Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.

Purification of skeletal muscle dihydropyridine binding sites has enabled protein complexes to be isolated from which Ca2+ currents have been reconstituted. Complementary DNAs encoding the five subunits of the dihydropyridine receptor, alpha 1, beta, gamma, alpha 2 and delta, have been cloned and it is now recognized that alpha 2 and delta are derived from a common precursor. The alpha 1 subunit can itself produce Ca2+ currents, as was demonstrated using mouse L cells lacking alpha 2 delta, beta and gamma (our unpublished results). In L cells, stable expression of skeletal muscle alpha 1 alone was sufficient to generate voltage-sensitive, high-threshold L-type Ca2+ channel currents which were dihydropyridine-sensitive and blocked by Cd2+, but the activation kinetics were about 100 times slower than expected for skeletal muscle Ca2+ channel currents. This could have been due to the cell type in which alpha 1 was being expressed or to the lack of a regulatory component particularly one of the subunits that copurifies with alpha 1. We show here that coexpression of skeletal muscle beta with skeletal muscle alpha 1 generates cell lines expressing Ca2+ channel currents with normal activation kinetics as evidence for the participation of the dihydropyridine-receptor beta subunits in the generation of skeletal muscle Ca2+ channel currents.     

Direct measurement of antiferromagnetic domain fluctuations

Measurements of magnetic noise emanating from ferromagnets owing to domain motion were first carried out nearly 100 years ago, and have underpinned much science and technology. Antiferromagnets, which carry no net external magnetic dipole moment, yet have a periodic arrangement of the electron spins extending over macroscopic distances, should also display magnetic noise. However, this must be sampled at spatial wavelengths of the order of several interatomic spacings, rather than the macroscopic scales characteristic of ferromagnets. Here we present a direct measurement of the fluctuations in the nanometre-scale superstructure of spin- and charge-density waves associated with antiferromagnetism in elemental chromium. The technique used is X-ray photon correlation spectroscopy, where coherent X-ray diffraction produces a speckle pattern that serves as a 'fingerprint' of a particular magnetic domain configuration. The temporal evolution of the patterns corresponds to domain walls advancing and retreating over micrometre distances. This work demonstrates a useful measurement tool for antiferromagnetic domain wall engineering, but also reveals a fundamental finding about spin dynamics in the simplest antiferromagnet: although the domain wall motion is thermally activated at temperatures above 100 K, it is not so at lower temperatures, and indeed has a rate that saturates at a finite value-consistent with quantum fluctuations-on cooling below 40 K.     

猪毛蒿对其他植物生长的他感作用

为测定猪毛蒿二次代谢物对其他植物产生的他感作用,利用气相色谱、质谱分析鉴定了猪毛蒿水溶性及挥发性二次代谢物,结果表明。猪毛蒿含有较多的水溶性及挥发性二次代谢物。这些物质对与其共生及与其非共生植物的种子发芽、幼苗生长具有较强的他感作用,这种作用随二次代谢物浓度的增高而增大,对非共生植物的作用远大于共生的植物。     

An allylic ketyl radical intermediate in clostridial amino-acid fermentation

The human pathogenic bacterium Clostridium difficile thrives by the fermentation of l-leucine to ammonia, CO(2), 3-methylbutanoate and 4-methylpentanoate under anaerobic conditions. The reductive branch to 4-methylpentanoate proceeds by means of the dehydration of (R)-2-hydroxy-4-methylpentanoyl-CoA to 4-methylpent-2-enoyl-CoA, which is chemically the most demanding step. Ketyl radicals have been proposed to mediate this reaction catalysed by an iron-sulphur-cluster-containing dehydratase, which requires activation by ATP-dependent electron transfer from a second iron-sulphur protein functionally similar to the iron protein of nitrogenase. Here we identify a kinetically competent product-related allylic ketyl radical bound to the enzyme by electron paramagnetic resonance spectroscopy employing isotope-labelled (R)-2-hydroxy-4-methylpentanoyl-CoA species. We also found that the enzyme generated the stabilized pentadienoyl ketyl radical from the substrate analogue 2-hydroxypent-4-enoyl-CoA, supporting the proposed mechanism. Our results imply that also other 2-hydroxyacyl-CoA dehydratases and the related benzoyl-CoA reductases-present in anaerobically living bacteria-employ ketyl radical intermediates. The absence of radical generators such as coenzyme B12, S-adenosylmethionine or oxygen makes these enzymes unprecedented in biochemistry.     

Prediction of Essential Proteins Using Topological Properties in GO-Pruned PPI Network Based on Machine Learning Methods

The prediction of essential proteins, the minimal set required for a living cell to support cellular life, is an important task to understand the cellular processes of an organism. Fast progress in high-throughput technologies and the production of large amounts of data enable the discovery of essential proteins at the system level by analyzing Protein-Protein Interaction (PPI) networks, and replacing biological or chemical experiments. Furthermore, additional gene-level annotation information, such as Gene Ontology (GO) terms, helps to detect essential proteins with higher accuracy. Various centrality algorithms have been used to determine essential proteins in a PPI network, and, recently motif centrality GO, which is based on network motifs and GO terms, works best in detecting essential proteins in a Baker’s yeast Saccharomyces cerevisiae PPI network, compared to other centrality algorithms. However, each centrality algorithm contributes to the detection of essential proteins with different properties, which makes the integration of them a logical next step. In this paper, we construct a new feature space, named CENT-ING-GO consisting of various centrality measures and GO terms, and provide a computational approach to predict essential proteins with various machine learning techniques. The experimental results show that CENT-ING-GO feature space improves performance over the INT-GO feature space in previous work by Acencio and Lemke in 2009. We also demonstrate that pruning a PPI with informative GO terms can improve the prediction performance further.     

The Lkb1 metabolic sensor maintains haematopoietic stem cell survival

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

Display of proteins on Bacillus subtilis endospores

The targeting and anchoring of heterologous proteins and peptides to the outer surface of bacteriophages and cells is becoming increasingly important, and has been employed as a tool for fundamental and applied research in microbiology, molecular biology, vaccinology, and biotechnology. Less known are endospores or spores produced by some Gram-positive species. Spores of Bacillus subtilis are surrounded by a spore coat on their outside, and a few proteins have been identified being located on the outside layer and have been successfully used to immobilize antigens and some other proteins and enzymes. The major advantage of spores over the other published systems is their synthesis within the cytoplasm of the bacterial cell. Therefore, any heterologous protein to be anchored on the outside does not have to cross any membrane. Furthermore, spores are extremely resistant against high temperature, irradiation and many chemicals, and can be stored for many years at room temperature.     

Identification of Lps2 as a key transducer of MyD88-independent TIR signalling

In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.     

Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes

Defining the molecular strategies that integrate diverse signalling pathways in the expression of specific gene programmes that are critical in homeostasis and disease remains a central issue in biology. This is particularly pertinent in cancer biology because downregulation of tumour metastasis suppressor genes is a common occurrence, and the underlying molecular mechanisms are not well established. Here we report that the downregulation of a metastasis suppressor gene, KAI1, in prostate cancer cells involves the inhibitory actions of beta-catenin, along with a reptin chromatin remodelling complex. This inhibitory function of beta-catenin-reptin requires both increased beta-catenin expression and recruitment of histone deacetylase activity. The coordinated actions of beta-catenin-reptin components that mediate the repressive state serve to antagonize a Tip60 coactivator complex that is required for activation; the balance of these opposing complexes controls the expression of KAI1 and metastatic potential. The molecular mechanisms underlying the antagonistic regulation of beta-catenin-reptin and the Tip60 coactivator complexes for the metastasis suppressor gene, KAI1, are likely to be prototypic of a selective downregulation strategy for many genes, including a subset of NF-kappaB target genes.