Signal transduction mediated by antigen receptors on teleost lymphocytes

To generate an adaptive response from the mammalian immune system requires that antigen bind to cognate receptors on T and B cells, a process which activates intracellular signaling pathways. Crosslinking the B cell antigen receptor (BCR) ultimately activates cell proliferation in both higher and lower vertebrates. Recent studies suggest that many functional components of these intracellular pathways were evolutionarily conserved among the vertebrates. Antibody-mediated crosslinking of surface immunoglobulin leads to tyrosine phosphorylation on presumptive accessory molecules of the teleost BCR as well as several intracellular proteins. Crosslinking the teleost BCR also triggers calcium influx and activation of protein kinase C (PKC) which are hallmark components of the phosphatidyl inositol signal transduction pathway in mammalian lymphocytes. The activation of teleost PKC ultimately generates dually-phosphorylated forms of mitogen activated protein kinase. The latter enzyme is viewed as a key cytoplasmic control point for integrating signals arriving from several kinase/phosphatase pathways in mammalian cells. Preliminary evidence suggests that intracellular signaling mediated through antigen receptor complexes may be very sensitive to external factors, including heavy metals such as mercuric chloride which can alter calcium flux and tyrosine phosphorylation patterns in teleost leukocytes. As the process of lymphocyte activation in teleost fish is better understood, it may be possible to provide aquaculturists, environmental regulators and fisheries managers with better information on those natural and man-made conditions which interfere with the development of protective immune responses in natural and captive finfish populations.     

Reproductive Endocrinology of Fishes

While evidence is currently lacking for the agnatha and elasmobranchs,the release of pituitary gonadotropin by the teleost pituitaryappears to be under stimulatory control by the hypothalamus. Gonadotropin has to date only been purified from teleost pituitaryglands. Bioassay and biochemical data suggest that the teleostpituitary gland elaborates only one gonadotropin; however, thereis some conflicting histological data on this point. Salmongonadotropin has a molecular weight of approximately 29,000at neutral pH and approximately 13,000 at low pH or after treatmentwith 8M urea or 1M propionic acid. Radioimmunossays have recently been developed for carp and salmongonadotropin. Immunological techniques have also been used toidentify pituitary gonadotrops. Pharmacological treatment of fish with methallibure has permittedinhibition of gonadal development while treatment with clomiphenecitrate has stimulated ovulation. The role of corticosteroidsand other steroid hormones in ovulation is still not fully elucidated.It is possible that the control of ovulation may differ betweenspecies. Experiments are described which aim to enhance natural stocksof pink salmon by endocrine manipulation of sexual developmentin the male.     

Molecular cloning of cellular apoptosis susceptibility (CAS) gene in Oreochromis niloticus and its proposed role in regulation of non-specific cytotoxic cell (NCC) functions

Cellular apoptosis susceptibility (CAS) gene is a homologue of the chromosome segregation gene (CSE) in yeast, involved in multiple cellular mechanisms associated with cell proliferation as well as cell death. CAS is highly expressed in proliferating cells but at a lower level in quiescent cells and tissues. Therefore it appears that CAS may play an important role in cancer development. We have previously identified CAS in tilapia non-specific cytotoxic cells (NCC) with a cross-reacting monoclonal antibody. Its expression was up-regulated in NCC in response to apoptosis regulatory factors. In the present report, the molecular cloning and expression of CAS in NCC is described, suggesting the importance of this protein in regulation of teleost immune functions. Furthermore, CAS expression is proposed as one of the mechanisms of regulation of activation induced programmed cell death (AIPCD) in these cytotoxic cells. As CAS expression is ubiquitous, we expect that these studies will help identify proliferating cells protected from apoptosis in additional tissues.     

Molecular and cellular mechanisms in the viral exacerbation of asthma

The aetiology of asthma associated with viral infection is complex. The dynamics that contribute to disease pathogenesis are multifactorial and involve overlapping molecular and cellular mechanisms, particularly the immune response to respiratory virus infection or allergen sensitization. This review summarizes the evidence associated with factors that may contribute to the development or exacerbation of asthma including age, host factors, genetic polymorphisms, altered immune responses, and aspects of viral antigen expression. This review also provides an important perspective of key events linked to the development of asthmatic disease and related pulmonary inflammation from human and animal studies, and discusses their relationship as targets for disease intervention strategies.     

Defining the molecular nexus of cancer, type 2 diabetes and cardiovascular disease

The metabolic syndrome is characterized by a state of metabolic dysfunction resulting in the development of several chronic diseases that are potentially deadly. These metabolic deregulations are complex and intertwined and it has been observed that many of the mechanisms and pathways responsible for diseases characterizing the metabolic syndrome such as type 2 diabetes and cardiovascular disease are linked with cancer development as well. Identification of molecular pathways common to these diverse diseases may prove to be a critical factor in disease prevention and development of potential targets for therapeutic treatments. This review focuses on several molecular pathways, including AMPK, PPARs and FASN that interconnect cancer development, type 2 diabetes and cardiovascular disease. AMPK, PPARs and FASN are crucial regulators involved in the maintenance of key metabolic processes necessary for proper homeostasis. It is critical to recognize and identify common pathways deregulated in interrelated diseases as it may provide further information and a much more global picture in regards to disease development and prevention. Thus, this review focuses on three key metabolic regulators, AMPK, PPARs and FASN, that may potentially serve as therapeutic targets.     

A comprehensive approach for drug safety assessment

A comprehensive, multidisciplinary approach is proposed here for the development of a drug with an acceptable safety profile. Key parameters to be considered for drug safety evaluation based on this comprehensive approach include the following: (1) Pharmacology: Possible toxicity due to drug-target interactions, including interactions with unintended molecular targets, or with molecular targets in unintended organs. (2) Chemistry: Chemical scaffolding and side-chains with safety concerns. (3) Toxicology: Toxicity in animals in vivo, and in relevant animal and human cells in culture. (4) Drug metabolism and pharmacokinetics: Safety concerns due to toxification or detoxification, organ distribution, clearance and pharmacokinetic drug-drug interactions. (5) Risk factors: Physiological, environmental and genetic factors that may enhance a patient's susceptibility. It is proposed that this integrated, multidisciplinary approach to safety evaluation may enhance the accuracy of the prediction of drug safety and thereby the efficiency of drug development.     

The medaka rs-3 locus required for scale development encodes ectodysplasin-A receptor

The bodies of most teleost fish species are covered with specialized subepithelial structures known as scales. The scale is an epithelial appendage that differentiates from the dermal mesenchyme. Mammals, on the other hand, have no scales, but instead their bodies are covered with hair. Although their appearances are quite different, scales and hair can be considered structurally similar in that both of them are epithelial appendages distributed over the body surface in an orderly pattern. This analogy suggests that they may have the same evolutionary origin. But, to date, no molecular evidence has been presented that links scales and hair. A mutation at the rs-3 locus of medaka (Oryzias latipes) leads to almost complete loss of scales. We demonstrated that the rs-3 locus encodes ectodysplasin-A receptor (EDAR), which is required for the initiation of hair development in mammals. We identified a novel transposon inserted in the first intron of EDAR, which causes aberrant splicing. This work shows that EDAR is required for scale development in fish and suggests that it is an evolutionarily conserved molecule that is required for the development of epithelial appendages in vertebrates.     

Molecular targets that link dioxin exposure to toxicity phenotypes

Many toxicology studies have elucidated health effects associated with exposure to various chemicals, but few have identified the molecular targets that cause specific endpoints of toxicity. Our understanding of the toxicity of dioxins, a group of chemicals capable of causing toxicity at environmentally relevant levels of exposure, is no exception. Dioxins are unique compared to most chemicals that we are exposed to in the environment because they activate a high affinity receptor, aryl hydrocarbon receptor (AhR), that was identified more than three decades ago. In recent years, several lines of experimental evidence have provided clues for opening the "black box" that contains the molecular mechanisms of dioxin action. These clues have emerged by toxicologists beginning to identify the molecular targets that link AhR signaling to tissue-specific toxicity phenotypes. Endpoints of dioxin toxicity for which downstream molecular targets have begun to be elucidated are observed in developmental or tissue regeneration processes, and include impaired prostate development and hydronephrosis in mouse fetuses and pups, reduced midbrain blood flow and jaw malformation in zebrafish embryos, and impaired fin regeneration in larval and adult zebrafish. Significant progress in identifying molecular targets for dioxin-induced hepatotoxicity in adult mice also has occurred. Misregulation of AhR downstream pathways, such as conversion of arachidonic acid to prostanoids via cyclooxygenase-2, and altered Wnt/β-catenin signaling downregulating Sox9, and signaling by receptors for inflammatory cytokines have been implicated in tissue-specific endpoints of dioxin toxicity. These findings may not only begin to clarify the molecular targets of dioxin action but shed light on new molecular events associated with development and disease.     

Role of apoptosis in congenital hematologic disorders and bone marrow failure

Apoptosis, the cell's intrinsic death program, plays a critical role in the regulation of tissue homeostasis, especially in cell systems with a high turnover rate such as hematopoiesis. Imbalances between survival, proliferation and death of precursor cells or mature cells may result in accelerated loss or impaired output or uncontrolled polyclonal or monoclonal expansion and may pave the way to the development of leukemia. Congenital hematologic disorders are characterized by disturbed growth control of hematopoietic cells. In the previous years, it has become clear that deregulated apoptosis contributes or is even a key determinator of the pathophysiology of diseases such as lymphoproliferation, aplastic anemia or chronic neutropenia. Hematopoietic growth factors have been shown not only to stimulate proliferation of hematopoietic stem cells and committed precursor cells, but also to act as survival factors protecting developing precursor cells from apoptotic signals. The molecular delineation of pathways of apoptosis signaling or survival in hematopoietic cells is expected to provide tools for molecular understanding of the pathophysiology of congenital and acquired hematopoietic disorders and to identify targets for therapeutic intervention strategies.     

Towards a molecular explanation of the high performance of the tuna heart

The tuna heart is capable of sustaining cardiac outputs that are high relative to other active teleost species. The question as to whether there are known molecular mechanisms to explain this phenomenon is examined. Unfortunately, the evidence at present is scant but the paper attempts to put existing knowledge into a contextual framework. It is known that the high cardiac outputs in tuna are due to the maximum heart rates that are high relative to other active teleost species. While the normalized stroke volume (ml/kg body weight) in tuna is in the same range as that of trout several important points are worth noting. The stroke volume in the tuna heart is generated in the face of an afterload that is two-fold higher (due to high ventral aortic pressure) than that observed in the trout or other teleosts. Since the stroke volume is predicated on the strength of ventricular contraction, the question then arises as to whether this is a consequence of factors intrinsic or extrinsic to the design of the cardiomyocytes that make up the myocardium. Two important extrinsic factors must be noted: the substantially higher normal operating temperatures and the apparent cardiac hyperplasia in the tuna. While the merit of these factors is discussed, the paper focuses on intrinsic factors. There is very little that has been determined to date that would indicate substantive changes in the design of the myocyte in the tuna heart compared to that of trout. Are these extrinsic factors alone then sufficient to account for the impressive cardiac output capabilities in the tuna?     

Recent findings on the structure and function of teleost IgT

As key effector molecules of jawed vertebrate’s adaptive immune system, immunoglobulins are produced by B lymphocytes, either as a secretory form (antibody) or as a membrane form (B cell receptor). Until recently, teleost fish B cells were thought to express only two classes of immunoglobulins, IgM and IgD. In addition, IgM in these species was thought to be the only immunoglobulin isotype responding to pathogens both in systemic or mucosal compartments. However, the unexpected discovery of IgT, a new teleost immunoglobulin unearthed in 2005, has provided for new opportunities to analyze further roles of teleost immunoglobulins in these two physiologically distinct compartments. The smoke about the potential function of IgT has cleared recently with the finding that this immunoglobulin appears to be specialized in gut mucosal immunity. Significantly, the new capability of measuring not only IgM but also IgT responses will greatly facilitate the evaluation and understanding of fish immune responses as well as the protective effects of fish vaccines. The purpose of this review is to summarize the molecular characterization of new IgT orthologs and subtypes in teleosts, as well as to describe the new findings concerning the protein structure of IgT, the B cells producing it, and its role in mucosal immunity.     

Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish

Evaluating the biological relevance of the myriad putative regulatory noncoding sequences in vertebrate genomes represents a huge challenge. Functional analyses in vivo have typically relied on costly and labor-intensive transgenic strategies in mice. Transgenesis has also been applied in nonrodent vertebrates, such as zebrafish, but until recently these efforts have been hampered by significant mosaicism and poor rates of germline transmission. We have developed a transgenic strategy in zebrafish based on the Tol2 transposon, a mobile element that was recently identified in another teleost, Medaka. This method takes advantage of the increased efficiency of genome integration that is afforded by this intact DNA transposon, activity that is mediated by the corresponding transposase protein. The approach described in this protocol uses a universal vector system that permits rapid incorporation of DNA that is tagged with sequence targets for site-specific recombination. To evaluate the regulatory potential of a candidate sequence, the desired interval is PCR-amplified using sequence-specific primers that are flanked by the requisite target sites for cloning, and recombined into a universal expression plasmid (pGW_cfosEGFP). Purified recombinant DNAs are then injected into 1-2-cell zebrafish embryos and the resulting reporter expression patterns are analyzed at desired timepoints during development. This system is amenable to large-scale application, facilitating rapid functional analysis of noncoding sequences from both mammalian and teleost species.     

Targeting signal transduction pathways by chemopreventive agents

Cancer is a dynamic process that involves many complex factors, which may explain why a "magic bullet" cure for cancer has not been found. Death rates are still rising for many types of cancers, which possibly contributes to the increased interest in chemoprevention as an alternative approach to the control of cancer. This strategy for cancer control is based on the presumption that because cancer develops through a multi-step process, each step may be a prospective target for reversing or suppressing the process. Thus, the design and development of chemopreventive agents that act on specific and/or multiple molecular and cellular targets is gaining support as a rational approach to control cancer. Nutritional or dietary factors have attracted a great deal of interest because of their perceived ability to act as highly effective chemopreventive agents. They are professed as being generally safe and may have efficacy as chemopreventive agents by preventing or reversing premalignant lesions and/or reducing second primary tumor incidence. Many of these dietary compounds appear to act on multiple target signaling pathways. Some of the most interesting and well documented are resveratrol and components of tea, including EGCG, theaflavins and caffeine. This review will focus on recent work regarding three well-accepted cellular/molecular mechanisms that may at least partially explain the effectiveness of selected food factors, including those indicated above, as chemopreventive anti-promotion agents. These food compounds may act by: (1) inducing apoptosis in cancer cells; (2) inhibiting neoplastic transformation through the inhibition of AP-1 and/or NF-kappaB activation; and/or (3) suppressing COX-2 overexpression in cancer cells.