Protein import into chloroplasts involves redox-regulated proteins

Pre-protein translocation into chloroplasts is accomplished by two distinct translocation machineries in the outer and inner envelope, respectively. We have isolated the translocon at the inner envelope membrane (Tic complex) by blue-native PAGE and describe a new Tic subunit, Tic62. Tic62, together with Tic110 and Tic55, forms a core translocation unit. The N-terminus of Tic62 shows strong homologies to NAD(H) dehydrogenases in eukaryotes and to Ycf39-like proteins present in cyanobacteria and non-green algae. The stromal-facing C-terminus of Tic62 contains a novel, repetitive module that interacts with a ferredoxin-NAD(P)(+) oxidoreductase. Ferredoxin-NAD(P)(+) oxidoreductase catalyses the final electron transfer of oxygenic photosynthesis from ferredoxin to NAD(P). Substrates that interfere with either NAD binding, such as deamino-NAD, or influence the ratio of NAD(P)/NAD(P)H, such as ruthenium hexamine trichloride, modulate the import characteristics of leaf-specific ferredoxin-NAD(P)(+) oxidoreductase isologues differently. We conclude that the Tic complex can regulate protein import into chloroplasts by sensing and reacting to the redox state of the organelle.     

Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells

The improvement of non-viral-based gene delivery systems is of prime importance for the future of gene and antisense therapies. We have previously described a peptide-based gene delivery system, MPG, derived from the fusion peptide domain of HIV-1 gp41 protein and the nuclear localisation sequence (NLS) of SV40 large T antigen. MPG forms stable non-covalent complexes with nucleic acids and improves their delivery. In the present work, we have investigated the mechanism through which MPG promotes gene delivery. We demonstrate that cell entry is independent of the endosomal pathway and that the NLS of MPG is involved in both electrostatic interactions with DNA and nuclear targeting. MPG/DNA particles interact with the nuclear import machinery, however, a mutation which affects the NLS of MPG disrupts these interactions and prevents nuclear delivery of DNA. Nevertheless, we show that this mutation yields a variant of MPG which is a powerful tool for delivery of siRNA into mammalian cells, enabling rapid release of the siRNA into the cytoplasm and promoting robust down-regulation of target mRNA. Taken together, these results support the potential of MPG-like peptides for therapeutic applications and suggest that specific variations in the sequence may yield carriers with distinct targeting features.     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

Insight into the biochemical characteristics of a novel glucokinase gene mutation

Glucokinase (GCK) acts as a glucose sensor and regulates β-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu³³⁹ → Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.     

Insight into somaclonal variation

ABSTRACT

Somaclonal variation (S.V.) refers to mutational events occurring in tissue culture, although some permanent methylation processes should possibly also be included under this name. In this review, the possible causes as well as the mechanisms implicated in the induction of mutation in cultured cells are discussed. The needs for an easy assay to assess S.V. is pointed out.     

Conversion of vascular endothelial cells into multipotent stem-like cells

Mesenchymal stem cells can give rise to several cell types, but varying results depending on isolation methods and tissue source have led to controversies about their usefulness in clinical medicine. Here we show that vascular endothelial cells can transform into multipotent stem-like cells by an activin-like kinase-2 (ALK2) receptor-dependent mechanism. In lesions from individuals with fibrodysplasia ossificans progressiva (FOP), a disease in which heterotopic ossification occurs as a result of activating ALK2 mutations, or from transgenic mice expressing constitutively active ALK2, chondrocytes and osteoblasts expressed endothelial markers. Lineage tracing of heterotopic ossification in mice using a Tie2-Cre construct also suggested an endothelial origin of these cell types. Expression of constitutively active ALK2 in endothelial cells caused endothelial-to-mesenchymal transition and acquisition of a stem cell-like phenotype. Similar results were obtained by treatment of untransfected endothelial cells with the ligands transforming growth factor-β2 (TGF-β2) or bone morphogenetic protein-4 (BMP4) in an ALK2-dependent manner. These stem-like cells could be triggered to differentiate into osteoblasts, chondrocytes or adipocytes. We suggest that conversion of endothelial cells to stem-like cells may provide a new approach to tissue engineering.     

Intraspecific gene genealogies: trees grafting into networks

Intraspecific gene evolution cannot always be represented by a bifurcating tree. Rather, population genealogies are often multifurcated, descendant genes coexist with persistent ancestors and recombination events produce reticulate relationships. Whereas traditional phylogenetic methods assume bifurcating trees, several networking approaches have recently been developed to estimate intraspecific genealogies that take into account these population-level phenomena.     

Mitochondrial disulfide relay: redox-regulated protein import into the intermembrane space

99% of all mitochondrial proteins are synthesized in the cytosol, from where they are imported into mitochondria. In contrast to matrix proteins, many proteins of the intermembrane space (IMS) lack presequences and are imported in an oxidation-driven reaction by the mitochondrial disulfide relay. Incoming polypeptides are recognized and oxidized by the IMS-located receptor Mia40. Reoxidation of Mia40 is facilitated by the sulfhydryl oxidase Erv1 and the respiratory chain. Although structurally unrelated, the mitochondrial disulfide relay functionally resembles the Dsb (disufide bond) system of the bacterial periplasm, the compartment from which the IMS was derived 2 billion years ago.     

Insight into biofilm-associated microbial life

Microbes cohabit our planet and are engaged in a struggle for survival though on a microscopic scale. This endeavor allows them to develop and devise means for survival and proliferation. One such strategy is the formation of biofilms leading to establishment of a protected community. Such multi-communities may consist of harmful and pathogenic microbes, and they may cause economic problems and threats to human health. Biofilms are formed when microorganisms are typically attached to support surfaces. Biofilm-associated cells are sessile and differentiated from their suspended counterparts by generation of an extracellular polymeric substance matrix, reduced growth rates, and the up- and downregulation of specific genes. Biofilm formation is a complex process regulated by diverse characteristics of the growth medium, substratum, and cell surface. Development of strategies to control or prevent biofilms requires a thorough understanding of the biofilm development process. Biofilm research has witnessed exponential growth, and exciting findings have been reported. This has led us to visualize some previously un-thought-of and fascinating events. This article aims to provide an overview of biofilm research and associated challenges.     

Insight into planar cell polarity

Planar cell polarity or PCP refers to a uniform cellular organization within the plan, typically orthogonal to the apico-basal polarity axis. As such, PCP provides directional cues that control and coordinate the integration of cells in tissues to build a living organism. Although dysfunctions of this fundamental cellular process have been convincingly linked to the etiology of various pathologies such as cancer and developmental defects, the molecular mechanisms governing its establishment and maintenance remain poorly understood. Here, we review some aspects of invertebrate and vertebrate PCPs, highlighting similarities and differences, and discuss the prevalence of the non-canonical Wnt signaling as a central PCP pathway, as well as recent findings on the importance of cell contractility and cilia as promising avenues of investigation.     

Aging of the cells: Insight into cellular senescence and detection Methods

Cellular theory of aging states that human aging is the result of cellular aging, in which an increasing proportion of cells reach senescence. Senescence, from the Latin word senex, means “growing old,” is an irreversible growth arrest which occurs in response to damaging stimuli, such as DNA damage, telomere shortening, telomere dysfunction and oncogenic stress leading to suppression of potentially dysfunctional, transformed, or aged cells. Cellular senescence is characterized by irreversible cell cycle arrest, flattened and enlarged morphology, resistance to apoptosis, alteration in gene expression and chromatin structure, expression of senescence associated- β-galactosidase (SA-β-gal) and acquisition of senescence associated secretory phenotype (SASP). In this review paper, different types of cellular senescence including replicative senescence (RS) which occurs due to telomere shortening and stress induced premature senescence (SIPS) which occurs in response to different types of stress in cells, are discussed. Biomarkers of cellular senescence and senescent assays including BrdU incorporation assay, senescence associated- β-galactosidase (SA-β-gal) and senescence-associated heterochromatin foci assays to detect senescent cells are also addressed.     

Structural Insight into Proteorhodopsin Oligomers

Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.     

独脚金内酯信号途径的新发现——抑制子也是转录因子

植物激素信号传导途径中的抑制子(repressor) DELLA、AUX/IAA、JAZ和D53/SMXL均结合下游转录因子并抑制其转录活性, 从而阻遏激素响应基因的表达; 激素分子则激活信号传导链降解抑制子、释放转录因子, 从而诱导响应基因表达并介导相应的生物学功能。中国科学院遗传与发育生物学研究所李家洋研究团队最新的研究发现, 独脚金内酯(SL)信号途径中的SMXL6、SMXL7和SMXL8是具有抑制子和转录因子双重功能的新型抑制子, 他们还通过研究SL转录调控网络发现了大量新的SL响应基因, 揭示了SL调控植物分枝、叶片伸长和花色素苷积累的分子机制。这些重要发现为探索植物激素作用机理提供了新思路, 具有重要科学意义和应用前景。     

Insight into cellular dedifferentiation in regenerative medicine

正The wonderful capacity of regeneration observed in some lower animals has been a focus of researchers for more than a century. However, decoding this regenerative potential and its governing mechanisms has been challenging. There are key questions that warrant investigation: how is regeneration elicited, initiated, and controlled; what are the cellular factors     

Thrombomodulin is a clock-controlled gene in vascular endothelial cells

Cardiovascular diseases are closely related to circadian rhythm, which is under the control of an internal biological clock mechanism. Although a biological clock exists not only in the hypothalamus but also in each peripheral tissue, the biological relevance of the peripheral clock remains to be elucidated. In this study we searched for clock-controlled genes in vascular endothelial cells using microarray technology. The expression of a total of 229 genes was up-regulated by CLOCK/BMAL2. Among the genes that we identified, we examined the thrombomodulin (TM) gene further, because TM is an integral membrane glycoprotein that is expressed primarily in vascular endothelial cells and plays a major role in the regulation of intravascular coagulation. TM mRNA and protein expression showed a clear circadian oscillation in the mouse lung and heart. Reporter analyses, gel shift assays, and chromatin immunoprecipitation analyses using the TM promoter revealed that a heterodimer of CLOCK and BMAL2 binds directly to the E-box of the TM promoter, resulting in TM promoter transactivation. Indeed, the oscillation of TM gene expression was abolished in clock mutant mice, suggesting that TM expression is regulated by the clock gene in vivo. Finally, the phase of circadian oscillation of TM mRNA expression was altered by temporal feeding restriction, suggesting TM gene expression is regulated by the peripheral clock system. In conclusion, these data suggest that the peripheral clock in vascular endothelial cells regulates TM gene expression and that the oscillation of TM expression may contribute to the circadian variation of cardiovascular events.     

Expression of interferon-stimulated gene 20 in vascular endothelial cells

ISG20 is an ribonuclease specific for single-stranded RNA and considered to play a role in innate immunity against virus infections. We herein show that both poly IC, an authentic double-stranded RNA, and IFN-gamma induced ISG20 expression in cultured HUVEC. Poly IC-induced ISG20 expression was inhibited by LY294002, an inhibitor of PI3K, or by RNA interference against IFN regulatory factor three. ISG20 expression was not induced by IFN-beta, loxoribine or CpG oligonucleotide. These results suggest that ISG20 induction by poly IC may not be dependent on the IRF-3-mediated type I IFN induction pathway in HUVEC. ISG20 may be involved in innate immunity against viral infection in vascular endothelial cells.