Phylogeny of the lizard subfamily Lygosominae (Reptilia: Scincidae), with special reference to the origin of the new world taxa

Phylogenetic relationships of the three lygosomine skink genera occurring both in the Old World and the New World (Mabuya, Scincella and Sphenomorphus) were inferred from mitochondrial DNA sequence of 12S and 16S rRNA genes. Results strongly suggested the non-monophyly for any of these three genera. Within the Mabuya group, Asian members appear to have diverged first, leaving the Neotropical and the Afro-Malagasy Mabuya as sister groups. These relationships, together with the absence of extant or fossil representatives of the Mabuya group from North America, strongly suggest the trans-Atlantic dispersals of Mabuya from Africa to Neotropics. Our results also indicated a closer affinity of the New World Scincella with the New World Sphenomorphus than with the Old World Scincella. Such relationships suggest the trans-Beringian dispersal of the common ancestor from Asia and its subsequent divergence into the North American Scincella and the Neotropical Sphenomorphus.     

Hybrid embryonic stem cell-derived tetraploid mice show apparently normal morphological,physiological, and neurological characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo.     

Bicarbonate actions on flagellar and Ca2+ -channel responses: initial events in sperm activation

At mating, mammalian sperm are diluted in the male and female reproductive fluids, which brings contact with HCO(3)(-) and initiates several cellular responses. We have identified and studied two of the most rapid of these responses. Stop-motion imaging and flagellar waveform analysis show that for mouse epididymal sperm in vitro, the resting flagellar beat frequency is 2-3 Hz at 22-25 degrees C. Local perfusion with HCO(3)(-) produces a robust, reversible acceleration to 7 Hz or more. At 15 mM the action of HCO(3)(-) begins within 5 seconds and is near-maximal by 30 seconds. The half-times of response are 8.8+/-0.2 seconds at 15 mM HCO(3)(-) and 17.5+/-0.4 seconds at 1 mM HCO(3)(-). Removal of external HCO(3)(-) allows a slow return to basal beat frequency over approximately 10 minutes. Increases in beat symmetry accompany the accelerating action of HCO(3)(-). As in our past work, HCO(3)(-) also facilitates opening of voltagegated Ca(2+) channels, increasing the depolarization-evoked rate of rise of intracellular Ca(2+) concentration by more than fivefold. This action also is detectable at 1 mM HCO(3)(-) and occurs with an apparent halftime of approximately 60 seconds at 15 mM HCO(3)(-). The dual actions of HCO(3)(-) respond similarly to pharmacological intervention. Thus, the phosphodiesterase inhibitor IBMX promotes the actions of HCO(3)(-) on flagellar and channel function, and the protein kinase A inhibitor H89 blocks these actions. In addition, a 30 minute incubation with 60 micro M cAMP acetoxylmethyl ester increases flagellar beat frequency to nearly 7 Hz and increases the evoked rates of rise of intracellular Ca(2+) concentration from 17+/-4 to 41+/-6 nM second(-1). However, treatment with several other analogs of cAMP produces only scant evidence of the expected mimicry or blockade of the actions of HCO(3)(-), perhaps as a consequence of limited permeation. Our findings indicate a requirement for cAMP-mediated protein phosphorylation in the enhancement of flagellar and channel functions that HCO(3)(-) produces during sperm activation.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis

Embryogenic and non-embryogenic callus lines derived from the same diploid Cyclamen persicum genotype (`Purple Flamed') were analyzed by flow cytometry and compared to the initial plant material. The DNA content of the diploid plant in the greenhouse was 1.12 pg DNA/2C as estimated in relation to the internal standards tomato nuclei and chicken erythrocytes. In both callus lines the majority of cells contained the same amount of DNA as the initial plant, indicating that no polyploidization has taken place after 5 years of culture on medium containing 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.8 mg/l 6-(γ-γ-dimethylallylamino)purine(zip). Thus, our data suggest that in Cyclamen callus lines there was no strict correlation between the ploidy level and the ability to produce somatic embryos. Furthermore, following the proportion of cells in the three phases of the cell cycle (G0/G1, S, G2/M) during one subculture period of 4 weeks revealed high division activity within the first 2 weeks for both callus lines cultured on the 2,4-D-containing medium. However, when transferred to hormone-free medium, the division activity of the embryogenic cell line decreased markedly, corresponding to the differentiation of somatic embryos. In contrast, for the non-embryogenic callus an increase in cells in the G2/M phase was observed. Received: 22 November 1996 / Revision received: 6 January 1997 / Accepted: 20 February 1997     

Chloride transporter KCC2-dependent neuroprotection depends on the N-terminal protein domain

Neurodegeneration is a serious issue of neurodegenerative diseases including epilepsy. Downregulation of the chloride transporter KCC2 in the epileptic tissue may not only affect regulation of the polarity of GABAergic synaptic transmission but also neuronal survival. Here, we addressed the mechanisms of KCC2-dependent neuroprotection by assessing truncated and mutated KCC2 variants in different neurotoxicity models. The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased chloride transport activity, but they also identify the KCC2 N-terminal domain (NTD) as the relevant minimal KCC2 protein domain that is sufficient for neuroprotection. As ectopic expression of the KCC2-NTD works independently of full-length KCC2-dependent regulation of Cl⁻ transport or structural KCC2 C-terminus-dependent regulation of synaptogenesis, our study may pave the way for a selective neuroprotective therapeutic strategy that will be applicable to a wide range of neurodegenerative diseases.Neurodegeneration restricts neuron numbers during development but can become a serious issue in disease conditions such as temporal lobe epilepsy (TLE).¹ GABA-activated Cl⁻ channels contribute to activity-dependent refinement of neural networks by triggering the so-called giant depolarizing potentials providing developing neurons with a sense of activity essential for neuronal survival and co-regulation of excitatory glutamatergic and (inhibitory) GABAergic synapses.² By regulating transmembrane Cl⁻ gradients KCC2 plays a vital role in development and disease.³ In addition, KCC2 plays a protein structural role in spine formation through its C-terminal protein domain (CTD).^{4, 5} Hence, regulation of KCC2 expression and function is relevant for development and disease-specific plasticity of neural networks.^{6, 7, 8, 9}GlyR α3K RNA editing leads to proline-to-leucine substitution (P185L) in the ligand-binding domain and generates gain-of-function neurotransmitter receptors.^{10, 11, 12, 13} GlyR RNA editing is upregulated in the hippocampus of patients with TLE and leads to GlyR α3K^185L-dependent tonic inhibition of neuronal excitability associated with neurodegeneration.¹⁴ KCC2 expression promotes neuroprotection^{14, 15} but whether this involves regulation of transmembrane Cl⁻ gradient or protein structural role is a matter of debate.^{14, 15}Here, we assessed neuroprotection through several KCC2 variants in two different models of neurodegeneration including chronic neuronal silencing (α3K^185L model) and acute neuronal overexcitation (NMDA model).^{14, 15} The results identify a threonine- and tyrosine-phosphorylation-resistant KCC2 variant with increased Cl⁻ transport activity, but they also demonstrate that the N-terminal KCC2 protein domain (NTD) is sufficient for neuroprotection.