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Biomedical subjects

N Tavernarakis

Publications and source records attributed to N Tavernarakis.

At least 19 recordsLinked to original sources
PubMed2006

Germ line transformation of the olive fly Bactrocera oleae using a versatile transgenesis marker.

The olive fruit fly (olive fly) Bactrocera oleae (Dacus), recently introduced in North America, is the most destructive pest of olives worldwide. The lack of an efficient gene transfer technology for olive fly has hampered molecular analysis, as well as development of genetic techniques for its control. We have developed a Minos-based transposon vector carrying a self-activating cassette which overexpresses the enhanced green fluorescent protein (EGFP). Efficient transposase-mediated integration of one to multiple copies of this vector was achieved in the germ line of B. oleae by coinjecting the vector along with in vitro synthesized Minos transposase mRNA into preblastoderm embryos. The self-activating gene construct combined with transposase mRNA present a system with potential for transgenesis of very diverse species.

Animals↗
PubMed2002

Ageing research in Greece.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece.

Aging↗
PubMed2001

Necrotic cell death in C. elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum.

In C. elegans, a hyperactivated MEC-4(d) ion channel induces necrotic-like neuronal death that is distinct from apoptosis. We report that null mutations in calreticulin suppress both mec-4(d)-induced cell death and the necrotic cell death induced by expression of a constitutively activated Galpha(S) subunit. RNAi-mediated knockdown of calnexin, mutations in the ER Ca(2+) release channels unc-68 (ryanodine receptor) or itr-1 (inositol 1,4,5 triphosphate receptor), and pharmacological manipulations that block ER Ca(2+) release also suppress death. Conversely, thapsigargin-induced ER Ca(2+) release can restore mec-4(d)-induced cell death when calreticulin is absent. We conclude that high [Ca(2+)](i) is a requirement for necrosis in C. elegans and suggest that an essential step in the death mechanism is release of ER-based Ca(2+) stores. ER-driven Ca(2+) release has previously been implicated in mammalian necrosis, suggesting necrotic death mechanisms may be conserved.

Amino Acid Sequence↗
PubMed2001

Mechanotransduction in Caenorhabditis elegans: the role of DEG/ENaC ion channels.

One of the looming mysteries in signal transduction today is the question of how mechanical signals, such as pressure or mechanical force delivered to a cell, are interpreted to direct biological responses. All living organisms, and probably all cells, have the ability to sense and respond to mechanical stimuli. At the single-cell level, mechanical signaling underlies cell-volume control and specialized responses such as the prevention of poly-spermy in fertilization. At the level of the whole organism, mechanotransduction underlies processes as diverse as stretch-activated reflexes in vascular epithelium and smooth muscle; gravitaxis and turgor control in plants; tissue development and morphogenesis; and the senses of touch, hearing, and balance. Intense genetic, molecular, and elecrophysiological studies in organisms ranging from nematodes to mammals have highlighted members of the recently discovered DEG/ENaC family of ion channels as strong candidates for the elusive metazoan mechanotransducer. Here, we discuss the evidence that links DEG/ENaC ion channels to mechanotransduction and review the function of Caenorhabditis elegans members of this family called degenerins and their role in mediating mechanosensitive behaviors in the worm.

Animals↗
PubMed2001

Degenerins. At the core of the metazoan mechanotransducer?

Mechanosensory signaling, believed to be mediated by mechanically gated ion channels, constitutes the basis for the senses of touch and hearing, and contributes fundamentally to the development and homeostasis of all organisms. Despite this profound importance in biology, little is known of the molecular identities or functional requirements of mechanically gated ion channels. Genetic analyses of touch sensation and locomotion in Caenorhabditis elegans have implicated a new class of ion channels, the degenerins (DEG) in nematode mechanotransduction. Related fly and vertebrate proteins, the epithelial sodium channel (ENaC) family, have been implicated in several important processes, including transduction of mechanical stimuli, pain sensation, gametogenesis, sodium reabsorption, and blood pressure regulation. Still-to-be-discovered DEG/ENaC proteins may compose the core of the elusive human mechanotransducer.

Animals↗
PubMed2000

Acquisition of a potential marker for insect transformation: isolation of a novel alcohol dehydrogenase gene from Bactrocera oleae by functional complementation in yeast.

The alcohol dehydrogenase genes make up one of the best studied gene families in Drosophila, both in terms of expression and evolution. Moreover, alcohol dehydrogenase genes constitute potential versatile markers in insect transformation experiments. However, due to their rapid evolution, these genes cannot be cloned from other insect genera by DNA hybridization or PCR-based strategies. We have therefore explored an alternative strategy: cloning by functional complementation of appropriate yeast mutants. Here we report that two alcohol dehydrogenase genes from the medfly Ceratitis capitata can functionally replace the yeast enzymes, even though the medfly and yeast genes have evolved independently, acquiring their enzymatic function convergently. Using this method, we have cloned an alcohol dehydrogenase gene from the olive pest Bactrocera oleae. We conclude that functional complementation in yeast can be used to clone alcohol dehydrogenase genes that are unrelated in sequence to those of yeast, thus providing a powerful tool for isolation of dominant insect transformation marker genes.

Alcohol Dehydrogenase↗
PubMed2000

Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes.

Double-stranded RNA interference (RNAi) is an effective method for disrupting expression of specific genes in Caenorhabditis elegans and other organisms. Applications of this reverse-genetics tool, however, are somewhat restricted in nematodes because introduced dsRNA is not stably inherited. Another difficulty is that RNAi disruption of late-acting genes has been generally less consistent than that of embryonically expressed genes, perhaps because the concentration of dsRNA becomes lower as cellular division proceeds or as developmental time advances. In particular, some neuronally expressed genes appear refractory to dsRNA-mediated interference. We sought to extend the applicability of RNAi by in vivo expression of heritable inverted-repeat (IR) genes. We assayed the efficacy of in vivo-driven RNAi in three situations for which heritable, inducible RNAi would be advantageous: (i) production of large numbers of animals deficient for gene activities required for viability or reproduction; (ii) generation of large populations of phenocopy mutants for biochemical analysis; and (iii) effective gene inactivation in the nervous system. We report that heritable IR genes confer potent and specific gene inactivation for each of these applications. We suggest that a similar strategy might be used to test for dsRNA interference effects in higher organisms in which it is feasible to construct transgenic animals, but impossible to directly or transiently introduce high concentrations of dsRNA.

Animals↗
PubMed2000

Caenorhabditis elegans degenerins and vertebrate ENaC ion channels contain an extracellular domain related to venom neurotoxins.

The DEG/ENaC (DEGenerin/Epithelial Na+ Channel) superfamily includes closely related ion channel subunits from divergent species ranging from the simple nematode Caenorhabditis elegans to humans. Members of this protein group play roles in several important processes including transduction of mechanical stimuli, sodium re-absorption and blood pressure regulation. Structure/function relationships in members of this superfamily are just beginning to be elaborated. Using a bio-informatics approach, we identified a novel structural element in the extracellular region of DEG/ENaC proteins that exhibits significant similarity to venom neurotoxins. Since venom neurotoxins bind to sodium channels at high affinity, we suggest that the related domain embedded in DEG/ENaC channels may interact with other regions of the channel or channel complex to modulate channel function.

Amino Acid Sequence↗
PubMed1999

UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans.

The UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. Here we show that UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes and that this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. We propose that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord.

Amino Acid Sequence↗
PubMed1997

Genetically targeted cell disruption in Caenorhabditis elegans.

The elimination of identified cells is a powerful tool for investigating development and system function. Here we report on genetically mediated cell disruption effected by the toxic Caenorhabditis elegans mec-4(d) allele. We found that ectopic expression of mec-4(d) in the nematode causes dysfunction of a wide range of nerve, muscle, and hypodermal cells. mec-4(d)-mediated toxicity is dependent on the activity of a second gene, mec-6, rendering cell disruption conditionally dependent on genetic background. We describe a set of mec-4(d) vectors that facilitate construction of cell-specific disruption reagents and note that genetic cell disruption can be used for functional analyses of specific neurons or neuronal classes, for confirmation of neuronal circuitry, for generation of nematode populations lacking defined classes of functional cells, and for genetic screens. We suggest that mec-4(d) and/or related genes may be effective general tools for cell inactivation that could be used toward similar purposes in higher organisms.

Animals↗
PubMed1997

The DNA target sequence influences the dependence of the yeast transcriptional activator Gcn4 on co-factors.

The yeast transcriptional activator Gcn4 requires the Ada2/Gcn5/Ada3 co-activator complex to exert part of its activation potential. Here we show that the sequence of the DNA target modulates the function of Gcn4 by modifying this requirement. Promoter configurations were generated that rendered Gcn4-induced transcription either completely dependent or completely independent of the Ada2/Gcn5/Ada3 complex. The topological constraints imposed by these configurations suggest that Gcn4 makes multiple contacts with the basic transcription machinery that are subject to modification by the incident DNA target. We propose that these modifications further determine the direction on the chromosome in which an otherwise symmetric, dimeric transcription factor will activate.

Binding Sites↗
PubMed1997

unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion.

Mechanically gated ion channels are important modulators of coordinated movement, yet little is known of their molecular properties. We report that C. elegans unc-8, originally identified by gain-of-function mutations that induce neuronal swelling and severe uncoordination, encodes a DEG/ENaC family member homologous to subunits of a candidate mechanically gated ion channel. unc-8 is expressed in several sensory neurons, interneurons, and motor neurons. unc-8 null mutants exhibit previously unrecognized but striking defects in the amplitude and wavelength of sinusoidal tracks inscribed as they move through an E. coli lawn. We hypothesize that UNC-8 channels could modulate coordinated movement in response to body stretch. del-1, a second DEG/ENaC family member coexpressed with unc-8 in a subset of motor neurons, might also participate in a channel that contributes to nematode proprioception.

Amino Acid Sequence↗
PubMed1997

Molecular modeling of mechanotransduction in the nematode Caenorhabditis elegans.

Genetic and molecular studies of touch avoidance in the nematode Caenorhabditis elegans have resulted in a molecular model for a mechanotransducing complex. mec-4 and mec-10 encode proteins hypothesized to be subunits of a mechanically gated ion channel that are related to subunits of the vertebrate amiloride-sensitive epithelial Na+ channel. Products of mec-5, a novel collagen, and mec-9, a protein that includes multiple Kunitz-type protease inhibitor repeats and EGF repeats, may interact with the channel in the extracellular matrix. Inside the cell, specialized 15-protofilament microtubules composed of mec-12 alpha-tubulin and mec-7 beta-tubulin may be linked to the mechanosensitive channel by stomatin-homologous MEC-2. MEC-4 and MEC-10 are members of a large family of C. elegans proteins, the degenerins. Two other degenerins, UNC-8 and DEL-1, are candidate components of a stretch-sensitive channel in motor neurons. Implications for advancing understanding of mechanotransduction in other systems are discussed.

Animals↗
PubMed1997

Molecules that mediate touch transduction in the nematode Caenorhabditis elegans.

Despite the widespread importance of mechanotransduction in biology, remarkably little is known about the nature of the molecules that mediate mechanical signaling. Mechanosensation in the nematode Caenorhabditis elegans is mediated by six mechanosensory neurons called touch receptor cells. Genetic analysis has resulted in the identification of over 400 mutations that disrupt the function of the touch receptors. Molecular characterization of the genes revealed has identified subunits of a candidate mechanosensory ion channel, tubulins expressed specifically in the touch receptors, and extracellular matrix proteins needed for mechanotransduction. mec-4 and mec-10 encode members of a C. elegans gene family related to the vertebrate epithelial Na+ channel that are hypothesized to encode subunits of a mechanosensory channel. mec-6 may encode another channel subunit. Inside the cell, alpha-tubulin MEC-12, beta-tubulin MEC-7 and a candidate linker protein MEC-2 may interact with the mechanotransducing channel to deliver gating tension. In the extracelluar matrix, collagen MEC-5 and MEC-9 and MEC-1 may interact with extracellular channel domains. A molecular model for mechanotransduction is discussed.

Animals↗
PubMed1996

Gene overexpression reveals alternative mechanisms that induce GCN4 mRNA translation.

The Saccharomyces cerevisiae GCN4 gene which encodes the transcription activator Gcn4, is under translational regulation. Derepression of GCN4 mRNA translation is mediated by the Gcn2 protein kinase which phosphorylates the alpha subunit of eIF-2, upon amino-acid starvation. Here, we report that overexpression of certain Saccharomyces cerevisiae genes generates intracellular conditions that alleviate the requirement for a functional Gcn2 kinase to induce GCN4 mRNA translation. Our findings, combined with the fact that Gcn2 kinase is dispensable during the initiation phase of the cellular response to amino-acid limitation, provide the grounds to further elucidate the mechanisms underlying the physiology of this homeostatic response.

DNA-Binding Proteins↗