Interaction of mutant genes Fgf5go-Y, we, and wal changes the duration of hair growth cycles in mice

Mutant gene wallhaarig (wa) was acting as a modifier of the mutant gene waved alopecia (wal), substantially increasing hair loss rate in mice, as was previously shown in our laboratory. The current paper is devoted to a study of mutant gene angora-Y (Fgf5 ^go-Y ), which had extended anagen stage of the first and second generations hair growth cycles in triple heterozygotes (Fgf5 ^go-Y /Fgf5 ^go-Y we/we wal/wal). First generation guard hair in triple homozygotes had their anagen stage 4 days longer than the same stage in double homozygotes (+/+ we/we wal/wal). Hair loss started at a catagen stage in double homozygotes, while it started in triple homozygotes at the end of the same stage or even in a telogen. Such mutant gene interaction in hair follicle morphogenesis led to a partial recovery of a body hair coat in triple homozygotes.     

Tunable Broadband Optical Responses of Substrate-Supported Metal/Dielectric/Metal Nanospheres

Using the image charge theory and finite element methods, we present the first comprehensive study on the optical properties of substrate-supported, three-layer, metal/dielectric/metal nanospheres. By adopting dipolar and quadrupolar approximations of the quasistatic image charge theory, we derive analytical expressions for the polarization-dependent polarizabilities of a three-layer nanosphere near a substrate and use them to find the nanosphere’s plasmon resonance wavelengths as functions of the geometric and material parameters of the nanosphere–substrate system. By calculating the resonance wavelength of substrate-supported gold/silica/gold nanosphere over a sufficiently large domain of the nanosphere’s dimensions, we show that this wavelength can be tuned from visible to infrared regions by altering only the size of the nanosphere’s core. We also show that the resonance position as well as the enhancement and confinement of the near-field can be dynamically tuned over broad ranges by changing the polarization of the excitation light. Of significance for the applicability of our results in practice is that we employ size-dependent permittivity of gold, which allows experimentalists to readily produce these substrate-supported nanospheres with desired optical responses. Upon comparing our analytical results with the results of numerical simulations, we reveal the range of the nanospheres’ outer radii within which the dipolar and quadrupolar approximations adequately describe the nanosphere–substrate interaction. Since majority of the optical functions are realized with light polarized parallel to the substrate, our results allow one to readily engineer the broadband optical responses of substrate-supported metal/dielectric/metal nanospheres for applications in resonance-enhanced sensing, in light harvesting, and in biomedicine.     

Phosphohexose isomerase/autocrine motility factor/neuroleukin/maturation factor is a multifunctional phosphoprotein

Phosphohexose isomerase (PHI) is a member of the ectoenzyme/exoenzyme family and plays a key role in both glycolysis and gluconeogenesis pathways. Upon secretion PHI acts as a cytokine with tumor autocrine motility factor (AMF), neuroleukin (NLK) and maturation factor (MF) functions. Signaling is initiated by its binding to a cell surface 78 kDa glycoprotein (gp78). However, since PHI protein is a 'leaderless' secretory protein, released from cells via a non-classical route(s), we questioned whether the molecule undergoes post-translation modification while retaining proper folding and maintaining intact enzymatic and motogenic activities. To address this, we have generated, expressed and isolated a recombinant human AMF (rhAMF). The rhAMF retained the biological activities of the native AMF, i.e., catalyzes phosphohexose isomerization and stimulated cell motility. Additionally, we show here that human PHI is phosphorylated at serine 185 by casein kinase II (CK II) and we provide experimental evidence suggesting that this phosphorylation is associated with secretion, thus providing insights for elucidating the intracellular signal transmission of cell response to stimulation by AMF/NLK/MF.     

Formal Demonstration of the Phosphorylation of Rat Brain Tryptophan Hydroxylase by Ca2+/Calmodulin-Dependent Protein Kinase

Tryptophan hydroxylase is activated in a crude extract by addition of ATP and Mg2+. This activation is reversible and requires in addition both Ca2+ and calmodulin. Thus, phosphorylation by an endogenous calmodulin-dependent protein kinase has long been suspected. Now that we have prepared a specific polyclonal antibody to rat brain tryptophan hydroxylase, we have been able to prove that this hypothesis is correct. After incubation of purified tryptophan hydroxylase with Ca2+/calmodulin-dependent protein kinase together with [gamma-32P]ATP, Mg2+, Ca2+, and calmodulin, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting of the enzymes onto nitrocellulose sheets, we could label the band of tryptophan hydroxylase by the antiserum and the peroxidase technique and show by autoradiography that 32P was incorporated into this band. By measuring the radioactivity, we calculated that about 1 mol of phosphate was incorporated per 8 mol of subunits of the enzyme (2 mol of native enzyme). Because the concentration of ATP which we employed (50 microM) gives about half-maximal activation in crude extract compared to saturating ATP conditions (about 1 mM), this result indicates that the incorporation of at least 1 mol of phosphate/mol of tetramer of native tryptophan hydroxylase is required for maximal activation.     

Protein Kinase FA/GSK-3 Phosphorylates on Ser235-Pro and Ser404-Pro that Are Abnormally Phosphorylated in Alzheimer's Disease Brain

Abstract— Previously, we identified protein kinase F_A/gly-cogen synthase kinase-3 (GSK-3) as a microtubule-associated protein kinase that can incorporate 4 mol of phosphates into 1 mol of protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological (PHF-) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase F_A/GSK-3 in using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain sequence, we further identified Ser²³⁵-Pro and Ser⁴⁰⁴-Pro as the two major phosphorylation sites according to the numbering of the longest isoform. Ser²³⁵ and Ser⁴⁰⁴ have been reported as two of the major abnormal phosphorylation sites in PHF-. Taken together, the results provide initial evidence that protein kinase F_A/GSK-3 may represent one of the Ser-Pro motif-directed kinases involved in the abnormal phosphorylation of pathological PHF- in Alzheimer's disease brain.     

口蹄疫病毒O/QYYS/s/06株感染性克隆的构建

本研究在完成FMDVO/QYYS/s/06株全基因组序列测定的基础上,分3段对全基因组进行克隆,其后将各片段克隆到载体P43中,从而获得携带O/QYYS/s/06株基因组全长cDNA的重组质粒P43C。将重组质粒P43C与表达RNA聚合酶的质粒T7共转染BHK-21细胞,48h后收获培养液接种2~3d乳鼠,取经乳鼠传代后的第4代病毒液,经反向间接血凝、中和试验和测序等方法证明拯救的病毒为O型FMDV。以上结果表明,O/QYYS/s/06株全长cDNA分子克隆的构建成功。     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

The ROSA26 LacZ-neo R insertion confers resistance to mammary tumors in Apc Min/+ mice

B6.129S7-Gtrosa26 (ROSA26) mice carry a LacZ-neo ^R insertion on Chromosome (Chr) 6, made by promoter trapping with AB1 129 ES cells. Female C57BL/6J Apc ^Min /+ (B6 Min/+) mice are very susceptible to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, ENU-treated B6 mice carrying both Apc ^Min and ROSA26 are resistant to mammary tumor formation. Thus, ROSA26 mice carry a modifier of Min-induced mammary tumor susceptibility. We have previously mapped the modifier to a 4-cM interval of 129-derived DNA that also contains the ROSA26 insertion. Here we report additional evidence for the effect of the ROSA26 insertion on mammary tumor formation. To test the hypothesis that the resistance was due to a linked modifier locus, we utilized two approaches. We have derived and tested two lines of mice that are congenic for 129-derived DNA within the minimal modifier interval and show that they are as susceptible to mammary tumors as are B6 mice. Additionally, we analyzed a backcross population segregating for the insertion and show that mice carrying the insertion are more resistant to mammary tumor development than are mice not carrying the insertion. Thus, the resistance is not due to a 129-derived modifier allele, but must be due to the ROSA26 insertion. In addition, the effect of the ROSA26 insertion can be detected in a backcross population segregating for other mammary modifiers. Received: 29 December 2000 / Accepted: 4 April 2001     

Mapping of SPARC/BM-40/osteonectin-binding sites on fibrillar collagens

The 33-kDa matrix protein SPARC (BM-40, osteonectin) binds several collagen types with moderate affinity. The collagen-binding site resides in helix alphaA of the extracellular calcium-binding domain of SPARC and is partially masked by helix alphaC. Previously, we found that the removal of helix alphaC caused a 10-fold increase in the affinity of SPARC for collagen, and we identified amino acids crucial for binding by site-directed mutagenesis. In this study, we used rotary shadowing, CNBr peptides, and synthetic peptides to map binding sites of SPARC onto collagens I, II, and III. Rotary shadowing and electron microscopy of SPARC-collagen complexes identified a major binding site approximately 180 nm from the C terminus of collagen. SPARC binding was also detected with lower frequency near the matrix metalloproteinase cleavage site. These data fit well with our analysis of SPARC binding to CNBr peptides, denaturation of which abolished binding, indicating triple-helical conformation of collagen to be essential. SPARC binding was substantially decreased in two of seven alpha2(I) mutant procollagen I samples and after N-acetylation of Lys/Hyl side chains in wild-type collagen. Synthetic peptides of collagen III were used to locate the binding sites, and we found SPARC binding activity in a synthetic triple-helical peptide containing the sequence GPOGPSGPRGQOGVMGFOGPKGNDGAO (where O indicates 4-hydroxyproline), with affinity for SPARC comparable with that of procollagen III. This sequence is conserved among alpha chains of collagens I, II, III, and V. In vitro collagen fibrillogenesis was delayed in the presence of SPARC, suggesting that SPARC might modulate collagen fibril assembly in vivo.     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Characterization of Gicerin/MUC18/CD146 in the rat nervous system

Gicerin is a cell adhesion molecule of an immunoglobulin (Ig) superfamily isolated from a chicken. It shows homophilic and heterophilic binding activities and has two isoforms. s-Gicerin which has small cytoplasmic domain and the same extracellular domain as l-gicerin shows stronger cell adhesion activity. In the chick nervous system, gicerin expression is only observed in the developmental stage when neurons extend neurites and migrate. In other tissues, gicerin participates in the tissue regeneration or oncogenesis. In this report, we identified two isoforms of rat gicerin corresponding to chicken and we concluded that gicerin is a homologue of human CD146/MUC18/MCAM. Next we generated antibody to characterize a rat gicerin in the nervous system. Gicerin is expressed in the hippocampal cells, Purkinje cells, and sensory neurons of a spinal chord of an adult rat, while expressed most abundantly in the lung. In addition to this, its expression in the hippocampus was increased by electroconvulsive shock, suggesting some role in the mature nervous system. And we also showed neurite promotion activity of gicerin from hippocampal neurons.     

Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator activity by I kappa B kinase

In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the I kappa B kinase (IKK). IKK is known to be responsible for the degradation of I kappa B and the subsequent activation of NF-kappa B. Since NF-kappa B plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-kappa B-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-kappa B-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-kappa B target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.     

Membrane lipid modifications: Biosynthesis and identification of phosphatidyl-N-methyl-N-isopropylethanolamine in rat liver microsomes

In previous studies on the modification of polar head groups of membrane phospholipids with the unnatural base analog, $\text{N-}\text{isopropylethanolamine}$, we reported an unidentified phospholipid in addition to $\text{phosphatidyl}\text{-N-}\text{isopropylethanolamine}$ in the various membrane fractions of rat liver. The structure of this phospholipid has now been identified as $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$ by nuclear magnetic resonance spectroscopy, and by chromatographic and enzymic analysis. In addition, we found that when rats were injected intraperitoneally with the $\text{N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$, 19% of teh liver microsomal phospholipid was $\text{phosphatidyl}\text{-N-}\text{methyl}\text{-N-}\text{isopropylethanolamine}$.     

Proliferation of Colorectal Cancer Is Promoted by Two Signaling Transduction Expression Patterns: ErbB2/ErbB3/AKT and MET/ErbB3/MAPK

One of the recent breakthroughs in cancer research is the identification of activating mutations in various receptor tyrosine kinase(RTK) pathways in many cancers including colorectal cancer(CRC). We hypothesize that, alternative to mutations, overexpression of various oncogenic RTKs may also underpin CRC pathogenesis, and different RTK may couple with distinct downstream signaling pathways in different subtypes of human CRC. By immunohistochemistry, we show here that RTK members ErbB2, ErbB3 and c-Met were in deed differentially overexpressed in colorectal cancer patient samples leading to constitutive activation of RTK signaling pathways. Using ErbB2 specific inhibitor Lapatinib and c-Met specific inhibitor PHA-665752, we further demonstrated that this constitutive activation of RTK signaling is necessary for the survival of colorectal cancer cells. Furthermore, we show that RTK overexpression pattern dictates the use of downstream AKT and/or MAPK pathways. Our data are important additions to current oncogenic mutation models, and further explain the clinical variation in therapeutic responses of colorectal cancer. Our findings advocate for more personalized therapy tailored to individual patients based on their type of RTK expression in addition to their mutation status.     

Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha

Wnt/beta-catenin signaling has been implicated in repressing adipogenesis. Several lines of evidence show that the possible mechanism is blockade of PPARgamma induction. However, the precise mechanisms remain to be elucidated. In this study, we demonstrated that Wnt3a conditioned medium suppresses C/EBPbeta/delta-induced adipogenesis of 3T3-L1 cells by inhibiting PPARgamma induction. In addition, the mutual activation of PPARgamma and C/EBPalpha was also repressed in the presence of Wnt3a. To further investigate the role of the canonical Wnt pathway in adipogenesis, we used mouse embryonic fibroblasts (MEFs) isolated from Lrp6-deficient embryos. Contrary to wild-type MEFs, Lrp6-deficient MEFs showed spontaneous adipogenesis and escaped the suppressive effect of exogenous Wnt3a. These findings suggest a critical role of Wnt/Lrp6/beta-catenin signaling in adipogenesis and cell fate decision of mesenchymal stem cells.     

中甸灯台报春与鹅黄灯台报春的系统演化

中甸灯台报春和鹅黄灯台报春在形态上较为相似,通常被认为是亲缘关系较近的物种。该研究选取核基因ITS序列和叶绿体trn L-F为分子标记,利用贝叶斯的方法构建系统树,在用两种分子标记构建的系统树中,两种报春均以0.81的支持率聚成一个分支,明确了两种报春的姐妹种关系。首先,通过选取两个物种的五个形态学性状,进行主成分分析,聚类结果中两种报春各自的个体分别聚在两块区域,表明两种报春的形态学性状产生了明显的分化,且中甸灯台报春的花部各性状值明显大于鹅黄灯台报春,表明花部综合征和繁育系统间的相关联系。然后,通过用8对SSR分子标记对两种报春进行STRUCTURE聚类分析,结果检测K的最适值为2,此时中甸灯台报春和鹅黄灯台报春的个体各自以不同颜色聚成界限清晰的两块区域,揭示了两种报春在分子水平上也产生了显著而稳定的分化,通过Ima2软件,依据叶绿体联合序列,计算了两种报春的分化时间大约是在更新世,推测这两个姐妹种的物种分化受更新世气候动荡和一些地质历史事件的影响。最后,运用maxent软件分析两个物种的生态位差异,结果表明最干季节降水量、年均降水量和季节性降水量对中甸灯台报春的分布有重要影响,最冷季节平均温度、最干月降水量、年均降水量和季节性降水量对鹅黄灯台报春的分布有重要影响,两个物种间的生态位产生了分化。由此推测,这种生态位的差异是物种分化的结果,同时也是物种间系统演化的重要驱动力。     

SOX9/miR-130a/CTR1 axis modulates DDP-resistance of cervical cancer cell

Cisplatin (DDP) -based chemotherapy is a standard strategy for cervical cancer, while chemoresistance remains a huge challenge. Copper transporter protein 1 (CTR1), a copper influx transporter required for high affinity copper (probably reduced Cu I) transport into the cell, reportedly promotes a significant fraction of DDP internalization in tumor cells. In the present study, we evaluated the function of CTR1 in the cell proliferation of cervical cancer upon DDP treatment. MicroRNAs (miRNAs) have been regarded as essential regulators of cell proliferation, apoptosis, migration, as well as chemoresistance. By using online tools, we screened for candidate miRNAs potentially regulate CTR1, among which miR-130a has been proved to promote cervical cancer cell proliferation through targeting PTEN in our previous study. In the present study, we investigated the role of miR-130a in cervical cancer chemoresistance to DDP, and confirmed the binding of miR-130a to CTR1. SOX9 also reportedly act on cancer chemoresistance. In the present study, we revealed that SOX9 inversely regulated miR-130a through direct targeting the promoter of miR-130a. Consistent with previous studies, SOX9 could affect cervical cancer chemoresistance to DDP. Taken together, we demonstrated a SOX9/miR-130a/CTR1 axis which modulated the chemoresistance of cervical cancer cell to DDP, and provided promising targets for dealing with the chemoresistance of cervical cancer.