The retromer component SNX6 interacts with dynactin p150Glued and mediates endosome-to-TGN transport

The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network （TGN）. It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins （SNXs）. Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNX1/2＇s dimerization partners, SNX6, interacts with the p150Gued subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50Gued interaction. Disruption of the SNX6-pl50Glued interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.

PRMT5 regulates Golgi apparatus structure through methylation of the golgin GM130

Maintenance of the Golgi apparatus （GA） structure and function depends on Golgi matrix proteins. The posttranslational modification of Golgi proteins such as phosphorylation of members of the golgin and GRASP families is important for determining Golgi architecture. Some Golgi proteins including golgin-84 are also known to be methylated, but the function of golgin methylation remains unclear. Here, we show that the protein arginine methyltransferase 5 （PRMT5） localizes to the GA and forms complexes with several components involved in GA ribbon formation and vesicle tethering. PRMT5 interacts with the golgin GM130, and depletion of PRMT5 causes defects in Golgi ribbon formation. Furthermore, PRMT5 methylates N-terminal arginines in GM130, and such arginine methylation appears critical for GA ribbon formation. Our findings reveal a molecular mechanism by which PRMT5-dependent arginine methylation of GM130 controls the maintenance of GA architecture.

Transgenic expression of DwMYB2 impairs iron transport from root to shoot in Arabidopsis thaliana

In plant, iron uptake and homeostasis are tightly regulated to ensure its absorption from soil and to avoid excess iron in the cell. Many genes involved in this process have been identified during past several years, but there are many problems remain unsolved in the genetic regulation of whole plant iron trafficking and allocation. MYB transcription factors contain tandem repeats of a -50 amino acid DNA-binding motif （R） and are involved in the regulation of many aspects of plant development, hormone signaling and metabolism. Here, we report that the ectopic expression of orchid R2R3-MYB gene DwMYB2 in Arabidopsis thaliana confers the transgenic plants hypersensitivity to iron deficiency. In DwMYB2 transgenic plants, the iron content in root is two-fold higher compared to that in wild-type root, while the reverse is true in shoot. This imbalance of iron content in root and shoot suggested that the translocation of iron from root to shoot was affected by the expression of DwMYB2 in the transgenic plants. Consistently, gene chip and reverse transcription-polymerase chain reaction analysis revealed that the ferric-chelate reductase gene, AtFRO2, and the iron transporter gene, AtlRT1 and AtlRT2, are up-regulated by DwMYB2 expression, while other potential iron transporters such as AtlREG1, AtFRD3 and NRAMP1 are down-regulated. In addition, the expression of several putative peptide transporters and transcription factors are also altered in the 35S：：DwMYB2 transgenic lines. These data provide us insight into the whole plant translocation of iron and identify candidate genes for iron homeostasis in plants despite the fact that a heterologous gene was expressed.

Transgenic analyses of TGIF family proteins in Drosophila imply their role in cell growth

TG-interacting factors （TGIFs） belong to a family of TALE-homeodomain proteins including TGIF, TGIF2, and TGIF2LX/Y （TGIF2 like on X or Y chromosome） in human. They potentially play important functions in various tissues during development. Mutations in TGIF are frequently associated with malformation of forebrain and facial structures; TGIF2 proteins are over-expressed in many ovarian cancer cell lines; and TGIF2LX/Y are specifically expressed in adult testis. The molecular functions of these proteins have been investigated mostly in cultured cells. TGIF and TGIF2 have been found as transcriptional repressors that modulate TGF-beta signaling. However these findings are far from sufficient to explain their mutant phenotypes or expression patterns, and the functions of TGIF2LX/Y have never been reported. Here we use Drosophila as a model system to explore the functions of TGIF family proteins in vivo. We observed in fly tissues such as fat body, epithelia, and neuronal cells, that expressing human TGIF2 or human TGIF2LX generally inhibited cell growth in size and number. Co-expressing Drosophila Myc, Cyclin E, or human c-MycS partially rescued the growth inhibition induced by human TGIFs, whereas activated insulin pathway signaling did not. Taken together, we provide in vivo evidence for the potential functions of human TGIF2 and TGIF2LX in growth control. Additionally, we confirmed that Drosophila TGIFs are transcriptional activators by assaying their activities in spermatogenesis.

Cbl negatively regulates JNK activation and cell death

Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apoptosis -- nerve growth factor （NGF） deprivation and DNA damage -- cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation （and most likely, activation） of c-Cbl. Targeting c-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase （JNK） signaling. We demonstrate that Cbl proteins interact with the JNK pathway components mixed lineage kinase （MLK） 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK activation and on cell death.

Adaptive evolution and structure modeling of rbcL gene in Ephedra

The adaptive evolution of the rbcL gene in 14 species of Ephedra and 9 other gymnosperms was tested by the PAML program. Three large-scale selections were found in the catalytic region of the RbcL protein by branch model. Species in Ephedra with positive selection at the 365th codon site in rbcL gene were detected by branch-site model. 3D structure modeling showed that the 365th codon could change the spatial structure of the β-sheet (βG, βH, or βE) of the RbcL protein. These changes may be associated with the adaptation of the protein in Ephedra to the environment.

A gene family-based method for interspecies comparisons of sequencing-based transcriptomes and its use in environmental adaptation analysis

We describe a new method for sequencing-based cross-species transcriptome comparisons and define a new metric for evaluating gene expression across species using protein-coding families as units of comparison. Using this measure transcriptomes from different species were evaluated by mapping them to gene families and integrating the mapping results with expression data. Statistical tests were applied to the transcriptome evaluation results to identify differentially expressed families. A Perl program named Pro-Diff was compiled to im- plement this method. To evaluate the method and provide an example of its use, two liver EST transcriptomes from two closely related fish that live in different temperature zones were compared. One EST library was from a recent sequencing project of Dissosticus maw- soni, a fish that lives in cold Antarctic sea waters, while the other was newly sequenced data （available at： http：//www.fishgenome.org/ polarbank/） from Notothenia angustata, a species that lives in temperate near-shore water of southern New Zealand. Results from the com- parison were consistent with results inferred from phenotype differences and also with our previously published Gene Ontology-based method. The Pro-Diffprogram and operation manual can be downloaded from： http：//www.fishgenome.org/download/Prodiff.rar.

Regulation of the protein stability of POSH and MLK family

Sequential activation of the JNK pathway components,including Rac1/Cdc42,MLKs(mixed-lineage kinases),MKK4/7 and JNKs,plays a required role in many cell death paradigms.Those components are organized by a scaffold protein,POSH(Plenty of SH3’s),to ensure the effective activation of the JNK pathway and cell death upon apoptotic stimuli.We have shown recently that the expression of POSH and MLK family proteins are regulated through protein stability.By generating a variety of mutants,we provide evidence here that the Nterminal half of POSH is accountable for its stability regulation and its over-expression-induced cell death.In addition,POSH’s ability to induce apoptosis is correlated with its stability as well as its MLK binding ability.MLK family’s stability,like that of POSH,requires activation of JNKs.However,we were surprised to find out that the widely used dominant negative(d/n)form of c-Jun could down-regulate MLK’s stability,indicating that peptide from d/n c-Jun can be potentially developed into a therapeutical drug.

Erectile dysfunction in Fragile X patients

Aim:To study a possible defect in spermatogenesis of Fragile X syndrome(FXS)patients.Methods:Two different polymerase chain reaction(PCR)based methods were used for the molecular diagnosis of FXS.Sperm collection was done mostly according to the laboratory manual of the World Health Organization.Results:We failed to collect sperm samples from five Fragile X subjects aged 18-60 years as a result of an unexpected erectile dysfunction(ED). Multiple examinations of the same subject at different times,and of different subjects from different provinces by different physicians,showed the same result consistently in all five subjects examined.Conclusion:Erectile reflex is an instinctive response in all healthy males.The absence of erection can be caused by hormonal,physical or neuronal malfunction.As hormonal profiles were reported to be generally normal in Fragile X men,we propose that an unknown physical factor or the neuronal circuit,or both,underlying the erection is compromised.The finding of ED in Fragile X patients may help better understand the clinical spectrum and pathogenesis of the disease.(Asian J Androl 2006 Jul;8:483-487)

Production of Brown/Yellow Patches in the SLC7A11 Transgenic Sheep via Testicular Injection of Transgene

The gene, SLC7All, which encodes the solute carrier family 7 member 11 （anionic amino acid transporter light chain, xCT）, has been reported to be implicated in multiple processes such as in pheomelanin production, cell proliferation and migration, Kaposi＇s sarcoma herpesvirus （KSHV） entry into the host cells, learning and memory. Its involvement in cancer cell proliferation and metastasis has been widely studied. Its role in pheomelanogenesis is likely conserved in sheep. The full-length cDNA of sheep SLC7A11 was cloned from sheep skin fibroblasts for evaluating its role in regulating sheep coat color. The complete open reading frame of sheep xCT （sxCT） is 1512 bp in length, encoding a 503 amino acid polypeptide. We explored its function on pheomelanogenesis in vitro and in vivo. In the melan-a non-agouti mouse melanocytes that mainly produce eumelanin, overexpressed sxCT reduced the content of eumelanin. Using a testicular injection transgenic method, sxCT-transgenic sheep were generated and exhibited patches of brown/yellow coat, suggesting that sxCT can be selectively expressed to increase the pheomelanin production in wool. Our studies suggest that testicular injection of transgene can be used to genetically modify sheep coat color.

GAMETOPHYTIC FACTOR 1,Involved in Pre-mRNA Splicing,Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 （GFA1）; a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homologs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogenesis and embryogenesis in plant.

A novel missense mutation of the TYR gene in a pedigree with oculocutaneous albinism type 1 from China

Background The mutation of the tyrosinase （TYR） gene results in oculocutaneous albinism type 1 （OCA1）, an autosomal recessive genetic disorder. OCA1 is the most common type of OCA in the Chinese population. Hence, the TYR gene was tested in this study. We also delineated the genetic analysis of OCA1 in a Chinese family. Methods Genomic DNA was isolated from the blood leukocytes of a proband and his family. Mutational analysis at the TYR locus by DNA sequencing was used to screen five exons, including the intron/exon junctions. A pedigree chart was drawn and the fundus of the eyes of the proband was also examined. Results A novel missense mutation p.1151S on exon 1, and homozygous TYR mutant alleles were identified in the proband. None of the mutants was identified among the 100 normal control subjects. Genetic analysis of the proband＇s wife showed normal alleles in the TYR gene. Thus, the fetus was predicated a carrier of OCA1 with a normal appearance. Conclusion This study provided new information about a novel mutation, p.1151S, in the TYR gene in a Chinese family with OCAI. Further investigation of the proband would be helpful to determine the effects of this mutation on TYR activity.

Multi-class cancer classification through gene expression profiles: microRNA versus mRNA

Both microRNA （miRNA） and mRNA expression profiles are important methods for cancer type classification. A comparative study of their classification performance will be helpful in choosing the means of classification. Here we evaluated the classification performance of miRNA and mRNA profiles using a new data mining approach based on a novel SVM （Support Vector Machines） based recursive fea- ture elimination （nRFE） algorithm. Computational experiments showed that information encoded in miRNAs is not sufficient to classify cancers; gut-derived samples cluster more accurately when using mRNA expression profiles compared with using miRNA profiles; and poorly differentiated tumors （PDT） could be classified by mRNA expression profiles at the accuracy of 100% versus 93.8% when using miRNA profiles. Furthermore, we showed that mRNA expression profiles have higher capacity in normal tissue classifications than miRNA. We concluded that classification performance using mRNA profiles is superior to that of miRNA profiles in multiple-class cancer classifications.

The Protein Arginine Methylase 5(PRMT5/SKB1) Gene Is Required for the Maintenance of Root Stem Cells in Response to DNA Damage

Plant root stem cells and their surrounding microenvironment,namely the stem cell niche,are hypersensitive to DNA damage.However,the molecular mechanisms that help maintain the genome stability of root stem cells remain elusive.Here we show that the root stem cells in the skbl（Shk1 kinase binding protein 1） mutant undergoes DNA damage-induced cell death,which is enhanced when combined with a lesion of the Ataxia-telangiectasia mutated（ATM） or the ATM/RAD3-related（ATR） genes,suggesting that the SKBI plays a synergistically effect with ATM and ATR in DNA damage pathway.We also provide evidence that SKBI is required for the maintenance of quiescent center（QC）,a root stem cell niche,under DNA damage treatments.Furthermore,we report decreased and ectopic expression of SHORTROOT（SHR） in response to DNA damage in the skbl root tips,while the expression of SCARECROW（SCR） remains unaffected.Our results uncover a new mechanism of plant root stem cell maintenance under DNA damage conditions that requires SKB1.

Novel Nuclear Protein ALC-INTERACTING PROTEIN1 is Expressed in Vascular and Mesocarp Cells in Arabidopsis

Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ （ALC）, a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTING PROTEIN1 （ACI1） via the yeast two-hybrid screen. ACll encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementaUon assay. Both ACI1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACll expression by RNA interference technology, suggesting that ACll may be redundant. Taken together, these data indicate that ALC may interact with ACI1 and its homologs to control cell separation during fruit dehiscence in Arabidopsis.

Regulation of neural stem cell by bone morphogenetic protein (BMP) signaling during brain development

Neurogenesis is the process in which neurons are generated from neural stem/progenitor cells(NSCs/NPCs).It involves the proliferation and neuronal fate specification/differentiation of NSCs,as well as migration,maturation and functional integration of the neuronal progeny into neuronal network.NSCs exhibit the two essential properties of stem cells:self-renewal and multi-potency.Contrary to previous dogma that neurogenesis happens only during development,it is generally accepted now that neurogenesis can take place throughout life in mammalian brains.This raises a new therapeutic potential of applying stem cell therapy for stroke,neurodegenerative diseases and other diseases.However,the maintenance and differentiation of NSCs/NPCs are tightly controlled by the extremely intricate molecular networks.Uncovering the underlying mechanisms that drive the differentiation,migration and maturation of specific neuronal lineages for use in regenerative medicine is,therefore,crucial for the application of stem cell for clinical therapy as well as for providing insight into the mechanisms of human neurogenesis.Here,we focus on the role of bone morphogenetic protein(BMP)signaling in NSCs during mammalian brain development.

Patterning the embryo in higher plants:Emerging pathways and challenges

Embryogenesis,which establishes the basic body plan for the post-embryonic organs after stereotyped cell divisions,initiates the first step of the plant life cycle.Studies in the last two decades indicate that embryogenesis is a precisely controlled process,and any defect would result in abnormalities.Here we discuss the recent progresses,with a focus on the cellular pathways governing early embryogenesis in the model species Arabidopsis.

miR-888 in MCF-7 Side Population Sphere Cells Directly Targets E-cadherin

Side population （SP） cells are a small subset of cells isolated from a cultured cancer cell line that exhibit characteristics similar to those of cancer stem cells （CSCs）, such as high metastatic and tumorigenic potential. The molecular mechanisms that give rise to the malignant properties of SP cells are not clear. We isolated SP cells from the MCF-7 breast cancer cell line and profiled microRNA （miRNA） expression patterns between SP cell-derived spheroids and non-SP cells. SP spheroids were found to possess 42 up-regulated miRNAs and 27 down-regulated ones （above 5-fold changes）. One of the up-regulated miRNAs, miR-888 computationally predicted to participate in the adherens junction （AJ） pathway, was investigated. Over-expression of miR-888 in MCF-7 cells reduced the mRNA levels of all four AJ pathway genes （E-cadherin, ACTG1, PTPRTand CDC42） that were selected for testing, whereas knocking down miR-888 reversed the trends. Western blot and flow cytometric quantitation of the membrane E-cadherin levels showed the same trend of change under these treatments. Luciferase reporter assay showed E-cadherin is a direct target of miR-888. As a potential role in intercellular adhesiveness and maintenance of malignant tissue architecture, the results indicate that miR-888 is a repressor of the AJ pathway in MCF-7 cells and that up-regulation of miR-888 contributes to aggressiveness in MCF-7 SP cells.

A lipid pathway for heat adaptation

The emergence of cell membrane is an essential step in the origin of life on earth. Cell membrane, a matrix with fatty acid derived lipids and proteins, separates the outside envi- ronment from enclosed cell internal. The composition of membrane lipids largely determines biophysical properties of cell membrane, such as fluidity, permeability and deformability, which are essential for cellular processes. Temperature is an important environmental factor. Different species live in a dramatic variation of temperature conditions; for example, bacterial Thermus Aquaticus （in which the Taq DNA polymerase for PCR was first isolated） grows in hot springs while polar bears reside in arctic area. Individual cell or organism can adapt to a limited range of temperatures, which is also known as thermal tolerance. Temperature changes affect membrane fluidity. It has been known for a long time that bacteria respond to temperature changes by changing the fatty acid saturation of cell mem- brane through altering the expression of lipid desaturase, a phenomenon named homeoviscous adaptation （HVA） [1]. Later on, the phenomenon of HVA was found conserved in evolution [2]. In bacteria, a two-component regulatory sys- tem regulates lipid desaturase expression and mediates HVA. However, the two-component system genes are not found in eukaryotes and the molecular mechanism of HVA remains elusive in eukaryotes.

TRIM-9 functions in the UNC-6/UNC-40 pathway to regulate ventral guidance

TRIpartite Motif （TRIM） family proteins are ring finger domain-containing, multi-domain proteins implicated in many biological processes. Members of the TRIM-9/C-I subfamily of TRIM proteins, including TRIM-9, MIDI and MID2, have neuronal functions and are associated with neurological diseases. To explore whether the functions of C-I TRIM proteins are conserved in invertebrates, we analyzed Caenorhabditis elegans and Drosophila trim-9 mutants. C. elegans trim-9 mutants exhibit defects in the ventral guidance of hermaphrodite specific neuron （HSN） and the touch neuron AVM. Further genetic analyses indicate that TRIM-9 participates in the UNC-6-UNC-40 attraction pathway. Asymmetric distribution of UNC-40 during HSN development is normal in trim-9 mutants. However, the asymmetric localization of MIG-10, a downstream effector of UNC-40, is abolished in trim-9 mutants. These results suggest that TRIM-9 functions upstream of MIG- 10 in the UNC- 40 pathway. Moreover, we showed that TRIM-9 exhibits E3 ubiquitin ligase activity in vitro and this activity is important for TRIM-9 function in vivo. Additionally, we found that Drosophila trim-9 is required for the midline attraction of a group of sensory neuron axons. Over-expression of the Netrin/UNC-6 receptor Frazzled suppresses the guidance defects in trim-9 mutants. Our study reveals an evolutionarily conserved function of TRIM-9 in the UNC-40/Frazzled-mediated UNC-6/Netrin attraction pathway.