Rejuvenation of aged progenitor cells by exposure to a young systemic environment

The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-alpha and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-alpha complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.     

The Caenorhabditis elegans heterochronic gene lin-14 encodes a nuclear protein that forms a temporal developmental switch

During wild-type development, a protein product of the Caenorhabditis elegans heterochronic gene lin-14 is localized to nuclei of specific somatic cells in embryos and early larvae, but is absent in late larvae and adult soma. Gain-of-function lin-14 mutations cause the level of lin-14 protein to remain high throughout development, resulting in developmental reiterations of early cell lineages. The normal down-regulation of the lin-14 nuclear protein level encodes a temporal switch between early and late cell fates.     

The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. Here we demonstrate that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals.     

The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans

The C. elegans heterochronic gene pathway consists of a cascade of regulatory genes that are temporally controlled to specify the timing of developmental events. Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated. Here we show that let-7 is a heterochronic switch gene. Loss of let-7 gene activity causes reiteration of larval cell fates during the adult stage, whereas increased let-7 gene dosage causes precocious expression of adult fates during larval stages. let-7 encodes a temporally regulated 21-nucleotide RNA that is complementary to elements in the 3' untranslated regions of the heterochronic genes lin-14, lin-28, lin-41, lin-42 and daf-12, indicating that expression of these genes may be directly controlled by let-7. A reporter gene bearing the lin-41 3' untranslated region is temporally regulated in a let-7-dependent manner. A second regulatory RNA, lin-4, negatively regulates lin-14 and lin-28 through RNA-RNA interactions with their 3' untranslated regions. We propose that the sequential stage-specific expression of the lin-4 and let-7 regulatory RNAs triggers transitions in the complement of heterochronic regulatory proteins to coordinate developmental timing.     

N-terminal of L protein of vesicular stomatitis virus contains a new signal sequence

The L protein (241kD) of vesicular stomatitis virus (VSV) is the most important snbnnit of the replication complex. The existence of specific localization signal in the L protein was investigated by making recombinant constructs expressing truncated mutants of the L protein fused to green fluorescent protein (GFP) in transient transfection assays. The chimeric genes encoding varied N-terminal of L and GFP gene were put under the control of T7 promoter or CMV promoter. The fusion proteins were transiently expressed in BHK-21, COS-7, CHO or Hep G2 cells. When more than 120 residues were deleted or only 96 residues were kept on the N-terminal, the fusion proteins were shown to be distributed throughout the cells, cytoplasm and nucleus under the confocal microscope. However, other chimeric proteins with 120 or more amino acids were dotted and distributed in the perinuclear regions. And the fusion protein with 96—120 aa has the similar distribution. A thirteen-residue peptide QGYSFLHEVDKEA (108—120) was identified as localization signal, whose function would be absolutely distributed with the deficiency of D or V. Our results show that there is an independent localizing signal in N-terminal domain of L protein of VSV and this functional signal is conserved in different cell lines.     

The ageing systemic milieu negatively regulates neurogenesis and cognitive function

In the central nervous system, ageing results in a precipitous decline in adult neural stem/progenitor cells and neurogenesis, with concomitant impairments in cognitive functions. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines--including CCL11 (also known as eotaxin)--the plasma levels of which correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and the levels of which are increased in the plasma and cerebrospinal fluid of healthy ageing humans. Lastly, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis and cognitive impairments observed during ageing can be in part attributed to changes in blood-borne factors.     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

Cnd2 has dual roles in mitotic condensation and interphase

Chromosome condensation requires condensin, which comprises five subunits. Two of these subunits--both being structural maintenance of chromosome (SMC) proteins-are coiled-coils with globular terminal domains that interact with ATP and DNA. The remaining three, non-SMC subunits also have essential, albeit undefined, roles in condensation. Here we report that Cnd2 (ref. 6), a non-SMC subunit of fission yeast similar to Drosophila Barren and the budding yeast protein Brn1 (refs 8, 9), is required for both interphase and mitotic condensation. In cnd2-1 mutants, ultraviolet-induced DNA damage is not repaired, and cells arrested by hydroxyurea do not recover. A definitive defect of interphase is abolishment of Cds1 (a checkpoint kinase) activation in the presence of hydroxyurea in both cnd2-1 mutant cells and in cells where other condensin subunits have been genetically disrupted. In the absence of hydroxyurea, a G2 checkpoint delay occurred in cnd2-1 mutants in a manner dependent on Cds1 and ATM-like Rad3, but not Chk1 (refs 10-13), before the mitotic condensation defect. Furthermore, cnd2-1 was synthetic-lethal with mutations of excision repair, RecQ helicase and DNA replication enzymes. These interphase and mitotic defects provide insight into the mechanistic role of non-SMC subunits that interact with the globular SMC domains in the heteropentameric holocomplex.     

A family of mitochondrial proteins involved in bioenergetICS and biogenesis

The respiratory chain complexes of mitochondria consist of many different subunits, of which only a few partake directly in electron transport. The functions of the subunits that do not contain prosthetic groups are largely unknown. The cytochrome reductase complex of Neurospora crassa, for examine, consists of nine different subunits, of which the peripheral membrane proteins I and II (ref.3) that are located on the matrix side of the mitochondrial inner membrane are the largest subunits devoid of redox centres. Significantly, a cytochrome reductase fraction lacking these two subunits was inactive in electron transfer, and in yeast mutants with defective genes for either of the two subunits, assembly of the reductase is disrupted. Most mitochondrial proteins are imported into the mitochondrion as precursor proteins, and two proteins are necessary for cleaving their presequences, namely the matrix processing peptidase (MPP) and the processing enhancing protein (PEP), the latter strongly stimulating the activity of the former. Temperature-sensitive yeast mutants, which are affected in PEP or MPP, accumulate precursors at the nonpermissive temperature. We report here that subunit I of the cytochrome reductase can be grouped as members of the same protein family.     

The molecular nature of the zebrafish tail organizer

Based on grafting experiments, Mangold and Spemann showed the dorsal blastopore lip of an amphibian gastrula to be able to induce a secondary body axis. The equivalent of this organizer region has been identified in different vertebrates including teleosts. However, whereas the graft can induce ectopic head and trunk, endogenous and ectopic axes fuse in the posterior part of the body, raising the question of whether a distinct organizer region is necessary for tail development. Here we reveal, by isochronic and heterochronic transplantation, the existence of a tail organizer deriving from the ventral margin of the zebrafish embryo, which is independent of the dorsal Spemann organizer. Loss-of-function experiments reveal that bone morphogenetic protein (BMP), Nodal and Wnt8 signalling pathways are required for tail development. Moreover, stimulation of naive cells by a combination of BMP, Nodal and Wnt8 mimics the tail-organizing activity of the ventral margin and induces surrounding tissues to become tail. In contrast to induction of the vertebrate head, known to result from the triple inhibition of BMP, Nodal and Wnt, here we show that induction of the tail results from the triple stimulation of BMP, Nodal and Wnt8 signalling pathways.     

Active-site mutants altering the cooperativity of E. coli phosphofructokinase

Crystal structures of the high- and low-activity states of the allosteric enzyme phosphofructokinase implicate three arginines in substrate binding, catalysis and cooperativity. Arginines 162 and 243 reach into the active site from an adjacent subunit and interact with the cooperative substrate fructose 6-phosphate. Mutation of these arginines to serine results in mutant enzymes with reduced substrate binding and lowered cooperativity, but with little change in their catalytic ability (kcat). Arg 72 bridges the two substrates fructose 6-phosphate and ATP, and interacts with the 1-phosphate of the product fructose 1,6-biphosphate. Mutation of this residue to serine reduces the catalytic activity, cooperativity and binding of fructose 6-phosphate and fructose 1,6-bisphosphate. In the reverse reaction, the kinetics of wild-type and the Ser 72 mutant with respect to fructose 1,6-bisphosphate are hyperbolic, whereas those of the Ser 162 and Ser 243 mutants are sigmoidal. These results show that each of the three arginines contributes to cooperativity and to the transmission of allosteric signals between the four subunit of the enzyme.     

Substantial increase of protein stability by multiple disulphide bonds

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.     

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.     

Conservation of the D-mannose-adhesion protein among type 1 fimbriated members of the family Enterobacteriaceae

A variety of genera and species of the family Enterobacteriaceae bear surface fimbriae that enable them to bind to D-mannose residues on eukaryotic cells. Until recently, it was thought that the D-mannose binding site was located in the major structural subunit (FimA), of relative molecular mass (Mr) 17,000 (17 K), of these organelles in Escherichia coli. New evidence indicates that this binding site resides instead in a minor protein Mr 28-31 K (FimH) located at the tips and at long intervals along the length of the fimbriae, and is reminiscent of the minor tip adhesion proteins of pyelonephritis-associated pili (Pap) and S fimbriae. In contrast to the antigenic heterogeneity of the major FimA subunit, the antigenic structure of FimH is conserved among different strains of E. coli. Here, we report an even broader conservation of this minor adhesion protein extending to other genera and species of type 1 fimbriated Enterobacteriaceae. Our results may have implications for the development of broadly protective vaccines against Gram-negative bacillary infections in animals and perhaps in man.     

人类乙型肝炎样病毒核心蛋白中第7位疏水性氨基酸重复肚段区域的突变可以抑制病毒核衣壳的组装

人类乙型肝炎病毒的核衣壳由核心蛋白的二聚体所组成．但是,核心蛋白亚单位与亚单位之间相互作用的机制至今尚不清楚．研究发现,在人类乙型肝炎样病毒──土拨鼠肝炎病毒（WHV）核心蛋白的氨基端,存在着4个保守的疏水氨基酸残基（氨基酸位置101～102）．它们分别是亮氨酸101,亮氨酸108,缬氨酸115和苯丙氨酸122．这4个疏水氨基酸残基以每隔6个氨基酸残基而重复出现1次．它们被称为“第7位疏水性氨基酸重复肽段（hhr）”．由于蛋白质中的疏水键往往在蛋白质的相互作用中起重要作用,因此就在培养细胞系统中研究WHV核心蛋白的hhr区域在…     

A model of synthetase/transfer RNA interaction as deduced by protein engineering

The recognition of transfer-RNA by their cognate aminoacyl-tRNA synthetases is the crucial step in the translation of the genetic code. In order to construct a structural model of the complex between the tyrosyl-tRNA synthetase (TyrTS) from Bacillus stearothermophilus and tRNATyr, 40 basic residues at the surface of the TyrTS dimer have been mutated by site-directed mutagenesis and heterodimers created in vitro by recombining subunits derived from different mutants. As reported here a cluster of basic residues (Arg 207-Lys 208) in the N-terminal domain of one TyrTS subunit interacts with the acceptor stem of tRNATyr and two separated clusters of basic residues (Arg 368-Arg 371; Arg 407-Arg 408-Lys 410-Lys 411) in the C-terminal domain of the other subunit interact with the anticodon arm. The TyrTS would thus clamp the tRNA in a fixed orientation. The precise alignment of the flexible... ACCA 3' end of the tRNA for attack on the tyrosyl adenylate is made by contacts closer to the catalytic groups of the enzyme, such as with Lys 151.     

钙调磷酸酶B亚基钙结合位点的突变对B亚基结构和功能的影响

应用远紫外CD光谱、紫外差光谱、内源荧光光谱以及ANS荧光光谱，探究了钙调磷酸酶B亚基钙结合位点的突变对B亚基结构和功能的影响．结果显示:钙结合位点突变体Y105W激活CNA的能力强于CNB；而E110Q活化CNA的能力弱于CNB；Y105W和E110Q与Ca2+亲和性低于CNB；CNB和它的突变体二级结构趋于一致，但三级结构存在明显差异，这种差异可能是它们功能差异的结构基础．      

Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.