Phylogenetic screening of the human genome: identification of differentially hybridizing repetitive sequence families

The phi-screen, a method of phylogenetic screening, can be employed to detect repetitive sequence families that differentially hybridize between closely related species. Such differences may involve sequence divergence or variations in copy number, including total presence versus absence of a family of repeated DNA. We present the results of a phi-screen comparing the human genome to that of the prosimian, Galago crassicaudatus. Three human repetitive families that are divergent or not present in galago have been detected. One of these families is described in detail; it is similar among the anthropoids but is present in a lower copy number and/or divergent form in prosimians. The family is clearly related to the transposon-like human element (THE) described by Paulson et al. (1985). THEs have long terminal repeats reminiscent of retroviruses but are unique in that they have no sequence similarity to known mammalian retroviruses. The sequence of a solo long terminal repeat, found unassociated with THE internal sequence, is presented. This family member, THE p2, is bordered by a 5-bp target-site repeat and is interrupted by the insertion of an Alu element. A solo THE element sequenced by Wiginton et al. (1986) contains an insertion of Alu at precisely the same position as does THE p2.      

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases

Mutations in DJ-1 cause an autosomal recessive, early onset familial form of Parkinson disease (PD). However, little is presently known about the role of DJ-1 in the more common sporadic form of PD and in other age-related neurodegenerative diseases, such as Alzheimer disease (AD). Here we report that DJ-1 is oxidatively damaged in the brains of patients with idiopathic PD and AD. By using a combination of two-dimensional gel electrophoresis and mass spectrometry, we have identified 10 different DJ-1 isoforms, of which the acidic isoforms (pI 5.5 and 5.7) of DJ-1 monomer and the basic isoforms (pI 8.0 and 8.4) of SDS-resistant DJ-1 dimer are selectively accumulated in PD and AD frontal cortex tissues compared with age-matched controls. Quantitative Western blot analysis shows that the total level of DJ-1 protein is significantly increased in PD and AD brains. Mass spectrometry analyses reveal that DJ-1 is not only susceptible to cysteine oxidation but also to previously unsuspected methionine oxidation. Furthermore, we show that DJ-1 protein is irreversibly oxidized by carbonylation as well as by methionine oxidation to methionine sulfone in PD and AD. Our study provides new insights into the oxidative modifications of DJ-1 and indicates association of oxidative damage to DJ-1 with sporadic PD and AD.     

Corneal Dystrophy-associated R124H Mutation Disrupts TGFBI Interaction with Periostin and Causes Mislocalization to the Lysosome

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-β-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.Corneal dystrophies are characterized by the progressive loss of corneal transparency as a result of extracellular amyloid and non-amyloid deposits, which accumulate in different layers of corneal tissues. 5q31-linked corneal dystrophies are pathologically heterogeneous, autosomal-dominant disorders caused by mutations in the TGFBI (transforming growth factor-β-induced) gene, which encodes the TGFBI protein (also known as keratoepithelin or Big-H3) (1, 2). To date, more than 30 different mutations leading to corneal dystrophies have been attributed to mutations in TGFBI, the most frequent of which are mutations within exons 4 and 12, which result in amino acid substitutions in Arg¹²⁴ and Arg⁵⁵⁵, respectively (3, 4). The different mutations in TGFBI cause clinically distinct types of corneal dystrophies, which are classified according to the accumulation patterns of the deposits, including lattice corneal dystrophies type I and IIIA, deep stromal lattice corneal dystrophy, granular corneal dystrophies (GCDs)² type I and II (also known as Avellino corneal dystrophy), Reis-Bucklers corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type I), or Thiel-Behnke corneal dystrophy (also known as corneal dystrophy of Bowman''s layer type II) (reviewed in Refs. 5 and 6). Histological examinations of corneal tissues demonstrate the presence of amyloid deposits in lattice corneal dystrophies and GCD II, hyaline accumulations in GCDs, and subepithelial fibrous material in Reis-Bucklers corneal dystrophy and Thiel-Behnke corneal dystrophy (7–14).TGFBI was originally identified as a gene induced by transforming growth factor-β stimulation in adenocarcinoma cells and is expressed in many tissues (15). The human TGFBI consists of 683 amino acids, with the mature protein predicted to have a molecular mass of ∼68 kDa. As shown in Fig. 1A, TGFBI contains an NH₂-terminal signal peptide that targets it for insertion into the lumen of the endoplasmic reticulum (ER) for eventual secretion, a cysteine-rich EMI domain, four tandem repeats of fasciclin-1 like (FAS1) domains, and a COOH-terminal RGD sequence (15–19). The FAS1 domains of TGFBI display homology to the cell adhesion protein fasciclin-I in Drosophila, an axon guidance protein that is involved in neuronal development (20). Based on the presence of multiple FAS1 domains, TGFBI has been assigned to a larger family of proteins, which includes periostin, stabilin-1, and stabilin-2 (16, 21). To date, many TGFBI homologues have been reported in various vertebrates, including mouse, chicken, pig, and zebrafish, but no homologues have been identified in invertebrates (16, 19, 21). TGFBI has been shown to interact with a number of extracellular matrix (ECM) proteins, including fibronectin, biglycan, decorin, and several types of collagen (19, 22–25). Furthermore, TGFBI also functions as a ligand for several integrins, including α3β1, αvβ5, αvβ3, and αmβ2 (26–29). The COOH-terminal RGD domain of TGFBI is the putative integrin-binding motif. However, several studies have suggested that the interactions between TGFBI and integrins are mediated via the YH (tyrosine-histidine) motifs and DI (aspartate-isoleucine) motifs present in the TGFBI FAS1 domains (30). Although the precise roles of TGFBI are not fully understood yet, emerging evidence suggests a role for TGFBI as a secreted factor involved in cell adhesion, proliferation, and migration.Open in a separate windowFIGURE 1.Periostin is expressed in human cornea. A, schematic representation of TGFBI and periostin. TGFBI and periostin contain NH₂-terminal signal peptides, followed by a cysteine-rich EMI domain and four tandem FAS1 domains. TGFBI also contains a COOH-terminal RGD motif that is not present in periostin, which instead contains a COOH-terminal hydrophilic region. The following domains of TGFBI and periostin are indicated: EMI, FAS1, fasciclin 1, and Arg-Gly-Asp (RGD). Antigenic regions of antibodies used in this study (ab14041 and C-20) are represented by black lines. B, cell and tissue lysates were separated by SDS-PAGE and Western blotted using the indicated antibodies. Anti-periostin antibody (C-20) recognizes an ∼85-kDa protein in several cell lines and corneal fibroblast cell lines. In contrast, anti-periostin antibody recognizes a ∼60-kDa form in corneal epithelial cell lines and corneal epithelium tissues (top, black arrow). The specificity of periostin antibody (C-20) was confirmed by preabsorption with 10 μg of C-20 antigen peptide (second panel). WB, Western blot.TGFBI and periostin show a high degree of similarity in amino acid sequence and in overall domain structure, diverging primarily at the COOH terminus (Fig. 1A) (16, 21). Similar to TGFBI, periostin contains a NH₂-terminal secretory signal peptide followed by a cysteine-rich EMI domain, four tandem repeats of FAS1 domains, and a hydrophilic region in its COOH terminus (Fig. 1A) (16, 17, 31, 32). Periostin has been found to be ubiquitously expressed in multiple tissues in mammals (31, 33, 34). In addition, the expression of periostin has been implicated in the development of variety of cancers, including neuroblastoma, head and neck cancer, and non-small cell lung cancer, possibly by regulating the metastatic growth (32, 35). Periostin is also associated with epithelial-mesenchymal transition during cardiac development (36) and is induced during the proliferation of cardiomyocytes, thereby promoting cardiac repair after heart failure (37, 38). In addition, interlukin-4 and -13 have been found to induce the secretion of periostin from lung fibroblasts, implicating periostin in subepithelial fibrosis in bronchial asthma (39).Despite the similarities between TGFBI and periostin, it is not known whether periostin is involved in the pathogenesis of 5q31-linked corneal dystrophies. In this study, we find that periostin specifically interacts with TGFBI via the NH₂-terminal cysteine-rich EMI domain and colocalizes with TGFBI in the trans-Golgi network of COS-7 and corneal fibroblast cells. In addition, corneal dystrophy-linked mutations in TGFBI disrupt its subcellular localization and impair its interaction with periostin. Furthermore, we find that periostin accumulates in extracellular corneal deposits in GCD II patients bearing homozygous R124H mutations in TGFBI. These findings provide new insights into the pathogenic mechanisms of TGFBI mutations in 5q31-linked corneal dystrophies and have important implications for understanding and treating corneal dystrophies.     

Germ band retraction as a landmark in glucose metabolism during <Emphasis Type="Italic">Aedes aegypti</Emphasis> embryogenesis

Background  

The mosquito A. aegypti is vector of dengue and other viruses. New methods of vector control are needed and can be achieved by a better understanding of the life cycle of this insect. Embryogenesis is a part of A. aegypty life cycle that is poorly understood. In insects in general and in mosquitoes in particular energetic metabolism is well studied during oogenesis, when the oocyte exhibits fast growth, accumulating carbohydrates, lipids and proteins that will meet the regulatory and metabolic needs of the developing embryo. On the other hand, events related with energetic metabolism during A. aegypti embryogenesis are unknown.     

大鼠心肌缺血再灌注损伤模型的实验研究

目的 制备一种新型的心肌急性缺血再灌注损伤模型,以探讨一种更符合临床实际需求的实验方法.方法 将20只雌性SD(Sprague-Dawley)大鼠随机分成2组(对照组、实验组),采用结扎主动脉根部引起心肌缺血5min再灌注30 min建立心肌急性缺血再灌注模型;通过应用透射电镜观察心肌细胞超微结构的改变,同时检测心肌组织匀浆丙二醛(Maleic Dialdehyde,MDA)含量、超氧化物歧化酶(Superoxide Dismutase,SOD)活力.结果 透射电镜下超微结构显示实验组较对照组明显加重了心肌组织结构和线粒体的损害;实验组心肌组织MDA明显高于对照组(P<0.01),而SOD明显低于对照组(P<0.01).结论 本实验成功建立了方法简便、易于操作、取材范围广泛的心肌缺血再灌注损伤模型,为心肌缺血再灌注损伤研究提供了一种更为可行的模型.     

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Thesaurus-based disambiguation of gene symbols

Background  

Massive text mining of the biological literature holds great promise of relating disparate information and discovering new knowledge. However, disambiguation of gene symbols is a major bottleneck.