Synthesis, crystal structure, magnetic properties and electrochemical behaviour of the mixed valence compound [Cu(CN)3Cu(C3H10N2)2]

The binuclear mixed valence copper(I/II) compound [Cu^I(CN)₃Cu^II(tn)₂] (1) (tn = propane-1,3-diamine) and its acetonitrile adduct [Cu^I(CN)₃Cu^II(tn)₂] · 2MeCN (2) have been synthesized. Complex 1 crystallizes triclinic, space group , a = 8.117(2) Å, b = 8.389(2) Å, c = 11.920(2) Å, α = 108.728(3)°, β = 100.024(3)°, γ = 104.888(4)°, Z = 2, and compound 2 monoclinic, space group P2₁/m, a = 8.752(2) Å, b = 13.243(3) Å, c = 9.549(2) Å, β = 114.678(4)°, Z = 2. In both crystal structures, the binuclear [Cu^I(CN)₃Cu^II(tn)₂] complex with slightly different bonding geometries is formed. One of the three nitrogen atoms of a Cu^I(CN)₃ moiety is coordinated to Cu(II) at the apex of a square-pyramid with two chelating ligands tn on its base. The shortest intramolecular Cu^II?Cu^II distance in 1 is 5.640(7) Å. The EPR behaviour of 1 has been investigated at room temperature and at 77 K. The magnetic properties were measured in the temperature range 1.8-300 K.     

Potentiometric sensor for the measurement of Cd2+ transport in yeast and plants

Research on heavy metals, and especially on transport of Cd(2+), has attracted much interest during the past decade. An optimized Cd(2+)-selective electrode for the continuous potentiometric monitoring of Cd(2+) fluxes in biological systems is presented. The selectivity of the electrode for Cd(2+) was further improved, and it now has very good long-term stability. The utility of this simple and inexpensive method is demonstrated by studying the Cd(2+) transport with model organisms, such as the yeast Saccharomyces cerevisiae and Arabidopsis cell cultures, frequently used in plant science. Its lower detection limits in the presence of commonly used growth media for yeast and plant cells are improved by approximately three orders of magnitude and are 10(-10) and 10(-8)M Cd(2+), respectively. Control experiments using atomic absorption spectrophotometry confirm that the decrease in Cd(2+) activities in the cell cultures is indeed due to the uptake of these metal ions by the cells. Both model systems can be easily transformed; therefore, in combination with the new electrode, they are very promising tools for the investigation of any protein of interest that might be involved in Cd(2+) transport.     

Moderate G6PD deficiency increases mutation rates in the brain of mice

Mice that harbored the x-ray-induced low efficiency allele of the major X-linked isozyme of glucose-6-phospate dehydrogenase (G6PD), Gpdx(a-m2Neu), and, in addition, harbored the transgenic shuttle vector for the determination of mutagenesis in vivo, pUR288, were employed to further our understanding of the interdependence of general metabolism, oxidative stress control, and somatic mutagenesis. The Gpdx(a-m2Neu) mutation conferred moderate G6PD deficiency in hemizygous males (Gpdx(a-m2Neu/y)) displaying residual enzyme activities of 27% in red blood cells and 13% in brain (compared to wild-type controls, Gpdx(a/y) males). In spite of this mild phenotype, the brains of G6PD-deficient males exhibited a significant distortion of redox control ( approximately 3-fold decrease in the ratio of reduced glutathione to oxidized glutathione), a considerable accumulation of promutagenic etheno DNA adducts ( approximately 13-fold increase in ethenodeoxyadenosine and approximately 5-fold increase in ethenodeoxycytidine), and a substantial elevation of somatic mutation rates ( approximately 3-fold increase in mutant frequencies in lacZ, the target and reporter gene of mutagenesis in the shuttle vector, pUR288). The mutation pattern in the brain was dominated by illegitimate genetic recombinations, a presumed hallmark of oxidative mutagenesis. These findings suggested a critical function for G6PD in limiting oxidative mutagenesis in the mouse brain.     

Tpi-1 and Gapd are linked very closely on mouse chromosome 6

Mutations in the structural genes for triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase activity in the mouse, selected after mutagen treatment, were used to estimate the map distance between the two loci. It is shown that Tpi-1 and Gapd are closely linked on chromosome 6, with a recombination frequency of 0.1 +/- 0.1%.     

Glucose phosphate isomerase enzyme-activity mutants inMus musculus: Genetical and biochemical characterization

Two glucose-6-phosphate isomerase (GPI) mutants with approximately 60% residual activity in blood compared to wild type have been independently detected in offspring derived from 1-ethyl-1-nitrosourea-treated male mice. Homozygous mutants with about 20% residual activity were recovered in progeny of inter se matings of heterozygotes. However, in both mutant lines the number of homozygous mutants was less than expected suggesting an increased lethality of these animals. Results of linkage studies and of investigations of physicochemical properties of the mutant enzymes indicate point mutations at theGpi-1s structural locus on chromosome 7. Based on these findings the two new alleles were designatedGpi-1s ^b-m1Neu andGpi-1s ^b-m2Neu, respectively. The b-m1Neu allele codes for an erythrocyte enzyme which, in the homodimeric form, exhibits a decreased stability toward heat and urea, an altered isoelectric point, normalpH dependence, an increasedK _m for fructose-6-phosphate, and increasedK _i's for 6-phosphogluconate and 2,3-diphosphoglycerate (2,3-DPG) compared to the wild-type enzyme. The GPI-1s^b-m2Neu homodimer, in contrast, is characterized by an even stronger instability, slightly alteredpH dependence, an increasedK _i for 2,3-DPG, normal other kinetics, and normal isoelectric point. The different degree of stability of the mutant homodimersin vitro seems to be reflected in a different degree of stabilityin vivo, since GPI deficiency in general is more strongly expressed in the tissues of the homozygousGpi-1s ^b-m2Neu mutant compared to the homozygousGpi-1s ^b-m1Neu mutant. The similarity of the mutant enzymes to the allozymes found in human GPI deficiencies indicates the GPI deficient mouse mutants to be excellent models for the human disease.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.     

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.     

A Mutation Affecting the Lactate Dehydrogenase Locus Ldh-1 in the Mouse. II. Mechanism of the Ldh-a Deficiency Associated with Hemolytic Anemia

A procarbazine hydrochloride-induced mutation at the Ldh-1 structural locus encoding the A subunit of lactate dehydrogenase (LDH) was used to study the molecular and metabolic basis of severe hemolytic anemia due to LDH-A deficiency in the mouse. The mutant allele designated Ldh-1(a-m1Neu) codes for an enzyme that as homotetramer differs from the wild-type enzyme by a marked instability, acidic shift of the pH profile, increased K(m) for pyruvate and altered inhibition by high concentrations of this substrate. Except for the latter, all these altered properties of the mutant protein contribute to the diminished LDH activity in heterozygous and homozygous mutant individuals. Impaired energy metabolism of erythrocytes indicated by a relatively low ATP concentration is suggested to result in cell death at the end of the reticulocyte stage leading to the expression of hemolytic anemia with extreme reticulocytosis and hyperbilirubinemia. Despite the severe anemia, affected homozygous mutants exhibit approximately normal body weight and do not show noticeable impairment of viability or fertility. To date no such condition is observed in man. This discrepancy is likely due to the fact that in human erythrocytes both LDH-A and LDH-B subunits are expressed such that homozygotes for a LDH-A or LDH-B deficiency would not result in a comparably extreme LDH activity deficiency.     

Genetic localization and phenotypic expression of X-linked cataract (Xcat) in Mus musculus

Linkage data relative to the markers tabby and glucose-6-phosphate dehydrogenase are presented to locate X-linked cataract (Xcat) in the distal portion of the mouse X-chromosome between jimpy and hypophosphatemia. The human X-linked cataract-dental syndrome, Nance-Horan Syndrome, also maps closely to human hypophosphatemia and would suggest homology between mouse Xcat and human Nance-Horan Syndrome genes. In hemizygous males and homozygous females penetrance is complete with only slight variation in the degree of expression. Phenotypic expression in Xcat heterozygous females ranges from totally clear to totally opaque lenses. The phenotypic expression between the two lenses of a heterozygous individual could also vary between totally clear and totally opaque lenses. However, a correlation in the degree of expression between the eyes of an individual was observed. A variegated pattern of lens opacity was evident in female heterozygotes. Based on these observations, the site of gene action for the Xcat locus is suggested to be endogenous to the lens cells and the precursor cell population of the lens is concluded to be small. The identification of an X-linked cataract locus is an important contribution to the estimate of the number of mutable loci resulting in cataract, an estimate required so that dominant cataract mutagenesis results may be expressed on a per locus basis. The Xcat mutation may be a useful marker for a distal region of the mouse X-chromosome which is relatively sparsely marked and the X-linked cataract mutation may be employed in gene expression and lens development studies.     

Induction of gene mutations in mice: The multiple endpoint approach

The multiple endpoint mammalian mutagenesis approach developed in our institute screens in the same animal for recessive specific-locus alleles at 7 loci, approximately 30 loci coding for dominant-cataract mutations, 23 loci controlling protein-charge changes and 12 loci for enzyme-activity alterations. Experiments to screen for the approximately 70 loci in the same offspring of treated male mice were performed with ethylnitrosourea (ENU), procarbazine and X-ray exposure. Mutations were recovered for each genetic endpoint in all treatment groups where a sufficient number of offspring was scored. ENU treatment is highly effective in inducing mutations to all genetic endpoints. The mutations were confirmed by breeding tests. The mutation rates to specific-locus and enzyme-activity alleles were both higher than the mutation rates to either dominant-cataract or protein-charge alleles. The advantages and possibilities of the multiple endpoint approach are discussed in detail.     

X-linked glucose-6-phosphate dehydrogenase deficiency inMus musculus

A mouse with X-linked glucose-6-phosphate dehydrogenase (G6PD) deficiency has been recovered in offspring of 1-ethyl-1-nitrosourea-treated male mice. The activity alteration was detected in blood but can also be observed in other tissue extracts. Hemizygous, heterozygous, and homozygous mutants have, respectively, about 15, 60, and 15% G6PD remaining activity in the blood as compared to the wild type. Erythrocyte indices did not show differences between mutants and wild types. The mutation does not affect the electrophoretic migration, the isoelectric point, or the thermal stability. Kinetic properties, such as theK _m for glucose-6-phosphate or for NADP and the relative utilization of substrate analogues, showed no differences between wild types and mutants with the exception of the relative utilization of deamino-NADP which was significantly lower in mutants. This is presently the only animal model for X-linked G6PD deficiency in humans.This research was supported in part by Contract BI6-156-D from the Commission of the European Communities.