Identification and expression patterns of members of the protease‐activated receptor (par) gene family during zebrafish development

Protease‐activated receptors (PARs) play critical roles in hemostasis in vertebrates including zebrafish. However, the zebrafish gene classification appears to be complex, and the expression patterns of par genes are not established. Based on analyses of genomic organization, phylogenetics, protein primary structure, and protein internalization, we report the identification of four zebrafish PARs: par1, par2a, par2b, and par3. This classification differs from one reported previously. We also show that these genes have distinct spatiotemporal expression profiles in embryos and larvae, with par1, par2a, and par2b expressed maternally and ubiquitously during gastrula stages and their expression patterns refined at later stages, and par3 expressed only in 3‐day‐old larvae. Notably, the expression patterns of zebrafish par1 and par2b resemble those of their mammalian counterparts, suggesting that receptor function is conserved among vertebrates. This conservation is supported by our findings that Par1 and Par2b are internalized following exposure to thrombin and trypsin, respectively. Developmental Dynamics, 2011. © 2010 Wiley‐Liss, Inc.     

Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD.

Listeria monocytogenes is a bacterial pathogen that is able to invade nonphagocytic cells. Two surface proteins, internalin, the inlA gene product, and InlB, play important roles in the entry into cultured mammalian cells. These proteins also have extensive sequence similarities. Previously, Southern hybridization predicted the existence of an internalin multigene family. Recently, InlC, a secreted protein of 30 kDa homologous to InlA and InlB, was identified. In this work, we identified and characterized four new members of the internalin multigene family, inlC2, inlD, inlE, and inlF which encode proteins of 548, 567, 499, and 821 amino acids respectively. inlC2, inlD, and inlE are contiguous on the chromosome of L. monocytogenes EGD, whereas inlF is located in a different chromosomal region. These four inl gene products display the principal features of internalin, namely, a signal sequence, two regions of repeats (or LRR and B repeats), and a putative cell wall anchor sequence containing the sorting motif LPXTG. The four inl genes were maximally expressed albeit at a low level during early exponential growth in bacterial medium at 37 degrees C. The role of these inl genes in L. monocytogenes invasion was assessed by constructing isogenic chromosomal deletion mutants and testing them for entry into various nonphagocytic cells. Unexpectedly, the inlC2, inlD, inlE, and inlF null mutants were not affected for entry into any of the cell lines tested, raising the possibility that these genes are needed for an aspect of pathogenicity other than invasion. The identity of such an aspect remains to be determined.     

Update on IgG4-mediated autoimmune diseases: New insights and new family members

Antibodies of IgG4 subclass are exceptional players of the immune system, as they are considered to be immunologically inert and functionally monovalent, and as such may be part of classical tolerance mechanisms. IgG4 antibodies are found in a range of different diseases, including IgG4-related diseases, allergy, cancer, rheumatoid arthritis, helminth infection and IgG4 autoimmune diseases, where they may be pathogenic or protective. IgG4 autoimmune diseases are an emerging new group of diseases that are characterized by pathogenic, antigen-specific autoantibodies of IgG4 subclass, such as MuSK myasthenia gravis, pemphigus vulgaris and thrombotic thrombocytopenic purpura. The list of IgG4 autoantigens is rapidly growing and to date contains 29 candidate antigens. Interestingly, IgG4 autoimmune diseases are restricted to four distinct organs: 1) the central and peripheral nervous system, 2) the kidney, 3) the skin and mucous membranes and 4) the vascular system and soluble antigens in the blood circulation. The pathogenicity of IgG4 can be validated using our classification system, and is usually excerted by functional blocking of protein-protein interaction.     

Fcgamma receptors: old friends and new family members

Although cellular receptors for immunoglobulins were first identified nearly 40 years ago, their central role in the immune response was discovered only in the last decade. They are key players in both the afferent and efferent phase of an immune response, setting thresholds for B cell activation, regulating the maturation of dendritic cells, and coupling the exquisite specificity of the antibody response to innate effector pathways, such as phagocytosis, antibody-dependent cellular cytotoxicity, and the recruitment and activation of inflammatory cells. Moreover, because of their general presence as receptor pairs consisting of activating and inhibitory molecules on the same cell, they have become a paradigm for studying the balance of positive and negative signals that ultimately determine the outcome of an immune response. This review will summarize recent results in Fc-receptor biology with an emphasis on data obtained in in vivo model systems.     

The human HTLV-3 and HTLV-4 retroviruses: new members of the HTLV family

Human T cell leukemia/lymphoma virus Type 1 and 2 (HTLV-1 and HTLV-2), together with their simian counterparts (STLV-1, STLV-2), belong to the Primate T lymphotropic viruses group (PTLV). HTLV-1 infects 15 to 20million people worldwide, while STLV-1 is endemic in a number of simian or ape species living in Africa or Asia. The high percentage of homologies between HTLV-1 and STLV-1 strains, led to the demonstration that most HTLV-1 subtypes arose from interspecies transmission between monkeys and humans. STLV-3 viruses belong to the third PTLV type and are equally divergent from HTLV-1 than from HTLV-2. They are endemic in several monkey species that live in West, Central, and East Africa. In 2005, we and others reported the discovery of the human homolog (HTLV-3) of STLV-3 in two asymptomatic inhabitants from South Cameroon whose sera exhibited HTLV indeterminate serologies. More recently, we reported a third case of HTLV-3 infection in Cameroon suggesting that this virus is not rare in the human population living in Central Africa. Together with STLV-3, these three human viral strains belong therefore to the PTLV-3 type. A fourth HTLV type (HTLV-4) was also discovered in the same geographical area. Current studies are aimed at determining the prevalence, distribution and modes of transmission of these viruses as well as their possible association with human diseases. Furthermore, molecular characterization of their viral transactivator Tax is ongoing in order to look for possible oncogenic properties.     

Prep2: cloning and expression of a new prep family member.

We describe Prep2, a new murine homeobox-containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain-containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes.     

Identification of a new HLA-DRB1 allele in three members of an Italian family

Abstract A new human leucocyte antigen (HLA)-DRB1 allele, HLA-DRB1*1149, has been identified in three members of an Italian family during routine sequence based typing. This new allele differs from HLA-DRB1*110101 only for a single nucleotide substitution at position 113 of exon 2 resulting in an amino acid change from Valine (GTG) to Alanine (GCG) at codon 38.     

Molecular structure and developmental expression of three muscle-type troponin T genes in zebrafish.

Troponin T (Tnnt), a troponin component, interacts with tropomyosin and is crucial to the regulation of striated muscle contraction. To gain insight into the molecular evolution and developmental regulation of Tnnt gene (Tnnt) in lower vertebrates, zebrafish Tnnt1 (slow Tnnt), Tnnt2 (cardiac Tnnt), and Tnnt3b (fast Tnnt isoform b) were characterized. The polypeptides of zebrafish Tnnt1, Tnnt2, and Tnnt3b were conserved in the central tropomyosin- and C-terminal troponin I-binding domains. However, the N-terminal hypervariable regions were highly extended and rich in glutamic acid in polypeptides of Tnnt1 and Tnnt2, but not Tnnt3b. The Tnnt2 and Tnnt3b contain introns, whereas Tnnt1 is intron-free. During development, large to small, alternatively spliced variants were detected in Tnnt2, but not in Tnnt1 or Tnnt3. Whole-mount in situ hybridization showed zebrafish Tnnt1 and Tnnt2 are activated during early somitogenesis (10 hr postfertilization, hpf) and cardiogenesis (14 hpf), respectively, but Tnnt3b is not activated until middle somitogenesis (18 hpf). Tnnt2 and Tnnt3b expression was cardiac- and fast-muscle specific, but Tnnt1 was expressed in both slow and fast muscles. We propose that three, distinct, muscle-type Tnnt evolved after the divergence of fish and deuterostome invertebrates. In zebrafish, the developmental regulation of Tnnt during somitogenesis and cardiogenesis is more restricted and simpler than in tetrapods. These new findings may provide insight into the developmental regulation and molecular evolution of vertebrate Tnnt.     

Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: II. The 6-O-sulfotransferase family.

Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 6-O-sulfotransferases (6-OST) catalyze the transfer of sulfate groups to the 6-O position of glucosamine residues of HS. We report here the characterization and developmental expression analysis of the 6-OST gene family in the zebrafish. The zebrafish 6-OST gene family consists of four conserved vertebrate orthologues, including a gene duplication specific to zebrafish. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, eyes, somites, brain, internal organ primordial, and pectoral fin development. Members of the 6-OST gene family have spatially and temporally distinct restricted expression, suggesting in vivo functional differences exist between members of this family.     

Combinatorial expression patterns of heparan sulfate sulfotransferases in zebrafish: I. The 3-O-sulfotransferase family.

Heparan sulfate (HS) is an unbranched chain of repetitive disaccharides, which specifically binds ligands when attached to the cell surface or secreted extracellularly. HS chains contain sulfated domains termed the HS fine structure, which gives HS specific binding affinities for extracellular ligands. HS 3-O-sulfotransferases (3-OST) catalyze the transfer of sulfate groups to the 3-O position of glucosamine residues of HS, a rare, but essential HS chain modification required for HS fine structure. We report here the first characterization and developmental expression analysis of the 3-OST gene family in a vertebrate. There are eight 3-OST genes in zebrafish: seven genes with homology to known 3-OST genes in mouse and human, as well as a novel, 3-OST-7. A phylogenetic comparison of human, mouse, and zebrafish indicates the 3-OST family can be subdivided into two distinct subgroups. We examined the mRNA expression patterns in several tissues/organs throughout early zebrafish development, including early cleavage stages, somites, brain, internal body organ primordial, and pectoral fin development. The 3-OST gene family has both specifically expressed and ubiquitously expressed genes, suggesting in vivo functional differences exist between members of this family.     

一寻常型银屑病家系的HLA等位基因分析

目的分析北方一寻常型银屑病家系的发病与HLA-A、B、DRB1等位基因的相关性。方法采用序列特异性寡核苷酸探针-聚合酶链反应(PCR-SSO)方法检测一北方家系16人的HLA-A、B、DRB1等位基因。结果家系中共有6人患寻常型银屑病,其中4个早发寻常型银屑病患者(Ⅱ-2、Ⅱ-4、Ⅲ-1、Ⅲ-5)发病均在30岁以前,B位点均为B*13、DRB1位点均为DRB1*07;家系中有6人携带单倍型11-15-15,其中4人发病(Ⅰ-2、Ⅱ-2、Ⅱ-4、Ⅲ-1),年龄稍小的2人未发病(Ⅲ-3、Ⅲ-4)。结论此家系中寻常型银屑病的发病涉及多个易感位点,携带B*13-DRB1*07基因的早发病者可能与对遗传因素和/或环境因素敏感性增加有关;易感单倍型携带者的发病受环境因素和/或其他易感因素的影响。     

Characterization of the Enigma family in zebrafish.

The three Enigma subfamily proteins, Enigma, Enigma homologue, and Cypher/ZASP belong to the PDZ and LIM encoding protein family, which is characterized by the presence of a PDZ- and one or more LIM domains. PDZ/LIM proteins play important biological roles, and all members have been shown to associate with the actin cytoskeleton. We describe here the splice form specific expression patterns for the three Enigma subfamily members during zebrafish embryogenesis. Whole-mount in situ hybridization revealed common and distinct expression patterns for the different PDZ or LIM domain encoding splice variants. We further studied the role of enigma in zebrafish development. Enigma knockdown appeared to be embryonic lethal shortly after the end of gastrulation and in few surviving embryos led to elongation defects and disorganized somites. In summary, we show here the temporal and spatial expression patterns of the three Enigma family members and their PDZ and LIM domain encoding splice forms during zebrafish embryogenesis. Our results suggest that enigma is important for the formation and organization of somites and might play an important role for actin cytoskeleton organization during development.