Ionomic responses of rice plants to the stresses of different arsenic species in hydroponics.

Different ionomic profiles of plants are associated with different external stresses to which they are exposed. Investigation of ionomic variation is necessary for understanding the migration and detoxification of toxic elements in plants. In the current study, rice plants were treated with arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid in hydroponics. The ionomic responses of the rice plants to different arsenic (As) species stresses were measured and analyzed. The multielement approach is more sensitive at detecting significant variations from external environmental stresses than the consideration of several individual elements. The pairs of significant correlations between elements varied based on the rice tissues and As species used in treatment, resulting in specific correlation networks. However, some pairs of correlations existed regardless of As species treatment used in this study. Positive correlations between P and Fe were observed in rice roots treated with any of the As species, implying that P and Fe share similar biological processes. The heatmap from hierarchical cluster analysis (HCA) agreed with the principal component analysis (PCA) results in ionomic differentiation between roots and shoots. Furthermore, ionomic differences between rice plants treated with different As species were identified through PCA. This study revealed that the ionomic profiles in rice plants are sufficient to detect responses to environmental perturbations. Association studies between ionomics and genomics are necessary to further understand the potential mechanisms that promote uptake or exclusion of elements in plants.

Phosphorus deficiency modifies As translocation in the halophyte plant species Atriplex atacamensis.

Most arsenic in surface soil and water exists primarily in its oxidized form, as arsenate (As(V); AsO43-), which is an analog of phosphate (PO43-). Arsenate can be taken up by phosphate transporters. Atriplex atacamensis Phil. is native to northern Chile (Atacama Desert), and this species can cope with high As concentrations and low P availability in its natural environment. To determine the impact of P on As accumulation and tolerance in A. atacamensis, the plants were cultivated in a hydroponic system under four treatments: no As(V) addition with 323µM phosphate (control); 1000µM As(V) addition with 323µM phosphate; no As(V) and no phosphate; 1000µM As(V) addition and no phosphate. Phosphate starvation decreased shoot fresh weight, while As(V) addition reduced stem and root fresh weights. Arsenate addition decreased the P concentrations in both roots and leaves, but to a lesser extent than for P starvation. Phosphorus starvation increased the As concentrations in roots, but decreased it in shoots, which suggests that P deficiency reduced As translocation from roots to shoots. Arsenate addition increased total glutathione, but P deficiency decreased oxidized and reduced glutathione in As(V)-treated plants. Arsenate also induced an increase in S accumulation and nonprotein thiol and ethylene synthesis, and a decrease in K concentrations, effects that were similar for the P-supplied and P-starved plants. In contrast, in As(V)-treated plants, P starvation dramatically decreased total soluble protein content and increased lipid peroxidation, compared to plants supplied with P. Phosphorus nutrition thus appears to be an important component of A. atacamensis response to As toxicity.

Trait-directed de novo population transcriptome dissects genetic regulation of a balanced polymorphism in phosphorus nutrition/arsenate tolerance in a wild grass, Holcus lanatus.

The aim of this study was to characterize the transcriptome of a balanced polymorphism, under the regulation of a single gene, for phosphate fertilizer responsiveness/arsenate tolerance in wild grass Holcus lanatus genotypes screened from the same habitat. De novo transcriptome sequencing, RNAseq (RNA sequencing) and single nucleotide polymorphism (SNP) calling were conducted on RNA extracted from H. lanatus. Roche 454 sequencing data were assembled into c. 22,000 isotigs, and paired-end Illumina reads for phosphorus-starved (P-) and phosphorus-treated (P+) genovars of tolerant (T) and nontolerant (N) phenotypes were mapped to this reference transcriptome. Heatmaps of the gene expression data showed strong clustering of each P+/P- treated genovar, as well as clustering by N/T phenotype. Statistical analysis identified 87 isotigs to be significantly differentially expressed between N and T phenotypes and 258 between P+ and P- treated plants. SNPs and transcript expression that systematically differed between N and T phenotypes had regulatory function, namely proteases, kinases and ribonuclear RNA-binding protein and transposable elements. A single gene for arsenate tolerance led to distinct phenotype transcriptomes and SNP profiles, with large differences in upstream post-translational and post-transcriptional regulatory genes rather than in genes directly involved in P nutrition transport and metabolism per se.

Novel phytase from Pteris vittata resistant to arsenate, high temperature, and soil deactivation.

Arsenate interferes with enzymatic processes and inhibits inorganic phosphorus (Pi) uptake in many plants. This study examined the role of phytase and phosphatase in arsenate tolerance and phosphorus (P) acquisition in the arsenic hyperaccumulator Pteris vittata . Enzyme-mediated hydrolysis of phytate in P. vittata extracts was not inhibited by arsenate at 5 mM or by heating at 100 °C for 10 min. Root exudates of P. vittata exhibited the highest phytase activity (18 nmol Pi mg(-1) protein min(-1)) when available P was low, allowing its growth on media amended with phytate as the sole source of P. Phosphorus concentration in P. vittata gametophyte tissue grown on phytate was equivalent to plants grown with inorganic phosphate at 2208 mg kg(-1), and arsenic was increased from 1777 to 2630 mg kg(-1). After 2 h of mixing with three soils, P. vittata phytase retained more activity, decreasing from ∼ 26 to ∼ 25 nmol Pi mg(-1) protein min(-1), whereas those from Pteris ensiformis and wheat decreased from ∼ 18 to ∼ 1 nmol Pi mg(-1) protein min(-1). These results suggest P. vittata has a uniquely stable phytase enabling its P acquisition in P-limiting soil environments. Furthermore, the P. vittata phytase has potential use as a soil amendment, a transgenic tool, or as a feed additive supplement, reducing the need for nonrenewable, polluting P fertilizers.

GFAJ-1 is an arsenate-resistant, phosphate-dependent organism.

The bacterial isolate GFAJ-1 has been proposed to substitute arsenic for phosphorus to sustain growth. We have shown that GFAJ-1 is able to grow at low phosphate concentrations (1.7 µM), even in the presence of high concentrations of arsenate (40 mM), but lacks the ability to grow in phosphorus-depleted (<0.3 µM), arsenate-containing medium. High-resolution mass spectrometry analyses revealed that phosphorylated central metabolites and phosphorylated nucleic acids predominated. A few arsenylated compounds, including C6 sugar arsenates, were detected in extracts of GFAJ-1, when GFAJ-1 was incubated with arsenate, but further experiments showed they formed abiotically. Inductively coupled plasma mass spectrometry confirmed the presence of phosphorus in nucleic acid extracts, while arsenic could not be detected and was below 1 per mil relative to phosphorus. Taken together, we conclude that GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent, bacterium.

Calcium supplementation modulates arsenic-induced alterations and augments arsenic accumulation in callus cultures of Indian mustard (Brassica juncea (L.) Czern.).

In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV (250 µM) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 µg g(-1) dry weight (DW)) than AsV without Ca (167 µg g(-1) DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24-47%) than calli subjected to AsV treatment (12-14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H(2)O(2)) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H(2)O(2), shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents', viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.

Indigenous soil bacteria with the combined potential for hydrocarbon consumption and heavy metal resistance.

INTRODUCTION: Transconjugant bacteria with combined potential for hydrocarbon utilization and heavy metal resistance were suggested by earlier investigators for bioremediation of soils co-contaminated with hydrocarbons and heavy metals. The purpose of this study was to offer evidence that such microorganisms are already part of the indigenous soil microflora. METHODS: Microorganisms in pristine and oily soils were counted on nutrient agar and a mineral medium with oil as a sole carbon source, in the absence and presence of either sodium arsenate (As V), sodium arsenite (As III) or cadmium sulfate, and characterized via 16S rRNA gene sequencing. The hydrocarbon-consumption potential of individual strains in the presence and absence of heavy metal salts was measured. RESULTS: Pristine and oil-contaminated soil samples harbored indigenous bacteria with the combined potential for hydrocarbon utilization and As and Cd resistance in numbers up to 4 × 105 CFU g⁻¹. Unicellular bacteria were affiliated to the following species arranged in decreasing order of predominance: Bacillus subtilis, Corynebacterium pseudotuberculosis, Brevibacterium linens, Alcaligenes faecalis, Enterobacter aerogenes, and Chromobacterium orangum. Filamentous forms were affiliated to Nocardia corallina, Streptomyces flavovirens, Micromonospora chalcea, and Nocardia paraffinea. All these isolates could grow on a wide range of pure aliphatic and aromatic hydrocarbons, as sole sources of carbon and energy, and could consume oil and pure hydrocarbons in batch cultures. Low As concentrations, and to a lesser extent Cd concentrations, enhanced the hydrocarbon-consumption potential by the individual isolates. CONCLUSION: There is no need for molecularly designing microorganisms with the combined potential for hydrocarbon utilization and heavy metal resistance, because they are already a part of the indigenous soil microflora.

Different effects of arsenate and phosphonoformate on P(i) transport adaptation in opossum kidney cells.

The main nonhormonal mechanism for controlling inorganic phosphate (P(i)) homeostasis is renal adaptation of the proximal tubular P(i) transport rate to changes in dietary phosphate content. Opossum kidney (OK) cell line is an in vitro renal model that maintains the ability of renal adaptation to the extracellular P(i) concentration. We have studied how two competitive inhibitors of P(i) transport, arsenate [As(V)] and phosphonoformate (PFA), affect adaptation to low and high P(i) concentrations. OK cells show very high affinity for As(V) (inhibitory constant, K(i) 0.12 mM) when compared with the rat kidney. As(V) very efficiently reversed the adaptation of OK cells to low P(i) (0.1 mM), whereas PFA induced adaptation similar to 0.1 mM P(i). Adaptation with 2 mM P(i) or As(V) was characterized by decreases in the maximal velocity (V(max)) of P(i) transport and an abundance of the NaPi-IIa P(i) transporter in the plasma membrane, shown by the protein biotinylation. Conversely, PFA and 0.1 mM P(i) increased the V(max) and transporter abundance. Changes in the V(max) were limited to a 50% variation, which was not paralleled by changes in the concentration of P(i) or of the inhibitor. OK cells are very sensitive to As(V), but the effects are reversible and noncytotoxic. These effects can be interpreted as As(V) being transported into the cell, thereby mimicking a high P(i) concentration. PFA blocks the uptake of P(i) but is not transported, and it therefore simulates a low P(i) concentration inside the cell. To conclude, a mathematical definition of the adaptation process is reported, thereby explaining the limited changes in P(i) transport V(max).

Accumulation, speciation, and coordination of arsenic in an inbred line and a wild type cultivar of the desert plant species Chilopsis linearis (Desert willow).

This study investigated the absorption of arsenic (As), sulfur (S), and phosphorus (P) in the desert plant Chilopsis linearis (Desert willow). A comparison between an inbred line (red flowered) and wild type (white flowered) plants was performed to look for differential responses to As treatment. One month old seedlings were treated for 7 days with arsenate (As(2)O(5), As(V)) at 0, 20, and 40 mg As(V)L(-1). Results from the ICP-OES analysis showed that at 20mg As(V)L(-1), red flowered plants had 280+/-11 and 98+/-7 mg As kg(-1) dry wt in roots and stems, respectively, while white flowered plants had 196+/-30 and 103+/-13 mg As kg(-1) dry wt for roots and stems. At this treatment level, the concentration of As in leaves was below detection limits for both plants. In red flowered plants treated with 40 mg As(V)L(-1), As was at 290+/-77 and 151+/-60 mg As kg(-1) in roots and stems, respectively, and not detected in leaves, whereas white flowered plants had 406+/-36, 213+/-12, and 177+/-40 mg As kg(-1) in roots, stems, and leaves. The concentration of S increased in all As treated plants, while the concentration of P decreased in roots and stems of both types of plants and in leaves of red flowered plants. X-ray absorption spectroscopy analyses demonstrated partial reduction of arsenate to arsenite in the form of As-(SX)(3) species in both types of plants.

Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library.

The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene (arsN) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.

Characterisation of inorganic phosphate transport in bovine articular chondrocytes.

In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.

Inorganic phosphate transport in matrix vesicles from bovine articular cartilage.

AIMS: In mineralizing tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV), possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. This study characterizes P(i) transport in MV derived from articular cartilage. METHODS: Matrix vesicles were harvested from collagenase digests of bovine articular cartilage by serial centrifugation. P(i) uptake by MV was measured using radioactive phosphate ((33)[P]HPO(4)(2-)). The Na(+) dependence, pH sensitivity and effects of P(i) analogues that inhibit P(i) transport were determined. RESULTS: P(i) uptake was temperature-sensitive and comprised Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high extracellular P(i) concentrations, with a K(m) of 0.16 mM. In Na(+)-free solutions, uptake did not fully saturate implying that carrier-mediated uptake is supplemented by a diffusive pathway. Uptake was inhibited by phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake persisted. Total P(i) uptake was maximal at pH 6.5, and reduced at more acidic or alkaline values, representing inhibition of both components. CONCLUSION: These properties are highly similar to those of P(i) uptake by chondrocytes, suggesting that MV inherit P(i) transporters of the chondrocyte membrane from which they are derived. Na(+)-independent P(i) uptake has not previously been described in MV from growth plate cartilage and is relatively uncharacterized, but warrants further attention in articular cartilage, given its likely role in initiating inappropriate mineral formation.

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus.

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.

Yield and arsenate uptake of arbuscular mycorrhizal tomato colonized by Glomus mosseae BEG167 in As spiked soil under glasshouse conditions.

A glasshouse pot experiment was conducted to study the effect of arbuscular mycorrhizal (AM) colonization by Glomus mosseae BEG167 on the yield and arsenate uptake of tomato plants in soil experimentally contaminated with five As levels (0, 25, 50, 75 and 150 mg kg(-1)). Mycorrhizal colonization (50-70% of root length) was little affected by As application and declined only in soil amended with 150 mg As kg(-1). Mycorrhizal colonization increased plant biomass at As application rates of 25, 50 and 75 mg kg(-1). Shoot As concentration increased with increasing As addition up to 50 mg kg(-1) but decreased with mycorrhizal colonization at As addition rates of 75 and 150 mg kg(-1). Shoot As uptake increased with mycorrhizal colonization at most As addition levels studied, but tended to decrease with addition of 150 mg As kg(-1). Total P uptake by mycorrhizal plants was elevated at As rates of 25, 50 and 75 mg kg(-1), and more P was allocated to the roots of mycorrhizal plants. Mycorrhizal plants had higher shoot and root P/As ratios at higher As application rates than did non-mycorrhizal controls. The soil of inoculated treatments had higher available As than uninoculated controls, and higher pH values at As addition levels of 25, 50 and 75 mg kg(-1). Mycorrhizal colonization may have increased plant resistance to potential As toxicity at the highest level of As contamination studied. Mycorrhizal tomato plants may have potential for phytoextraction of As from moderately contaminated soils or phytostabilization of more highly polluted sites.

Insertional mutagenesis in a homologue of a Pi transporter gene confers arsenate resistance on chlamydomonas.

An arsenate-resistant mutant AR3 of Chlamydomonas reinhardtii is a recessive mutant generated by random insertional mutagenesis using the ARG7 gene. AR3 shows about 10-fold resistance against arsenate toxicity compared with the wild type. By using a flanking region of an inserted tag as a probe, we cloned the corresponding wild-type allele (PTB1) of a mutated gene, which could completely complement the arsenate-resistance phenotype of AR3. The size of PTB1 cDNA is about 6.0 kb and it encodes a putative protein comprising 1666 amino acid residues. This protein exhibits significant sequence similarity with the yeast Pho89 protein, which is known to be a Na(+)/Pi co-transporter, although the PTB1 protein carries an additional Gln- and Gly-rich large hydrophilic region in the middle of its primary structure. Analyses of arsenic accumulation and release revealed that PTB1-disrupted cells show arsenate resistance due to low arsenate uptake. These results suggest that the PTB1 protein is a factor involved in arsenate (or Pi) uptake. Kinetics of Pi uptake revealed that the activity of high-affinity Pi transport component in AR3 is more activated than that in the wild type.

Tetraarsenic oxide, a novel orally administrable angiogenesis inhibitor.

Arsenic compounds have been used to treat angiogenic diseases such as cancer, psoriasis, and rheumatoid arthritis in traditional oriental medicine. In recent years, arsenic trioxide (As2O3, diarsenic oxide) has been successfully used to treat acute promyelocytic leukemia. We investigated the antiangiogenic properties of tetraarsenic oxide (As4O6), another trivalent arsenic compound. In in vitro studies, tetraarsenic oxide inhibited the proliferation (IC50 = 99.7 nM), migration into the denuded area (IC50 = 27.4 nM), and invasion through a layer of Matrigel (IC50 = 73.5 nM) of basic fibroblast growth factor (bFGF)-stimulated bovine capillary endothelial (BCE) cells in a dose-dependent manner. Tetraarsenic oxide also inhibited the tube formation of human umbilical vein endothelial cells. Tetraarsenic oxide induced cell cycle arrest of bFGF-stimulated BCE cells in the G2/M phase and inhibited the secretion of matrix metalloproteinase-2 from BCE cells. Orally administered tetraarsenic oxide (50 mg/kg/day) inhibited bFGF-induced new-vessel formation in a rat corneal micropocket assay, and reduced by about 54% the number of experimental pulmonary metastatic nodules in mice implanted with B16F10 melanoma cells. Thus, we provide evidence that tetraarsenic oxide has effective antiangiogenic activities.

Isolation and characterization of mitochondrial manganese superoxide dismutase (MnSOD) from Capsicum annuum L.

A cDNA clone for a mitochondrial manganese superoxide dismutase (MnSOD) was isolated and characterized from red pepper (Capsicum annuum L.). The clone consisted of 941 bp containing one open reading frame (ORF) of 687 bp, 34 bp/220 bp of 5'/3'-untranslated region. Amino acid sequence of the ORF showed the highest homology (86%) with that of Nicotiana plumbaginifolia. It encodes for a polypeptide of 228 amino acids with a molecular mass of 25.5 kDa and a pI value of 8.39. Genomic Southern hybridization suggested that more than one copy are present. Northern hybridization showed that the MnSOD transcript was more abundant in stems than in leaves and roots. When seedlings were treated with arsenate (0.1-10 mM), the MnSOD transcript level increased slightly at 0.1 mM and then dropped, while the Cu/ZnSOD transcript level increased at 1 mM, and also dropped at higher concentrations.

Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria.

The two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451-10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coli and reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule.

[Activation of endogenous nucleases as affected by sodium arsenate and cisplatin]. / Aktivirovanie éndogennykh nukleaz pod deistviem arsenata natriia i tsisplatina.

Screening of pharmaceutical agents releasing active nucleases from inactive complexes was performed. Cisplatin and sodium arsenate were shown in vitro to have such properties. Sodium arsenate which is more expedient for the treatment of human patients was studied in vivo. It was administered to albino mice in a dose of 0.2 mg/kg once a day for 6 days. Due to the release, the activity of free serum DNAse and RNAse markedly increased (1.5-2 times). Activation of endogenic nucleases is more promising than the use of exogenic enzymes in complex therapy of viral affections and rickettsiosis.

Transport characteristics of a murine renal Na/Pi-cotransporter.

A complementary deoxyribonucleic acid (cDNA) corresponding to a murine renal cortical Na/phosphate-(Na/Pi-) cotransporter was isolated and its transport properties characterized by electrophysiological techniques after expression in Xenopus laevis oocytes. A Na-dependent inward movement of positive charges ("short-circuit current") was observed upon superfusion with Pi (and with arsenate). Increasing the Na concentration led to a sigmoidal elevation in Pi-induced short-circuit current; the apparent Michaelis constant, Km, (around 40 mM Na) was increased by lowering the pH of the superfusate but was not influenced by altering the Pi concentration. Increasing the Pi (and arsenate) concentration led to a hyperbolic elevation in Na-dependent short-circuit current (apparent Km for Pi at 100 mM Na was around 0.1 mM; apparent Km for arsenate was around 1 mM); lowering the Na concentration decreased the apparent affinity for Pi. The Pi-induced short-circuit current was lower at more acidic pH values (at pH 6.3 it was about 50% of the value at pH 7.8); this pH dependence was similar if the Pi concentration was calculated in total, or if distinction was made between its mono- and divalent forms. Thus, the pH dependence of Na-dependent Pi transport (total Pi) may not be related primarily to a pH-dependent alteration in the availability of divalent Pi, but includes also a competitive interaction of Na with protons. The effect of Pi and Na concentration on the apparent Km values for Na or Pi, respectively, provides evidence for an ordered interaction of "cosubstrate" (Na first) and "substrate" (Pi or arsenate second).