The major amino acid transporter superfamily has a similar core structure as Na+-galactose and Na+-leucine transporters

The sodium solute symporters (SSS) and neurotransmitter sodium symporters (NSS) are two families of secondary transporters that are not related in amino acid sequence. Nonetheless, recent crystal structures showed that the Na⁺/galactose (SSS) and Na⁺/leucine (NSS) transporters have similar core structures. The structural relatedness highlights the need for classification methods for membrane protein structures based on other criteria than amino acid similarity. Here, we demonstrate that a method based on hydropathy profile alignments convincingly identifies structural similarity between the NSS and SSS families. Most importantly, the method shows that one of the largest transporter families for which a crystal structure is elusive (the amino acid/polyamine/organocation or APC superfamily), also shares the similar core structure observed for the Na⁺/galactose and Na⁺/leucine transporters. The APC superfamily contains the major amino acid transporter families that are found throughout life. Insight into their structure will significantly facilitate the studies of this important group of transporters.     

Compartmentation of NAD+-dependent signalling

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.     

An analysis of biphasic time courses: the inactivation of (Na+ + K+)-ATPase and Ca2+-ATPase by ATP-analogs

The inactivation of (NA+ + K+)-ATPase and Ca2+-ATPase brought about by the substitution of ATP by covalently binding analogs is studied. Most of the analogs cause biphasic courses of inactivation. The families of time courses obtained for different concentrations of the analog exhibit a characteristic feature that is common to both ATPases. The times of transition from one branch to the other of the biphasic curves are practically independent of the concentration of the analog. An analysis of the eigenvalues from different reaction models shows that for these time evolutions the enzyme exists necessarily in two states, only one of which is active for the analog. As a preliminary attempt, the models have been fitted to the experimental data of three different sets of families of curves. It is demonstrated that a two-sites model of inactivation of (Na+ + K+)-ATPase postulated in the literature cannot be valid.     

Blocks+: a non-redundant database of protein alignment blocks derived from multiple compilations.

MOTIVATION: As databanks grow, sequence classification and prediction of function by searching protein family databases becomes increasingly valuable. The original Blocks Database, which contains ungapped multiple alignments for families documented in Prosite, can be searched to classify new sequences. However, Prosite is incomplete, and families from other databases are now available to expand coverage of the Blocks Database. RESULTS: To take advantage of protein family information present in several existing compilations, we have used five databases to construct Blocks+, a unified database that is built on the PROTOMAT/BLOSUM scoring model and that can be searched using a single algorithm for consistent sequence classification. The LAMA blocks-versus-blocks searching program identifies overlapping protein families, making possible a non-redundant hierarchical compilation. Blocks+ consists of all blocks derived from PROSITE, blocks from Prints not present in PROSITE, blocks from Pfam-A not present in PROSITE or Prints, and so on for ProDom and Domo, for a total of 1995 protein families represented by 8909 blocks, doubling the coverage of the original Blocks Database. A challenge for any procedure aimed at non-redundancy is to retain related but distinct families while discarding those that are duplicates. We illustrate how using multiple compilations can minimize this potential problem by examining the SNF2 family of ATPases, which is detectably similar to distinct families of helicases and ATPases. AVAILABILITY: http://blocks.fhcrc.org/     

The age of major monocot groups inferred from 800+ rbcL sequences

Phylogenetic research on monocots has been extraordinarily active over the past years. With the familial interrelationships being sufficiently understood, the question of divergence times and crown node ages of major lineages comes into focus. In this study we present the first attempt to estimate crown and stem node ages for most orders and families of monocots, based on rbcL sequence data and comprehensive taxon sampling. From our analysis it is obvious that considerable monocot diversification took place during the Early Cretaceous, with most families already present at the Cretaceous–Tertiary boundary. Araceae, Arecaceae and Orchidaceae are among the oldest families with crown node ages reaching back into the Early Cretaceous. We comment on possible error sources and the necessity for methodological improvement in molecular dating.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 385–398.     

脊髓性肌萎缩症SMN1基因2+0基因型携带者的家系研究

脊髓性肌萎缩症(spinal muscular atrophy, SMA)是一种儿童时期较为常见的神经肌肉病,属于常染色体隐性遗传。绝大多数SMA由运动神经元存活基因1 (survival motor neuron 1, SMN1)的纯合缺失突变所致。而SMN1的2+0基因型个体作为一种特殊的SMA携带者,给携带者筛查以及家系的遗传咨询带来了巨大的挑战。已有研究表明,g.27134T>G和g.27706₂7707delAT多态位点变异对于Ashkenazi犹太人群中的2+0基因型个体具有提示作用。为进一步探究这两个多态位点是否在中国人群也具有特异性,本研究纳入了44例家系成员和204例已知SMN1基因拷贝数的对照样本。44例家系成员来自于9个无关的SMN1基因纯合缺失的SMA家系,先证者双亲之一疑似为2+0基因型携带者。利用多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)和短串联重复(short tandem repeat, STR)连锁分析进行基因型的鉴定以及多态位点的筛查,最终...     

K+ channel activity in plants: genes, regulations and functions

Potassium (K(+)) is the most abundant cation in the cytosol, and plant growth requires that large amounts of K(+) are transported from the soil to the growing organs. K(+) uptake and fluxes within the plant are mediated by several families of transporters and channels. Here, we describe the different families of K(+)-selective channels that have been identified in plants, the so-called Shaker, TPK and Kir-like channels, and what is known so far on their regulations and physiological functions in the plant.     

梭梭Na+/H+逆向转运蛋白基因克隆及分析

采用RT-PCR、RACE方法从超旱生、耐盐植物梭梭中扩增出Na+/H+逆向转运蛋白基因的开放阅读框架,其核苷酸序列长1 683bp,推测的氨基酸序列全长为560个氨基酸残基。含有多个物种Na+/H+逆向转运蛋白基因的高度保守序列氨氯砒嗪脒的结合位点(LFFIYLIPPI)。序列一致性分析结果显示,该cDNA片段与同科植物NHX基因的一致性为70%～80%,但与不同科植物的一致性较低,仅为60%,表明该基因在进化上存在多样性,但它们都具有氨氯砒嗪脒结合位点,对Na+具有高度专一性,对植物的耐盐性起着重要作用。     

澳门青洲山白楸+假苹婆+破布叶群落特征研究

采用样方调查法对澳门青洲山白楸（Mallotus paniculatus）＋假苹婆（Sterculia lanceolata）＋破布叶（Microcos paniculata）群落的种类组成、外貌、结构特征、区系和物种多样性进行了分析,结果表明,1700m2样方中有维管植物70种,隶属于42科65属,其中热带性分布属占90.32%,群落属于南亚热带常绿阔叶林植被类型。群落外貌常绿,生活型以藤本高位芽为主（占22.86%）。群落的物种丰富度Magarlef指数为8.78,Shannon-Wienner指数为2.22,Simpson指数为0.98,均匀度Pielou指数为0.52。群落各层次的丰富度、Simpson指数以及Shannon-Wienner指数均表现为下木层〉乔木层〉藤本植物〉草本层,均匀度表现为藤本植物〉乔木层〉下木层〉草本层,表明下木层中组成的种类较乔木层多,优势树种较乔木层明显。     

On the correlation between genomic G+C content and optimal growth temperature in prokaryotes: data quality and confounding factors

The correlation between genomic G+C content and optimal growth temperature in prokaryotes has gained renewed interest after Musto et al. [H. Musto, H. Naya, A. Zavala, H. Romero, F. Alvarex-Valin, G. Bernardi, Correlations between genomic GC levels and optimal growth temperatures in prokaryotes, FEBS Lett. 573 (2004) 73-77], reported that positive correlations exist in 15 families studied. We have reanalyzed their data and found that when genome size and data quality were adjusted for, there was no significant evidence of relationship between optimal temperature and GC content for two of the families that had previously shown strongly significant correlations. Using updated temperature optima for Halobacteriaceae species we found the correlation is insignificant in this family. For the family Enterobacteriaceae when genome size and optimal temperature are included in a multiple linear regression, only genome size is significant as a predictor of GC content. We showed that more profound statistical methods than simple two factor correlation analysis should be used for analyzing complex intrinsic and extrinsic factors that affect genomic GC content. We further found that a positive correlation between temperature and genomic GC is only evident in free-living species of low optimal growth temperatures.     

CluSTr: a database of clusters of SWISS-PROT+TrEMBL proteins

The CluSTr (Clusters of SWISS-PROT and TrEMBL proteins) database offers an automatic classification of SWISS-PROT and TrEMBL proteins into groups of related proteins. The clustering is based on analysis of all pairwise comparisons between protein sequences. Analysis has been carried out for different levels of protein similarity, yielding a hierarchical organisation of clusters. The database provides links to InterPro, which integrates information on protein families, domains and functional sites from PROSITE, PRINTS, Pfam and ProDom. Links to the InterPro graphical interface allow users to see at a glance whether proteins from the cluster share particular functional sites. CluSTr also provides cross-references to HSSP and PDB. The database is available for querying and browsing at http://www.ebi.ac.uk/clustr.     

Involvement of extracellular Ca2+ influx through voltage-independent Ca2+ channels in endothelin-1 function

This article reviews the types and roles of voltage-independent Ca(2+) channels involved in the endothelin-1 (ET-1)-induced functional responses such as vascular contraction, cell proliferation, and intracellular Ca(2+)-dependent signaling pathways and discusses the molecular mechanisms for the activation of voltage-independent Ca(2+) channels by ET-1. ET-1 activates some types of voltage-independent Ca(2+) channels, such as Ca(2+)-permeable nonselective cation channels (NSCCs) and store-operated Ca(2+) channels (SOCC). Extracellular Ca(2+) influx through these voltage-independent Ca(2+) channels plays essential roles in ET-1-induced vascular contraction, cell proliferation, activation of epidermal growth factor receptor tyrosine kinase, regulation of proline-rich tyrosine kinase, and release of arachidonic acid. The experiments using various constructs of endothelin receptors reveal the importance of G(q) and G(12) families in activation of these Ca(2+) channels by ET-1. These findings provide a potential therapeutic mechanism of a functional interrelationship between G(q)/G(12) proteins and voltage-independent Ca(2+) channels in the pathophysiology of ET-1, such as in chronic heart failure, hypertension, and cerebral vasospasm.     

Functional features of human CD25+ regulatory thymocytes

Two families of Treg cells have been described and named "naturally occurring" and "adaptive" regulatory T cells. The naturally occurring Treg cells arise in the thymus, where they achieve their complete functional maturation, whereas the adaptive Treg cells derive from the thymus too, but achieve their functional maturation in the periphery. This review discusses the phenotype and the functional features of the human naturally occurring CD25+ regulatory thymocytes.     

Formation of the killer Ig-like receptor repertoire on CD4+CD28null T cells

Killer Ig-like receptors (KIRs) are expressed on CD4(+)CD28(null) T cells, a highly oligoclonal subset of T cells that is expanded in patients with rheumatoid arthritis. It is unclear at what stage of development these T cells acquire KIR expression. To determine whether KIR expression is a consequence of clonal expansion and replicative senescence, multiple CD4(+)CD28(null) T cell clones expressing the in vivo dominant TCR beta-chain sequences were identified in three patients and analyzed for their KIR gene expression pattern. Based on sharing of TCR sequences, the clones were grouped into five clone families. The repertoire of KIRs was diverse, even within each clone family; however, the gene expression was not random. Three particular receptors, KIR2DS2, KIR2DL2, and KIR3DL2, had significant differences in gene expression frequencies between the clone families. These data suggest that KIRs are successively acquired after TCR rearrangement, with each clone family developing a dominant expression pattern. The patterns did not segregate with the individual from whom the clones were derived, indicating that peripheral selection in the host environment was not a major shaping force. Several models were examined using a computer algorithm that was designed to simulate the expression of KIRs at various times during T cell proliferation. The computer simulations favored a model in which KIR gene expression is inducible for a limited time during the initial stages of clonal expansion.     

Determining K+ channel activation curves from K+ channel currents

Potassium ion channels are generally believed to have current-voltage (IV) relations which are linearly related to driving force ( V - E(K)), where V is membrane potential and E(K) is the potassium ion equilibrium potential. Consequently, activation curves for K+ channels have often been measured by normalizing voltage-clamp families of macroscopic K+ currents with (V - E(K)), where V is the potential of each successive step in the voltage clamp sequence. However, the IV relation for many types of K+ channels actually has a non-linear dependence upon driving force which is well described by the Goldman-Hodgkin-Katz relation. When the GHK dependence on (V - E(K)) is used in the normalization procedure, a very different voltage dependence of the activation curve is obtained which may more accurately reflect this feature of channel gating. Novel insights into the voltage dependence of the rapidly inactivating I(A) channels Kv1.4 and Kv4.2 have been obtained when this procedure was applied to recently published results.     

Unicellular Ca2+ signaling 'toolkit' at the origin of metazoa

Ca(2+) signaling pathways control many physiological processes in almost all types of animal cells such as fertilization, muscle contraction, hormone release, and learning and memory. Each animal cell type expresses a unique group of molecules from the Ca(2+) signaling 'toolkit' to control spatiotemporal patterns of Ca(2+) signaling. It is generally believed that the complex Ca(2+) signaling 'toolkit' has arisen from the ancestral multicellular organisms to fit unique physiological roles of specialized cell types. Here, we demonstrate for the first time the presence of an extensive Ca(2+) signaling 'toolkit' in the unicellular choanoflagellate Monosiga brevicollis. Choanoflagellates possess homologues of various types of animal plasma membrane Ca(2+) channels including the store-operated channel, ligand-operated channels, voltage-operated channels, second messenger-operated channels, and 5 out of 6 animal transient receptor potential channel families. Choanoflagellates also contain homologues of inositol 1,4,5-trisphosphate receptors. Furthermore, choanoflagellates master a complete set of Ca(2+) removal systems including plasma membrane and sarco/endoplasmic reticulum Ca(2+) ATPases and homologues of 3 animal cation/Ca(2+) exchanger families. Therefore, a complex Ca(2+) signaling 'toolkit' might have evolved before the emergence of multicellular animals.     

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria

Background  

The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide.     

Phosphorylation of (Na+ + K+)-ATPase; stimulation and inhibition by substituted and unsubstituted amines

(1) In view of a previously established stimulation of steady-state phosphorylation of (Na+ + K+)-ATPase by imidazole and its inhibition by tris(hydroxymethyl)aminomethane, the effect of (structure, chemical composition and charge of) a number of primary, secondary and tertiary amines (including imidazole derivatives) has been investigated. (2) Primary amines are predominantly inhibitory and diamines are more inhibitory than monoamines. The strongest inhibition is exerted by ethylenediamine (I50 in 50 mM imidazole = 25 microM, vs. 60 mM for n-propylamine). Increasing the distance between the two amino groups from 3.7 to 8.7 A increases the I50 180-fold. The optimal distance of 3-4 A indicates a similar distance between two ligand(presumably Na+)-binding sites on the enzyme. (3) Screening or substitution of the central N-atom decreases inhibition by the nitrogen compound. Triple substitution by propyl or allyl groups leads to maximal activation, amounting to about 90% of the Na+-activation level. Triethyl substitution gives suboptimal activation and tributyl substitution leads to inhibition. Substitution by polar or negatively charged carboxyl groups diminishes or even abolishes inhibition and also diminishes or abolishes activation. (4) Although occasionally positive charge is not required for inhibition, it is prerequisite for activation. Within certain families of compounds (e.g., ethylenediamine and imidazole derivatives) inhibition or activation increases with pKa, hence with positive charge. (5) The above data are interpreted in terms of inhibition, which is competitive to Na+, being governed by Coulomb interaction. Activation, on the other hand, is predominantly determined by lipophilic (van der Waals or pi-pi electron) interactions, excluding water from the phosphorylation site, hence decreasing phosphoenzyme hydrolysis and increasing the phosphoenzyme level. The requirement of charge (though hidden by substitution) implies weak additional electrostatic interaction.     

Mapping protein family interactions: intramolecular and intermolecular protein family interaction repertoires in the PDB and yeast

In the postgenomic era, one of the most interesting and important challenges is to understand protein interactions on a large scale. The physical interactions between protein domains are fundamental to the workings of a cell: in multi-domain polypeptide chains, in multi-subunit proteins and in transient complexes between proteins that also exist independently. To study the large-scale patterns and evolution of interactions between protein domains, we view interactions between protein domains in terms of the interactions between structural families of evolutionarily related domains. This allows us to classify 8151 interactions between individual domains in the Protein Data Bank and the yeast Saccharomyces cerevisiae in terms of 664 types of interactions, between protein families. At least 51 interactions do not occur in the Protein Data Bank and can only be derived from the yeast data. The map of interactions between protein families has the form of a scale-free network, meaning that most protein families only interact with one or two other families, while a few families are extremely versatile in their interactions and are connected to many families. We observe that almost half of all known families engage in interactions with domains from their own family. We also see that the repertoires of interactions of domains within and between polypeptide chains overlap mostly for two specific types of protein families: enzymes and same-family interactions. This suggests that different types of protein interaction repertoires exist for structural, functional and regulatory reasons. Copyright 12001 Academic Press.