Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Molecular cloning, characterization and expression of atpA and atpB genes from Ginkgo biloba

The chloroplast ATP synthase (ATPase) utilizes the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. The chloroplast ATPase α and β subunits are the essential components of multisubunit protein complex. In this paper, the full-length cDNA and genomic DNA of ATPase α (designated as GbatpA) and β (designated as GbatpB) subunit genes were isolated from Ginkgo biloba. The GbatpA and GbatpB genes were both intronless. The coding regions of GbatpA and GbatpB were 1530 bp and 1497 bp long, respectively, and their deduced amino acid sequences showed high degrees of identity to those of other plant ATPase α and β proteins, respectively. The expression analysis by RT-PCR revealed that GbatpA and GbatpB both expressed in tissue-specific manners in G. biloba and might involve in leaf development. The recombinant GbATPB protein was successfully expressed in E. coli strain using pET28a vector with ATPase activity as three times high as the control, and the results showed that the molecular weight of the recombinant protein was about 54 kDa, a size that was in agreement with that predicted by bioinformatics analysis. This study provides useful information for further studying on overall structure, function and regulation of the chloroplast ATPase in G. biloba, the so-called “living fossil” plant as one of the oldest gymnosperm species. These authors contributed equally to this work     

A novel role for the mating type (MAT) locus in the maintenance of cell wall integrity in Saccharomyces cerevisiae.

The cell wall and stress response component (Wsc) protein family in the yeast Saccharomyces cerevisiae is encoded by at least three genes, WSC1, WSC2, and WSC3. The Wsc proteins are putative upstream activators of the RHO1-regulated PKC1-MAP kinase cascade, and are required for maintenance of cell wall integrity and the stress response. Deletion of WSC1 causes a cell lysis defect that is exacerbated by deleting WSC2 or WSC3. This cell lysis defect can be rescued by adding osmotic stabilizers, such as 1 M sorbitol, to the medium, and by overexpressing PKC1 or RHO1. To advance our understanding of the function of the WSC genes, we performed a genetic screen to identify other components of the pathways they regulate. Here we report our findings. MAT a 1 and MATα2 were identified as dosage-dependent suppressors of the lysis defect of a wscΔ mutant. Overexpression of MAT a 1 or MATα2 was found to suppress the heat shock sensitivity, in addition to the lysis defect, of the wscΔ mutant. Phenotypic suppression by these two genes, MAT a 1 and MATα2, is significantly stronger when they are overexpressed in cells of the opposite mating type. Deletion of MAT a 1 exacerbates the lysis defect of haploid and diploid wscΔ strains. Our results suggest that the MAT locus plays a role in responses similar to those regulated by WSC and provide evidence for a regulatory effect of the MAT locus outside the realm of cell type determination. Received: 24 September 1998 / Accepted: 22 February 1999     

Subunit Regulation of the Human Brain α1E Calcium Channel

The α₁ subunit coding for the human brain type E calcium channel (Schneider et al., 1994) was expressed in Xenopus oocytes in the absence, and in combination with auxiliary α₂δ and β subunits. α_1E channels directed with the expression of Ba²⁺ whole-cell currents that completely inactivated after a 2-sec membrane pulse. Coexpression of α_1E with α_2bδ shifted the peak current by +10 mV but had no significant effect on whole-cell current inactivation. Coexpression of α_1E with β_2a shifted the peak current relationship by −10 mV, and strongly reduced Ba²⁺ current inactivation. This slower rate of inactivation explains that a sizable fraction (40 ± 10%, n= 8) of the Ba²⁺ current failed to inactivate completely after a 5-sec prepulse. Coinjection with both the cardiac/brain β_2a and the neuronal α_2bδ subunits increased by ≈10-fold whole-cell Ba²⁺ currents although coinjection with either β_2a or α_2bδ alone failed to significantly increase α_1E peak currents. Coexpression with β_2a and α_2bδ yielded Ba²⁺ currents with inactivation kinetics similar to the β_2a induced currents, indicating that the neuronal α_2bδ subunit has little effect on α_1E inactivation kinetics. The subunit specificity of the changes in current properties were analyzed for all four β subunit genes. The slower inactivation was unique to α_1E/β_2a currents. Coexpression with β_1a, β_1b, β₃, and β₄, yielded faster-inactivating Ba²⁺ currents than currents recorded from the α_1E subunit alone. Furthermore, α_1E/α_2bδ/β_1a; α_1E/α_2bδ/β_1b; α_1E/α_2bδ/β₃; α_1E/α_2bδ/β₄ channels elicited whole-cell currents with steady-state inactivation curves shifted in the hyperpolarized direction. The β subunit-induced changes in the properties of α_1E channel were comparable to modulation effects reported for α_1C and α_1A channels with β₃≈β_1b > β_1a≈β₄≫β_2a inducing fastest to slowest rate of whole-cell inactivation. Received: 27 March 1997/Revised: 10 July 1997     

Minichromosome stability induced by partial genome duplication in Arabidopsis thaliana

Two partially reconstructed karyotypes (RK1 and RK2) of Arabidopsis thaliana have been established from a transformant, in which four structurally changed chromosomes (α, β, γ, and δ) were involved. Both karyotypes are composed of 12 chromosomes, 2n = 1￠￠+ 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ g￠￠ = 12 {2}n = {1}\prime \prime + {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \gamma \prime \prime = {12} for RK1 and 2n = 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ b￠￠+ g￠￠ = 12 {2}n = {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \beta \prime \prime + \gamma \prime \prime = {12} for RK2, and these chromosome constitutions were relatively stable at least for three generations. Pairing at meiosis was limited to the homologues (1, 3, 4, 5, α, β, or γ), and no pairing occurred among non-homologous chromosomes in both karyotypes. For minichromosome α (mini α), precocious separation at metaphase I was frequently observed in RK2, as found for other minichromosomes, but was rare in RK1. This stable paring of mini α was possibly caused by duplication of the terminal tip of chromosome 1 that is characteristic of RK1.     

Tissue-specific expression of hormonal carcinogenesis target genes in rats treated with polycyclic aromatic hydrocarbons

We have investigated the effect of polycyclic aromatic hydrocarbons (PAHs) on expression of the estrogen-metabolizing genes CYP1A1, CYP1B1, CYP19 and also ERα, and cyclinD1 genes, regulating cell division in estrogen-depended tissues. Treatment of rats with benzo(a)pyrene (BP) or 3-methylcholantrene (MCA) significantly up-regulated CYP1A1, CYP1B1 gene expression in liver, uterus and ovary, whereas α-naphthoflavone (α-NF) did not have any effect. The high level of aromatase gene (CYP19) expression was detected in ovary only. Treatment of rats with BP or MCA significantly down-regulated expression of this gene (15- and 5,5-fold, respectively), whereas α-NF was ineffective. Administration of BP but not MCA or α-NF increased ERα and cyclinD1 gene expression in rat liver. The levels of ERα and cyclinD1 mRNA levels remained unchanged in uterus of after treatment of rats with these PAHs. BP administration increased ERα and cyclinD1 mRNA levels (3,5- and 2,5-fold, respectively) in ovary, while MCA and α-NF were ineffective. Thus, our results give evidence for tissue-specific effects of PAHs on expression of genes, which participate in hormonal carcinogenesis. On the other hand, the fact that BP and MCA treatments influenced the expression of estrogen-metabolizing genes and genes, which control cell division, supports the viewpoint that PAHs may be one of the causes of endocrine disorders and subsequent hormonal carcinogenesis.     

Potential for evolutionary change in the seasonal timing of germination in sea beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> ssp. <Emphasis Type="Italic">maritima</Emphasis>) mediated by seed dormancy

In sea beet (Beta vulgaris ssp. maritima), germination occurs in autumn or spring and is mediated by dormancy which can be released by cold or dry periods. Environmental change such as current climate change may require evolutionary response in seasonal timing. Here, we explore the potential for such evolutionary change. Seed dormancy was studied in a composite population based on seeds from all over the species range in France together with several generations of reciprocal crosses. We found high, repeatable variability for dormancy rate among individuals under greenhouse conditions and confirmed its relevance for germination phenology in the field. Our data fitted best with an exclusively maternal determination of the dormancy phenotype. Narrow-sense heritability, h2 ≈ 0.5 in the composite population and ≈0.4 in the original local populations, was such that rapid evolutionary change in the relative proportions of autumn and spring germination may be possible.     

Stability of the recombinant plasmid carrying theBacillus amyloliquefaciens α-amylase gene inB. subtilis

Summary Theα-amylase gene ofBacillus amyloliquefaciens has previously been cloned into pUB110 to give the recombinant plasmid, pKTH10 (Palva 1982. Gene 19:81–87). Strains transformed by this plasmid are promising candidates for industrialα-amylase production. The stability of pKTH10 was determined in variousB. subtilis strains possessing specific alleles which affect the level ofα-amylase secretion.B. subtilis strains carrying pKTH10 were cultivated in starch-containing medium for up to 50 generations without antibiotic selection and then screened for the presence of pKTH10. The plasmid proved stable enough (< 1.0% cured after 50 generations) for industrial batchwise enzyme production in two strains, but in asacU9 strain (thesacU9 mutation increases concominantly the production ofα-amylase levansucrase and proteases) 99.9% of cells had lost pKTH10 after 50 generations, although the parental plasmid (pUB110) was stable in this strain (0.09% cured after 50 generations). The instability of pKTH10 in thesacU9 strain seems somehow to be related to high expression of the clonedα-amylase gene: when grown in a medium restrictingα-amylase production, only 0.53% ofsacU9 cells had lost pKTH10 after 50 generations.     

Alpha and beta subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ⁻) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ⁻ and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ɛ subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ⁻ or ρ^o mutations in S. cerevisiae. Received: 20 March 1999 / Accepted: 18 July 1999