Non-mammalian Systems Research Articles

Neural and molecular mechanisms of microcognition inLimax

Various non-mammalian model systems are being explored in the search for mechanisms of learning and memory storage of sufficient generality to contribute to the understanding of mammalian learning mechanisms. The terrestrial mollusk Limax maximus is one such model system in which mammalian-quality learning has been documented using odors as conditioned stimuli. The Limax odor information-processing circuits incorporate several system design features also found in mammalian odor-processing circuits, such as the use of cellular and network oscillations for making odor computations and the use of nitric oxide to control network oscillations. Learning and memory formation has been localized to a particular central circuit, the procerebral lobe, in which selective gene activation occurs through odor learning. Since the isolated Limax brain can perform odor learning in vitro, the circuits and synapses causally linked to learning and memory formation are assessable for further detailed analysis.

Honoring contributions of Richard A Lockshin to the field of cell death

On 10 December 2007, a scientific symposium was organized at The Rockefeller University by Zahra Zakeri and Hermann Steller to honor Dr. Richard Lockshin for his contributions to the field of cell death. Over 300 scientists attended this meeting. The speakers and chairs included distinguished scientists in the field, from 20 countries, who have contributed to this special issue. Richard Lockshin is one of the pioneers in the field of cell death contributing to the field for over the past 40 years and still remains one of the field’s primary scientists. Rick was born in Columbus, Ohio in December 1937. His fascination and interest in the natural sciences from an early age led him to become a scientist. He obtained his bachelor’s degree at Harvard University (1959), where he continued his doctoral studies with Carroll M Williams and received his PhD in 1963. The topic of his dissertation was published in a series of five landmark papers, beginning with ‘Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths’ coining the term ‘programmed cell death’ in 1964. H Robert Horvitz (one of the recipients of the Nobel Prize in 2002) and Lockshin acknowledged the possibility that cell death may be regulated using nonmammalian model systems such as Caenorhabditis elegans and moths. This idea led to the elucidation of the genetic pathways of programmed cell death and has proven operative in mammals as well and has become a major area of scientific endeavor, as evidenced by the existence of this journal. Cell death is now the focus of much research not only in the area of basic science but also in understanding and controlling many diseases such as cancer, degenerative diseases, infection, and many others. Richard has studied the underlying mechanisms involved in programmed cell death using insects, and more recently, zebrafish, as model systems. He was among the first to report on alternative ways of cell death and the involvement of different proteases in cell killing. This finding now has blossomed into a major area of research in the field of cell death. His current research is a continuation of this in the developing embryo. Collaborating with several individuals, he was among the first to recognize and seek new proteins synthesized during programmed cell death; to study the physiology of dying cells and to question why they shrank; to recognize that the regulation of oncogenes was important in cell death; and to emphasize that not all deaths were by classical apoptosis. He continued to work on cell death and development, with a brief excursion into embryology, where he was among the first to initiate studies on gene activity in early embryos as a postdoctoral fellow at the University of Edinburgh until 1965. Upon his return to the United States, he took the position of Assistant Professor at the University of Rochester School of Medicine and Dentistry. He moved to St. John’s University in 1975 as an associate professor, becoming a professor 1 year later, and served for 9 years as the chair of the department. He has mentored more than 30 graduate students and many undergraduates. He has been and remains a role model and a great help to young researchers and faculty all over the world. He has a tremendous love for teaching and it is this love that has made him the author of a book entitled ‘The Joy of Science’ to enlighten the students about the process of scientific inquiry. For his teaching, he has been honored with the Outstanding Achievement Award, St. John’s University, 1987; the Presidential Award, Long Island University, 1987; and the Outstanding Achievement Award, New York City, 1982. Another dimension of Richard’s contribution to the field is his cofounding of the International Cell Death Society, for which he also served as President from 1998 to 2002. He has received the Society’s award for ‘great contributions to the field’ given to a few others including Drs. John Kerr, Shigekazu Nagata, Peter Krammer, Junying Yuan, and Robert Horvitz. Richard, who is also the founding Chair of the first Gordon Conference on Cell Death, is on the Editorial Boards of Cell Death and Differentiation, Apoptosis, and the Journal of Biological Chemistry. He has served as the organizer and chair of numerous meetings in the field. In collaboration with Dr. Zahra Zakeri, he has established a focus group of researchers that work in enhancing scientific communication and knowledge to the third world countries and less privileged areas by the creation of the organization of Scientists Without Borders (Frontiers), whose aim is to advance teaching and scientific communication. A quotation from Richard Lockshin is the banner for the Scientists Without Borders group, which is as follows: ‘Science should be like a monarch butterfly. It

The evolution of the C‐terminal domain of soluble epoxide hydrolase

The mammalian soluble epoxide hydrolase is a multidomain enzyme composed of N‐ and C‐terminal regions that contain active sites for epoxide hydrolase and phosphatase activities, respectively. The spatial separation and differing homologies of the N‐ and C‐terminal domains have led to the hypothesis that the mammalian enzyme is the result of a gene fusion event between two ancestral genes. Although several plant and mammalian enzymes have been identified, little is known about sEH epoxide hydrolase activity in non‐mammalian animal systems. Analysis of genomic databases identified chicken, nematode and urchin sEH homologs which were subsequently cloned and expressed. The chicken and urchin enzymes contained both the N‐ and C‐terminal domains, while two nematode sEH homologs contained only the C‐terminal domain. All enzymes displayed sEH‐like epoxide hydrolase activity, which was described in terms of the ability to hydrolyze trans‐1,3‐diphenylpropene oxide and fatty acid epoxides such as the epoxyeicosatrienoic acids, and susceptibility to inhibition by urea‐based sEH inhibitors. The identification of sEH homologs with similar substrate selectivity as the mammalian enzyme suggests a conserved role for sEH epoxide hydrolase activity, perhaps involving the hydrolysis of epoxide lipid signaling molecules. This work has been supported by NIEHS Grant R37 ES02710 and NIEHS Grant P42 ES004699.

Telomere length inversely correlates with radiosensitivity in human carcinoma cells with the same tissue background

A relationship between telomeres and radiosensitivity has been established by several studies based on non-mammalian model systems, mouse models, and few human genetic diseases. However, the relationship has not been proven in human carcinoma cells, which have more clinical significance than these other models. The present study aims to determine whether telomere length is related to radiosensitivity in human carcinoma cells, and to examine the influence of tissue or genetic background. Two HEp-2 larynx squamous carcinoma cell lines, eight hepatocellular carcinoma cell lines, and five breast cancer cell lines were used. Telomere length was determined by terminal restriction fragment (TRF) Southern blot analysis and cell survival was measured by a colony-forming assay. Our results indicated that there was a significant negative correlation of telomere length and radiosensitivity in the same tissue-derived cell lines, with or without the same genetic background. Thus, telomere length may be used as a promising tool to predict the radiosensitivity of human carcinomas.

Retinoic acid is a negative regulator of matrix Gla protein gene expression in teleost fish Sparus aurata

Matrix Gla protein (MGP) is an extracellular mineral-binding protein expressed in several tissues while accumulated only in bone and cartilage under physiological conditions. Although the precise molecular mechanism of action of MGP remains unknown, all available evidence indicates that it acts as a physiological inhibitor of mineralization. This work presents the cloning of gilthead seabream MGP gene (SaMGP) and the functional analysis of its promoter. SaMGP gene was found to be organized in five exons and to be under control of a distal and a proximal promoter, both, capable of activating SaMGP transcription in transient transfections. Furthermore, we present strong evidence that retinoic acid down-regulates SaMGP gene transcription by interacting, through binding of its receptor, with a specific region within distal promoter. Interestingly, the presence of repetitive motifs in the proximity of SaMGP gene regulatory regions suggests that they may modulate promoter accessibility to transcription machinery, as already seen for other genes. This work provides additional evidence of the usefulness of non-mammalian model systems to elucidate the complex regulation of MGP gene transcription.

Fishing for an ECG: a student-directed electrocardiographic laboratory using rainbow trout

Cardiac physiology is emphasized in many undergraduate physiology courses, but few nonmammalian vertebrate model systems exist that 1) can be studied fairly noninvasively, 2) are well suited for controlled experimentation, and 3) emphasize principles characteristic of the vertebrate heart. We have developed an inquiry-based undergraduate/graduate-level laboratory in cardiac physiology and electrocardiography using rainbow trout (Oncorhynchus mykiss Walbaum) and the BioPac MP30 data-acquisition system (other fish species and/or electrocardiographic recording devices can be substituted). This laboratory facilitates intensive study of vertebrate electrocardiograms (ECGs) under a variety of environmental and physiological perturbations and is ideal for use in multi-session, inquiry-based laboratory projects in animal physiology. Furthermore, students gain valuable experience in scientific inquiry, study design, following and/or developing scientific protocols, and animal care. This laboratory requires the ability to keep captive fish of at least 100 g and equipment to record ECGs. Departments meeting these requirements can adopt this technique at modest expense. Student enthusiasm and feedback were positive, and several students commented that the nonlethal methods used added to the laboratory's perceived value.

Actions of Epidermal Growth Factor Receptor/Mitogen-Activated Protein Kinase and Protein Kinase C Signaling in Granulosa Cells from Gallus gallus Are Dependent upon Stage of Differentiation1

Studies in both mammalian and nonmammalian ovarian model systems have demonstrated that activation of the mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) signaling pathways modulates steroid biosynthesis during follicle development, yet the collective evidence for facilitory versus inhibitory roles of these pathways is inconsistent. The present studies in the hen ovary describe the changing role of MAPK and PKC signaling in the regulation of steroidogenic acute regulatory protein (STAR) expression and progesterone production in undifferentiated granulosa cells collected from prehierarchal follicles prior to follicle selection versus differentiated granulosa from preovulatory follicles subsequent to selection. Treatment of undifferentiated granulosa cells with a selective epidermal growth factor receptor (EGFR) and ERBB4 receptor tyrosine kinase inhibitor (AG1478) both augments FSH receptor (Fshr) mRNA expression and initiates progesterone production. Conversely, selective inhibitors of both EGFR/ERBB4 and MAPK activity attenuate steroidogenesis in differentiated granulosa cells subsequent to follicle selection. In addition, inhibition of PKC signaling with GF109203X augments FSH-induced Fshr mRNA plus STAR protein expression and initiates progesterone synthesis in undifferentiated granulosa cells, but inhibits both gonadotropin-induced STAR expression and progesterone production in differentiated granulosa. Granulosa cells from the most recently selected (9- to 12-mm) follicle represent a stage of transition as inhibition of MAPK signaling promotes, while inhibition of PKC signaling blocks gonadotropin-induced progesterone production. Collectively, these data describe stage-of-development-related changes in cell signaling whereby the differentiation-inhibiting actions of MAPK and PKC signaling in prehierarchal follicle granulosa cells undergo a transition at the time of follicle selection to become obligatory for gonadotropin-stimulated progesterone production in differentiated granulosa from preovulatory follicles.

Pillar cell and erythrocyte localization of fugu ET A receptor and its implication

Endothelin, a vasoconstrictor peptide, plays important roles not only in the mammalian circulatory system but also in non-mammalian systems, such as the gill lamellar vascular network with complex structural characteristics. Here, we show that (i) the contraction of pillar cells that delimit the lamellar vasculature is controlled by endothelin through the type A endothelin receptor (ET A) linked to the intracellular calcium signaling system and (ii) ET A receptor is also highly expressed on fugu erythrocytes, a hitherto unexpected finding. Database mining revealed the presence of five endothelin receptor (ETR) sequences in the fugu genome. By Northern blotting, cDNA cloning, and fura-2 monitoring, the branchial ETR subtype was shown to be ET A able to induce a Ca 2+ transit. Immunohistochemistry revealed its pillar cell and erythrocyte localization. These results suggest an endothelin/ET A-mediated coordinated regulation of the pillar cell shape and erythrocyte membrane flexibility.

Cardiovascular regeneration in non-mammalian model systems: What are the differences between newts and man?

The mammalian heart cannot regenerate substantial cardiac injuries, while certain non-mammalian vertebrates such as certain fish (Danio rerio) and amphibiae (Notophthalmus viridescens) are able to repair the heart without functional impairment. In mammalians, the prevailing repair process is accompanied by fibrosis and scarring, while zebrafish and newts can replace lost contractile tissue by newly formed cardiac muscle with only little or no scar formation. A better understanding of cardiac regeneration in non-mammalian vertebrates might provide new insights for the manipulation of regenerative pathways in the human heart. Here, we summarize the current knowledge in cardiac regeneration of newts and the principal differences to repair processes in mammalian hearts.

Inhibition of nucleotide excision repair (NER) by microcystin-LR in CHO-K1 cells

Microcystin-LR (MC-LR), a potent inhibitor of PP1 and PP2A protein phosphatases, is related to tumor promotion and initiation. Although the genotoxic properties of this toxin have been extensively investigated with a variety of non-mammalian and mammalian test systems, the existing results are contradictory. Based on our previous results regarding the impact of MC-LR on the processes of DNA repair we decided to examine in greater detail its effect on the capacity of nucleotide excision repair (NER). CHO-K1 cells were pre-treated with increasing doses of MC-LR (1, 10 and 20 μg/ml) and then exposed to UV radiation (25 J/m 2). Apoptosis was analyzed to exclude the possibility of false positive results in the comet assay. The results suggest that MC-LR targets the nucleotide excision repair mechanisms by interference with the incision/excision phase as well as the rejoining phase of NER and leads to an increased level of UV-induced cytogenetic DNA damage in CHO-K1 cells.

Kinesin 5–independent poleward flux of kinetochore microtubules in PtK1 cells

Forces in the spindle that align and segregate chromosomes produce a steady poleward flux of kinetochore microtubules (MTs [kMTs]) in higher eukaryotes. In several nonmammalian systems, flux is driven by the tetrameric kinesin Eg5 (kinesin 5), which slides antiparallel MTs toward their minus ends. However, we find that the inhibition of kinesin 5 in mammalian cultured cells (PtK1) results in only minor reduction in the rate of kMT flux from ∼0.7 to ∼0.5 μm/min, the same rate measured in monopolar spindles that lack antiparallel MTs. These data reveal that the majority of poleward flux of kMTs in these cells is not driven by Eg5. Instead, we favor a polar “pulling-in” mechanism in which a depolymerase localized at kinetochore fiber minus ends makes a major contribution to poleward flux. One candidate, Kif2a (kinesin 13), was detected at minus ends of fluxing kinetochore fibers. Kif2a remains associated with the ends of K fibers upon disruption of the spindle by dynein/dynactin inhibition, and these K fibers flux.

Enhanced DNA Transfer Into Fish Bone Cells Using Polyethylenimine

The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA into a wide range of cell types, including non-mammalian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection.

Developmental hematopoiesis from fly to human

Developmental hematopoiesis is an exciting multi-disciplinary field at the crossroads of developmental biology, hematopoiesis, stem cell biology and genetics. This field began nearly a century ago with detailed observations on the formation of the first blood cells, the primitive erythrocytes, in

BAPTA Induces Frequency Shifts in vivo of Spontaneous Otoacoustic Emissions of the Bobtail Lizard

Spontaneous otoacoustic emissions (SOAEs) are indicators of active processes in the inner ear and are found in all classes of land vertebrates. In the Australian bobtail lizard, earlier work showed that otoacoustic emissions are generated by an active motility process in the hair-cell bundle. This is likely to be driven by calcium-sensitive mechanisms implicated in other non-mammalian hair cell systems. If so, it should be fundamentally influenced by the extracellular calcium concentration. In in vitro studies, the rate of force generation in hair cell stereovilli is linked to the extracellular calcium concentration. In such preparations, low-calcium solutions, buffered by the calcium chelator BAPTA, were reported to change the frequency of hair cell bundle oscillations. In the present study, BAPTA was iontophoresed into the endolymph of the bobtail skink in vivo, and SOAEs were monitored. Application of BAPTA resulted in a prolonged downward shift in the frequency of individual SOAE spectral peaks. Recovery took more than 1 h, consistent with a slow clearance of BAPTA from endolymph. SOAE peak amplitudes were most often enhanced, suggesting there was no functional disruption of tip links. The direction and degree of frequency shifts were consistent with in vitroand in vivo data showing the effects of changing calcium concentrations in the endolymph directly.

Identification of alternative promoter usage for the matrix Gla protein gene

Recent cloning of the Xenopus laevis (Xl) matrix Gla protein (MGP) gene indicated the presence of a conserved overall structure for this gene between mammals and amphibians but identified an additional 5'-exon, not detected in mammals, flanked by a functional, calcium-sensitive promoter, 3042 bp distant from the ATG initiation codon. DNA sequence analysis identified a second TATA-like DNA motif located at the 3' end of intron 1 and adjacent to the ATG-containing second exon. This putative proximal promoter was found to direct transcription of the luciferase reporter gene in the X. laevis A6 cell line, a result confirmed by subsequent deletion mutant analysis. RT-PCR analysis of XlMGP gene expression during early development identified a different temporal expression of the two transcripts, strongly suggesting differential promoter activation under the control of either maternally inherited or developmentally induced regulatory factors. Our results provide further evidence of the usefulness of nonmammalian model systems to elucidate the complex regulation of MGP gene transcription and raise the possibility that a similar mechanism of regulation may also exist in mammals.

Calcium Modulates the Frequency and Amplitude of Spontaneous Otoacoustic Emissions in the Bobtail Skink

Active processes in the inner ear of lizards can be monitored using spontaneous otoacoustic emissions (SOAE) measured outside the eardrum. In the Australian bobtail lizard, SOAE are generated by an active motility process in the hair-cell bundle. This mechanism has been shown to be sensitive to the calcium-chelating agent 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and is presumed to be related to the calcium-sensitive transduction-channel motor implicated in other nonmammalian hair cell systems. In studies of frog saccular and turtle auditory papillar hair cells in vitro, the frequency and amplitude of bundle oscillations depend on the concentration of calcium in the bathing solutions. In the present study, the calcium concentration in the endolymph was changed in vivo in the Australian bobtail lizard Tiliqua rugosa, and SOAE were monitored. Glass pipettes with large tips and containing different calcium concentrations in their fluids were introduced into scala media, and their contents were allowed to passively flow into the endolymph. Low calcium concentrations resulted in a downward shift in the frequency of SOAE spectral peaks and generally an increase in their amplitudes. Calcium concentrations > 2 mM resulted in increases in frequency of SOAE peaks and generally a loss in amplitude. These frequency shifts were consistent with in vitro data on the frequencies and amplitudes of spontaneous oscillation of hair cell bundles and thus also implicate calcium ions in the generation of active motility in nonmammalian hair cells. The data also suggest that in this lizard species, the ionic calcium concentration in the cochlear endolymph is > or = 1 mM.

Apoptotic cell and phagocyte interplay: recognition and consequences in different cell systems

Cell death by apoptosis is characterized by specific biochemical changes, including the exposure of multiple ligands, expected to tag the dying cell for prompt recognition by phagocytes. In non-pathological conditions, an efficient clearance is assured by the redundant interaction between apoptotic cell ligands and multiple receptor molecules present on the engulfing cell surface. This review concentrates on the molecular interactions operating in mammalian and non-mammalian systems for apoptotic cell recognition, as well as on the consequences of their signaling. Furthermore, some cellular models where the exposure of the phosphatidylserine (PS) phospholipid, a classical hallmark of the apoptotic phenotype, is not followed by cell death will be discussed.

Is there a link between telomere maintenance and radiosensitivity?

Several recent studies point to the possibility that telomere maintenance may constitute a potential genetic marker of radiosensitivity. For example, the human diseases ataxia telangiectasia and Nijmegen breakage syndrome, which are characterized by clinical radiosensitivity, show alterations in telomere maintenance. In addition, Fanconi's anemia patients, who are characterized by mild cellular radiosensitivity and in some cases marked clinical radiosensitivity, have altered telomere maintenance. Similarly, a correlation between telomere maintenance and cellular radiosensitivity was reported in a group of breast cancer patients. Another study demonstrated that radiosensitivity may be more pronounced in human fibroblasts with short telomeres than in their counterparts with long telomeres. Several mouse models including mice deficient in Ku, DNA-PKcs (Prkdc), Parp and Atm, all of which are radiosensitive in vivo, show clear telomere alterations. The link between telomere maintenance and radiosensitivity is also apparent in mice genetically engineered to have dysfunctional telomeres. Finally, studies using non-mammalian model systems such as C. elegans and yeast point to the link between radiosensitivity and telomere maintenance. These results warrant further investigation to identify the extent to which these two phenotypes, namely radiosensitivity and telomere maintenance, are linked.

Het up mould unleashes a sporekiller prion.

het-s/het-S incompatibility is one of only a handful of fungal heterokaryon incompatibility systems that have begun to be molecularly analysed (for a review see Saupe 2000). The spore killer phenotype also is one of only a few segregation ratio distortions that has been studied in any detail. Finally the [HET-s] prion is one of only three examples known in non-mammalian systems (for a review see Wickneret al 1999). Clearly thehet-s/het-S story intersects with several exciting areas of research that offer many opportunities for young scientists.

Non-mammalian models for studying neural development and function.

Early neuroscientists scoured the animal kingdom for the ideal preparation with which to study specific problems of interest. Today, non-mammalian nervous systems continue to provide ideal platforms for the study of fundamental problems in neuroscience. Indeed, the peculiarities of body plan and nervous systems that have evolved to carry out precise tasks in unique ecological niches enable investigators not only to pose specific scientific questions, but also to uncover principles that are general to all nervous systems.