Expression profiling of reciprocal maize hybrids divergent for cold germination and desiccation tolerance

Recombinant inbred lines (RILs) derived from B73 x M017 were screened for cold germination (CG) and desiccation tolerance (DT) phenotypes. Reciprocal F(1) hybrids were made between divergent RILs, and hybrids that showed differential phenotypes (parent-of-origin effect) for CG or DT were selected for profiling mRNA and protein expression. mRNA and proteins were extracted from embryo axes of seed germinated for 11 d at 12.5 degrees C in the dark and developing embryos at 40% seed moisture (R5 stage) for CG and DT, respectively. GeneCalling analysis, an open-ended mRNA profiling method, identified 336 of 32,496 and 656 of 32,940 cDNA fragments that showed >or=1.5-fold change in expression between the reciprocal F(1) hybrids for CG and DT, respectively. Protein expression map (PEM) analysis, an open-ended two-dimensional polyacrylamide gel electrophoresis, identified 117 of 2,641 and 205 of 1,876 detected proteins to be differentially expressed with >or=1.5-fold change between the reciprocal F(1) hybrids in CG and DT samples, respectively. A subset of these proteins was identified by tandem mass spectrometry followed by database query of the spectra. The differentially expressed genes/proteins were classified into various functional groups including carbohydrate and amino acid metabolism, ion transporters, stress and defense response, polyamine metabolism, chaperonins, cytoskeleton associated, etc. Phenotypic analysis of seed from self-pollinated ears of the reciprocal F(1) hybrids displayed small differences compared with the reciprocal hybrids themselves, suggesting a negligible effect of cytoplasmic factors on CG and DT traits. The results provide leads to improving our understanding of the genes involved in stress response during seed maturation and germination.     

Functional analysis of genes differentially expressed in the Drosophila wing disc: role of transcripts enriched in the wing region

Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.     

Isolation and characterization of sea urchin egg cortical granules

A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface- labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.     

TGF-β regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins

We investigated putative roles of transforming growth factor (TGF)-β expressed in peripheral ganglia in the regulation of neuronal cell survival during the period of ontogenetic neuron death (OD). The chick ciliary ganglion (CG), where OD occurs between embryonic days (E) 6 and 10, was employed as a model system. We show that CG neurons (E8) are immunoreactive (ir) for TGF-β2 and -β3 as well as the TGF-β receptor TβR-II, but are not ir for TGF-β1. Ciliary neurotrophic factor (CNTF) and fibroblast growth factor (FGF)-2, established neurotrophic molecules for CG neurons, up-regulate TGF-β3 mRNA and TGF-β biological activity in cultures of E8 CG neurons. None of the TGF-β isoforms—β1, β2, or β3—has a trophic, survival-promoting effect on cultured CG neurons. However, all isoforms enhance CG neuron survival mediated by CNTF or FGF-2, significantly and over a wide range of concentrations. In combination with the neurotrophins (NT) nerve growth factor (NGF) and NT-3, which are not neurotrophic for CG neurons, TGF-β significantly promotes CG neuron survival. However, TGF-β does not act synergistically with the neuropoietic cytokines oncostatin M, leukemia inhibiting factor, or interleukin-6. Immunoneutralization of endogenous TGF-β released from CG neurons using an antibody to TGF-β1/-β2/-β3 significantly reduces the potency of CNTF or FGF-2 to promote CG neuron survival. The blocking effect of the anti–pan-TGF-β antibody could be rescued by adding exogenous TGF-β. Together, these data suggest that para-/autocrine TGF-β signaling has an important effect on the regulation of neuron survival in a model system of peripheral neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 563–572, 1998     

A Potential Role for Epigenetic Modulatory Drugs in the Enhancement of Cancer/Germ-Line Antigen Vaccine Efficacy

The discovery of epigenetic silencing as a key mechanism of tumor suppressor gene inactivation in human cancer has led to great interest in utilizing epigenetic modulatory drugs as cancer therapeutics. It is less appreciated that medically important tumor-associated antigens, particularly the Cancer Testis or Cancer/Germ-line family of antigens (CG antigens), which are being actively tested as cancer vaccine targets, are epigenetically activated in many human cancers. However, a major limitation to the therapeutic value of CG antigen-directed vaccines is the limited and heterogeneous expression of CG antigens in tumors. Recent work has begun to dissect the specific epigenetic mechanisms controlling differential expression of CG antigen genes in human cancers. From a clinical perspective, convincing data indicate that epigenetic modulatory agents, including DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, robustly promote the expression of CG antigens, as well as class I major histocompatibility complex (MHC I) and other immune co-stimulatory molecules, in tumors. Importantly, the effects of these agents on CG antigen gene expression often show marked specificity for tumor cells as compared to normal cells. Taken together, these data encourage clinical evaluation of combination therapies involving epigenetic modulatory drugs and CG antigen-directed tumor vaccines for the treatment of human malignancies.     

Structural and Biochemical Characterization of the Two Drosophila Low Molecular Weight-Protein Tyrosine Phosphatases DARP and Primo-1

The Drosophila genome contains four low molecular weight-protein tyrosine phosphatase (LMW-PTP) members: Primo-1, Primo-2, CG14297, and CG31469. The lack of intensive biochemical analysis has limited our understanding of these proteins. Primo-1 and CG31469 were previously classified as pseudophosphatases, but CG31469 was also suggested to be a putative protein arginine phosphatase. Herein, we present the crystal structures of CG31469 and Primo-1, which are the first Drosophila LMW-PTP structures. Structural analysis showed that the two proteins adopt the typical LMW-PTP fold and have a canonically arranged P-loop. Intriguingly, while Primo-1 is presumed to be a canonical LMW-PTP, CG31469 is unique as it contains a threonine residue at the fifth position of the P-loop motif instead of highly conserved isoleucine and a characteristically narrow active site pocket, which should facilitate the accommodation of phosphoarginine. Subsequent biochemical analysis revealed that Primo-1 and CG31469 are enzymatically active on phosphotyrosine and phosphoarginine, respectively, refuting their classification as pseudophosphatases. Collectively, we provide structural and biochemical data on two Drosophila proteins: Primo-1, the canonical LMW-PTP protein, and CG31469, the first investigated eukaryotic protein arginine phosphatase. We named CG31469 as DARP, which stands for Drosophila ARginine Phosphatase.     

A fast conformational search strategy for finding low energy structures of model proteins.

We describe a new computer algorithm for finding low-energy conformations of proteins. It is a chain-growth method that uses a heuristic bias function to help assemble a hydrophobic core. We call it the Core-directed chain Growth method (CG). We test the CG method on several well-known literature examples of HP lattice model proteins [in which proteins are modeled as sequences of hydrophobic (H) and polar (P) monomers], ranging from 20-64 monomers in two dimensions, and up to 88-mers in three dimensions. Previous nonexhaustive methods--Monte Carlo, a Genetic Algorithm, Hydrophobic Zippers, and Contact Interactions--have been tried on these same model sequences. CG is substantially better at finding the global optima, and avoiding local optima, and it does so in comparable or shorter times. CG finds the global minimum energy of the longest HP lattice model chain for which the global optimum is known, a 3D 88-mer that has only been reachable before by the CHCC complete search method. CG has the potential advantage that it should have nonexponential scaling with chain length. We believe this is a promising method for conformational searching in protein folding algorithms.     

Effects of chorionic gonadotropin on dynamics of primary immune response]

The effect of chorionic gonadotropin (CG) on primary immune response was estimated according to the level of direct and indirect plaque-forming cells (PFC) on day 5, 8 and 12 after immunization of non-castrated and ovariectomized female mice of CBA strain. It was established, that on the 5th day CG (40-200 IU) did not influence the direct PFC level in ovariectomized animals, but stimulated them in non-ovariectomized mice (40 IU). In ovariectomized animals the selective immunodepressive effect of hormone on the IgG-PFC formation processes has been revealed. The CG effect depended on the time of PFC number examination as well as on the hormone dose. In non-castrated animals, where immunomodulating CG effects are partially mediated by ovarian hormones, the injection of hormone only in the dose of 200 IU significantly lowered the number of IgM and IgG-PFC. It is suggested, that sex steroids on the late stages of PFC formation, when the processes of isotype antibody synthesis switch take place, appear to be synergists of CG immunodepressive effect.     

Duplication-dependent CG suppression of the seed storage protein genes of maize

This study investigates the prevalence of CG and CNG suppression in single- vs. multicopy DNA regions of the maize genome. The analysis includes the single- and multicopy seed storage proteins (zeins), the miniature inverted-repeat transposable elements (MITEs), and long terminal repeat (LTR) retrotransposons. Zein genes are clustered on specific chromosomal regions, whereas MITEs and LTRs are dispersed in the genome. The multicopy zein genes are CG suppressed and exhibit large variations in CG suppression. The variation observed correlates with the extent of duplication each zein gene has undergone, indicating that gene duplication results in an increased turnover of cytosine residues. Alignment of individual zein genes confirms this observation and demonstrates that CG depletion results primarily from polarized C:T and G:A transition mutations from a less to a more extensively duplicated gene. In addition, transition mutations occur primarily in a CG or CNG context suggesting that CG suppression may result from deamination of methylated cytosine residues. Duplication-dependent CG depletion is likely to occur at other loci as duplicated MITEs and LTR elements, or elements inserted into duplicated gene regions, also exhibit CG depletion.     

Phenogenetic analysis of testicular responsiveness to chorionic gonadotropin in inbred mouse strains

Genetic differences in the testicular hormonal responsiveness to in vivo administration of chorionic gonadotropin (CG) between adult male mice of eight inbred strains (A/Sn, CBA/Lac, CC57Br, C57Bl/6J, DBA/2J, GR, PT, and YT) were determined. In addition, the genetic variation of the body and testis weights was estimated as related to the responsiveness to stimulation of steroidogenesis with CG. Adult males were subcutaneously injected with 10 IU of CG or physiological saline 120 min before decapitation. It was found that the baseline testosterone level in the blood serum and its content in the testes only slightly varied in males of the strains studied. Administration of CG increased these parameters by a factor of 3–45, depending on the strain. The results of the study indicate genetic differences in the testicular reactivity to CG. In addition, it has been found that the response to administration of CG, as compared to the baseline levels, provides the most reliable information on the genetic characteristics of the hormonal potential of the testes. The given set of inbred mouse strains may be a promising genetic model for studying the physiological and hereditary variations of testicular steroidogenesis.     

Silicon membrane interface for the direct analysis of Kathon CG in aqueous solutions and cosmetic emulsions.

An easy determination of Kathon CG, an antimicrobial agent widely used in cosmetic and toiletry products, has been achieved by means of silicone membrane interfaced mass spectrometry. Low levels of the above preservative (up to p.p.b.) could be easily detected in both aqueous solutions and cosmetic emulsions. Valuable information has been obtained on the stabilization and decomposition processes to which the organic components of Kathon CG are subject. The role of magnesium ions, which are present as a stabilizing agent in Kathon CG formulation, in these processes has been investigated, leading to the identification of some degradation products.     

How Well Do Computer-Generated Faces Tap Face Expertise?

The use of computer-generated (CG) stimuli in face processing research is proliferating due to the ease with which faces can be generated, standardised and manipulated. However there has been surprisingly little research into whether CG faces are processed in the same way as photographs of real faces. The present study assessed how well CG faces tap face identity expertise by investigating whether two indicators of face expertise are reduced for CG faces when compared to face photographs. These indicators were accuracy for identification of own-race faces and the other-race effect (ORE)–the well-established finding that own-race faces are recognised more accurately than other-race faces. In Experiment 1 Caucasian and Asian participants completed a recognition memory task for own- and other-race real and CG faces. Overall accuracy for own-race faces was dramatically reduced for CG compared to real faces and the ORE was significantly and substantially attenuated for CG faces. Experiment 2 investigated perceptual discrimination for own- and other-race real and CG faces with Caucasian and Asian participants. Here again, accuracy for own-race faces was significantly reduced for CG compared to real faces. However the ORE was not affected by format. Together these results signal that CG faces of the type tested here do not fully tap face expertise. Technological advancement may, in the future, produce CG faces that are equivalent to real photographs. Until then caution is advised when interpreting results obtained using CG faces.     

Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA.

Using MA-10 Leydig tumor cells as a model system we have examined the possibility that the lutropin/choriogonadotropin (LH/CG)-induced down-regulation of the LH/CG receptor is accompanied by changes in LH/CG receptor mRNA. We show that LH or CG are indeed capable of reducing the levels of LH/CG receptor mRNA, but that the time course and magnitude of the reduction in receptor mRNA are such that this phenomenon cannot account entirely for the down-regulation of the receptor. In fact, we estimate that LH/CG can reduce the number of LH/CG receptors by at least 80% with little or no change in the levels of LH/CG receptor mRNA. These data are consistent with our previous hypothesis that the LH/CG-induced down-regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor that occurs as a result of the receptor-mediated endocytosis of LH/CG. Our studies also show that the LH/CG-induced down-regulation of the LH/CG receptor mRNA is mediated by cAMP. Thus, addition of 8-bromo-cAMP to MA-10 cells leads to a similar reduction in the levels of LH/CG receptor and receptor mRNA; while deglycosylated human CG, a hormone derivative that binds to the LH/CG receptor but has a reduced ability to stimulate cAMP synthesis, does not reduce the levels of LH/CG receptor mRNA. Last, human CG or 8-bromo-cAMP are unable to reduce LH/Cg receptor mRNA in a mutant MA-10 cell line that express a cAMP-resistant phenotype.     

Bio-hydrogen production by biodiesel-derived crude glycerol bioconversion: a techno-economic evaluation

Global biodiesel production is continuously increasing and it is proportionally accompanied by a huge amount of crude glycerol (CG) as by-product. Due to its crude nature, CG has very less commercial interest; although its pure counterpart has different industrial applications. Alternatively, CG is a very good carbon source and can be used as a feedstock for fermentative hydrogen production. Further, a move of this kind has dual benefits, namely it offers a sustainable method for disposal of biodiesel manufacturing waste as well as produces biofuels and contributes in greenhouse gas (GHG) reduction. Two-stage fermentation, comprising dark and photo-fermentation is one of the most promising options available for bio-hydrogen production. In the present study, techno-economic feasibility of such a two-stage process has been evaluated. The analysis has been made based on the recent advances in fermentative hydrogen production using CG as a feedstock. The study has been carried out with special reference to North American biodiesel market; and more specifically, data available for Canadian province, Québec City have been used. Based on our techno-economic analysis, higher production cost was found to be the major bottleneck in commercial production of fermentative hydrogen. However, certain achievable alternative options for reduction of process cost have been identified. Further, the process was found to be capable in reducing GHG emissions. Bioconversion of 1 kg of crude glycerol (70 % w/v) was found to reduce 7.66 kg CO₂ eq (equivalent) GHG emission, and the process also offers additional environmental benefits.     

基因组序列8-mer频次使用规律及与物种进化的关系

基因组序列k-mer的非随机使用规律及包含的生物学意义一直是人们关注的问题,目前还没有根本性进展。本文以七个物种的全部基因序列为样本,得到各物种基因组序列的8-mer频谱分布。发现狗和牛的频谱有三个峰,而斑马鱼、青鳉鱼、秀丽线虫和酿酒酵母的频谱只有一个峰,鸡的频谱分布形状介于两者之间。将8-mer集合按照XY二核苷含量分类,结果显示只有CG二核苷分类下0CG、1CG和2CG三类子集的频谱形成各自独立的单峰分布。对照随机序列,发现0CG模体是随机进化的,1CG和2CG模体是定向进化的,它们的使用频次远小于随机频次,且这种独立进化分离规律具有物种普适性。三个CG子集频谱之间的距离是产生单峰或多峰现象的根本原因。将七个物种基因组序列标准化到109bp,比较发现1CG和2CG子集频谱与物种进化显著相关,0CG子集频谱与物种进化无显著关系。可以认为三种CG模体各自执行着不同的生物学功能。基因组序列8-mer的独立分离规律为揭示基因组结构、基因组进化以及模体的生物功能提供了一种新的思维方式。