already been foud in the domestic chicken. Structurally important features in mammalian class II B genes were generally conserved also in the pheasant sequences, but the loss of a potential salt bridge constituent (Arg₇₂) in several sequences may suggest a slightly different structure of the adjacent parts of the peptide-binding groove. The pheasant genes are most closely related to the so called B-LBII family in the chicken, indicating that this represents a major line of development among avian class II B genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X75403-X75407. Correspondence to: H. Wittzell, Department of Theoretical Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden.     

Prediction of effective genome size in metagenomic samples

We introduce a novel computational approach to predict effective genome size (EGS; a measure that includes multiple plasmid copies, inserted sequences, and associated phages and viruses) from short sequencing reads of environmental genomics (or metagenomics) projects. We observe considerable EGS differences between environments and link this with ecologic complexity as well as species composition (for instance, the presence of eukaryotes). For example, we estimate EGS in a complex, organism-dense farm soil sample at about 6.3 megabases (Mb) whereas that of the bacteria therein is only 4.7 Mb; for bacteria in a nutrient-poor, organism-sparse ocean surface water sample, EGS is as low as 1.6 Mb. The method also permits evaluation of completion status and assembly bias in single-genome sequencing projects.     

Photoinhibition of photosynthesis represents a mechanism for the long-term regulation of photosystem II

The obligate shade plant, Tradescantia albiflora Kunth grown at 50 mol photons · m^–2 s^–1 and Pisum sativum L. acclimated to two photon fluence rates, 50 and 300 mol · m^–2 · s^–1, were exposed to photoinhibitory light conditions of 1700 mol · m^–2 · s^–1 for 4 h at 22° C. Photosynthesis was assayed by measurement of CO₂-saturated O₂ evolution, and photosystem II (PSII) was assayed using modulated chlorophyll fluorescence and flash-yield determinations of functional reaction centres. Tradescantia was most sensitive to photoinhibition, while pea grown at 300 mol · m^–2 · s^–1 was most resistant, with pea grown at 50 mol · m^–2 · s^–1 showing an intermediate sensitivity. A very good correlation was found between the decrease of functional PSII reaction centres and both the inhibition of photosynthesis and PSII photochemistry. Photoinhibition caused a decline in the maximum quantum yield for PSII electron transport as determined by the product of photochemical quenching (q_p) and the yield of open PSII reaction centres as given by the steady-state fluorescence ratio, F_vF_m, according to Genty et al. (1989, Biochim. Biophys. Acta 990, 81–92). The decrease in the quantum yield for PSII electron transport was fully accounted for by a decrease in F_vF_m, since q_p at a given photon fluence rate was similar for photoinhibited and noninhibited plants. Under lightsaturating conditions, the quantum yield of PSII electron transport was similar in photoinhibited and noninhibited plants. The data give support for the view that photoinhibition of the reaction centres of PSII represents a stable, long-term, down-regulation of photochemistry, which occurs in plants under sustained high-light conditions, and replaces part of the regulation usually exerted by the transthylakoid pH gradient. Furthermore, by investigating the susceptibility of differently lightacclimated sun and shade species to photoinhibition in relation to q_p, i.e. the fraction of open-to-closed PSII reaction centres, we also show that irrespective of light acclimation, plants become susceptible to photoinhibition when the majority of their PSII reaction centres are still open (i.e. primary quinone acceptor oxidized). Photoinhibition appears to be an unavoidable consequence of PSII function when light causes sustained closure of more than 40% of PSII reaction centres.Abbreviations F_o and F_o minimal fluorescence when all PSII reaction centres are open in darkness and steady-state light, respectively - F_m and F_m maximal fluorescence when all PSII reaction centres are closed in darkand light-acclimated leaves, respectively - F_v variable fluorescence - (F_m-F_o) under steady-state light con-ditions - F_s steady-state fluorescence in light - Q_A the primary,stable quinone acceptor of PSII - q_Ne non-photochemical quench-ing of fluorescence due to high energy state - (pH); q_Ni non-photochemical quenching of fluorescence due to photoinhibition - q_p photochemical quenching of fluorescence To whom correspondence should be addressedThis work was supported by the Swedish Natural Science Research Council (G.Ö.) and the award of a National Research Fellowship to J.M.A and W.S.C. We thank Dr. Paul Kriedemann, Division of Forestry and Forest Products, CSIRO, Canberra, Australia, for helpful discussions.     

Contrast-enhancement cardiac magnetic resonance imaging beyond the scope of viability

The clinical applications of cardiovascular magnetic resonance imaging with contrast enhancement are expanding. Besides the direct visualisation of viable and non-viable myocardium, this technique is increasingly used in a variety of cardiac disorders to determine the exact aetiology, guide proper treatment, and predict outcome and prognosis. In this review, we discuss the value of cardiovascular magnetic resonance imaging with contrast enhancement in a range of cardiac disorders, in which this technique may provide insights beyond the scope of myocardial viability.     

Covalent labelling of histones with aurothiomalate. Gold-labelled H2A-H2B dimers assemble to octamers, which form crystalline helical tubes

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A-H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3-H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.     

Laminin alters cell shape and stimulates motility and proliferation of murine skeletal myoblasts

Proliferating skeletal myoblasts show multiple specific responses to laminin, one of the major glycoprotein components of basement membranes. Using MM14Dy myoblasts, a myogenic cell strain derived from a normal adult mouse skeletal muscle, we show in this study that substrate-bound laminin but not other matrix proteins such as collagens or fibronectin specifically and rapidly induces the outgrowth of cell processes, resulting in bipolar, spindle-shaped cells. This effect is independent from the presence of collagens or serum, and was also observed in primary cultures of fetal mouse skeletal myoblasts. The outgrowth of cell processes on laminin is associated with a dramatic stimulation of cell motility: MM14 myoblasts migrate about five times faster on laminin than on fibronectin. In another series of experiments the effect of laminin and fibronectin on thymidine uptake and proliferation of myoblasts was tested. On top of a type I collagen substrate which was provided to ensure complete adhesion even at low doses of laminin or fibronectin, laminin stimulated myoblast proliferation and incorporation of [3H]thymidine in a dose-dependent manner. The stimulation is two- to threefold higher than on dishes coated with equivalent amounts of fibronectin and is observed both in the presence and in the absence of serum. These results suggest that laminin, a major component of the muscle basal lamina, may be actively involved in the development and regeneration of skeletal muscle.     

Transcending the impenetrable: how proteins come to terms with membranes

In the living cell, proteins are efficiently sorted to a whole range of subcellular compartments. In many cases, sorting specificity is mediated by short 'sorting signals' attached either permanently or transiently to the protein. At long last, a fairly coherent picture of the design and function of many such sorting signals is beginning to emerge.