镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达

本文在荧光定量PCR优化的基础上,利用该技术考察了不同浓度的重金属镉和铜对嗜热四膜虫金属硫蛋白基因(MTT1)诱导表达的变化规律。结果表明MTT1基因的表达对镉离子的诱导更灵敏,且在一定阈值浓度(≤35.2μmol/L)范围内,镉离子浓度升高会增加MTT1基因表达量,超过该阈值后表达量迅速下降;镉与铜同时诱导时MTT1基因的表达情况与镉单独诱导的类似,但阈值浓度减小为22μmol/L,表明二者的联合毒性为协同作用。镉离子浓度低于22μmol/L时,与铜离子的共同作用会大大增加MTT1基因的表达量,从而增强了四膜虫的解毒能力。     

On metallothionein,cadmium, copper and zinc relationships in the liver and kidney of adult rats

1. A short-term exposure of adult Wistar rats to Cu (50 μg/ml) and Cd (10.0 μg/ml drinking water) caused significant changes in the subcellular concentrations of Cd, Cu, Zn and metallothionein (MT) in the liver and kidney; the concentrations were close to the physiological values, however.2. To establish a relationship between these changes in the subcellular concentrations of Cd, Cu, Zn and the level of MT in the post-mitochondrial fraction of the liver and kidney, the analytical data (N = 42) were subjected to the multiple regression analysis.3. The analysis showed that MT synthesis in the liver was principally induced by small amounts of Cd (0.32–1.4 μg/g wet wt) whereas in the kidney a level of MT in the post-mitochondrial fraction correlated positively with the renal Cd and Cu, as well as with the level of this protein in the liver.4. The above results together with the positive correlation between the level of MT in the post-mitochondrial fraction and the concentration of Cu in this fraction, as well as the fact that under normal physiological conditions the capacity of MT (β-domain) in the liver and kidney was sufficient to bind 50–100% of the total post-mitochondrial Cu suggest that MT, first induced by small amounts of Cd, may be involved in the metabolism of Cu.     

Xanthine dehydrogenase activity in the clawed frog, Xenopus laevis

Xanthine dehydrogenase (XDH; EC 1.2.1.37) activity in the clawed frog, Xenopus laevis, was detected in kidney tissue homogenates, but not in skin, liver, ovaries or gut tissues. The enzyme migrated as a single band of activity on both polyacrylamide and starch gel electropherograms, exhibited substrate inhibition, and did not appear developmentally until feeding larval stages. The tissue specificity, post-fertilization stage of appearance and single isozymic form make this a useful enzyme marker for further study concerning its developmental appearance and maintenance as a kidney-specific protein.     

Discriminative uptake of metals by the liver and its relation to induction of metallothionein by cadmium, copper and zinc

1. Disappearance from plasma and uptake by the liver of cadmium (Cd), copper (Cu) and zinc (Zn) were examined with a view to studying the biological discrimination between essential and non-essential heavy metals. 2. Cd injected intravenously at a single dose of 0.8 mg/kg body wt disappeared from rat plasma rapidly within about 10 min, while Cu and Zn injected at the same dose disappeared slowly in plasma and decreased to the control level after about 3 hr. 3. Uptake of Cd by the liver corresponded well with the rapid disappearance from plasma, while Cu and Zn accumulated slowly in the liver and their concentrations started to increase after their plasma concentrations had decreased. 4. Metallothionein was induced in the liver at a similar time course for the three metals, suggesting the presence of discriminative uptake processes by the liver with similar or the same detoxification mechanisms through induction of metallothionein.     

Nephroblastoma in the clawed frog Xenopus laevis.

One 2.5-3 year old female clawed frog (Xenopus laevis), out of a consignment of 4,000 frogs, was found to have an abnormal abdominal growth, weighing 7.9 g. The growth was examined histologically and on the basis of the abundant stroma and serially-arranged tubules nephroblastoma was diagnosed. The growth is not considered to be transmissible.     

Biophysics of underwater hearing in the clawed frog,Xenopus laevis

Anesthetized clawed frogs (Xenopus laevis) were stimulated with underwater sound and the tympanic disk vibrations were studied using laser vibrometry. The tympanic disk velocities ranged from 0.01 to 0.5 mm/s (at a sound pressure of 2 Pa) in the frequency range of 0.4–4 kHz and were 20–40 dB higher than those of the surrounding tissue. The frequency response of the disk had two peaks, in the range of 0.6–1.1 kHz and 1.6–2.2 kHz, respectively. The first peak corresponded to the peak vibrations of the body wall overlying the lung. The second peak matched model predictions of the pulsations of the air bubble in the middle ear cavity. Filling the middle ear cavity with water lowered the disk vibrations by 10–30 dB in the frequency range of 0.5–3 kHz.Inflating the lungs shifted the low-frequency peak downwards, but did not change the high-frequency peak. Thus, the disk vibrations in the frequency range of the mating call (main energy at 1.7–1.9 kHz) were mainly caused by pulsations of the air in the middle ear cavity; sound transmission via the lungs was more important at low frequencies (below 1 kHz). Furthermore, the low-frequency peak could be reversibly reduced in amplitude by loading the larynx with metal or tissue glue. This shows that the sound-induced vibrations of the lungs are probably coupled to the middle ear cavities via the larynx. Also, anatomical observations show that the two middle ear cavities and the larynx are connected in an air-filled recess in submerged animals.This arrangement is unique to pipid frogs and may be a structural adaptation to connect all the air spaces of the frog and improve low-frequency underwater hearing. Another function of the recess may be to allow cross-talk between the two middle ear cavities. Thus, the ear might be directional. Our pilot experiments show up to 10 dB difference between ipsi- and contralateral stimulus directions in a narrow frequency range around 2 kHz.     

Concentrations of cadmium,zinc, copper,iron, and metallothionein in liver and kidney of nonhuman primates

To evaluate the species specificity of Cd accumulation and the relationship of Cd with other essential metals and metallothionein (MT), the concentrations of Cd, Zn, Cu, and Fe in the liver and kidney and the MT concentrations in the soluble fractions of the liver and kidney were determined in Cd-uncontaminated nonhuman primates (11 species, 26 individuals) kept in a zoo and two wild-caught Japanese macaques. The compositions of metal-binding proteins in the soluble fractions were also investigated by high-performance liquid chromatography (HPLC). The hepatic Cd concentration was 0.03–14.0 μg/g and the renal Cd concentration was 0.35–99.0 μg/g, both varying greatly and being higher in nonhuman primates, which were more closely related to man. The hepatic Zn concentration was 24.0–176 μg/g and the renal Zn concentration was 13.5–138 μg/g, showing 7- to 10-fold differences, and a correlation (r=0.558, p<0.01) was found between renal Zn and renal Cd concentrations. It was proved that in the liver, MT is more closely correlated with Zn (r=0.795, p<0.001) than with Cd (r=0.492, p<0.01) and that in the kidney MT is correlated with both Cd (r=0.784, p<0.001) and Zn (r=0.742, p<0.001). HPLC analysis of metals bound to MT-like protein in chimpanzees, de Brazza’s monkeys, and Bolivian squirrel monkeys showed that more than 90% of Cd in both the liver and kidney, approx 40% of Zn in liver and 28–69% of Zn in kidney were bound to MT-like protein. The higher percentage Zn was bound to high-molecular protein.     

The longitudinal distribution of cadmium,zinc, copper,iron, and metallothionein in the small-intestinal mucosa of rats after administration of cadmium chloride

Different routes of Cd intake may influence the intestinal distribution of Cd, metallothionein (MT), and trace metals differently. Therefore, we compared the effects of parenteral and enteral administration of Cd on the distribution of trace metals and MT along the small intestine. In a first experiment three groups of rats were employed: a control, one receiving CdCl₂ within the drinking water, and another receiving sc injections of CdCl₂. In a second experiment, rats were fed three different diets with either 0, 0.3, or 1 mmol CdCl₂/kg for one and two weeks to study the time- and dose-dependent effects of orally administered Cd. Metal concentrations (Cd, Zn, Cu, Fe) were measured by atomic emission spectrometry and MT was determined by radioimmunoassay. Intestinal MT levels did not show proximodistal gradients in controls or after sc administration of Cd, but orally administered Cd increased mucosal MT levels longitudinally from the duodenum to the ileum. Cd levels paralleled those of MT. Compared with the metal concentrations in the controls, sc administration of Cd did not change intestinal Zn, Cu, and Fe levels. Oral administration of Cd, however, increased Cu and decreased Fe levels in the intestinal mucosa significantly. The second experiment revealed that only high dietary concentrations of Cd increase intestinal Cd and MT levels longitudinally toward the distal parts, whereas at lower dietary concentration the longitudinal distribution was reversed. This shows that different routes and doses of Cd intake lead to a different trace metal and MT distribution and emphasizes the role of dietary Cd in the local induction of small-intestinal MT.     

Nitric oxide synthase in the spinal cord of the frog, Xenopus laevis

The expression of nitric oxide synthase was investigated in the spinal cord of the South African clawed frog by NADPH diaphorase histochemistry and immunohistochemistry. The dorsal field contained many strongly positive neurons and a dense plexus of processes. Only few nitric oxide synthase-positive cells occurred in the lateral and central field. Motoneurons were negative. A dense accumulation of stained neurons was located dorsal and dorsomedial to the motoneurons. The white matter harbored many positive fibers. These were most abundant in the dorsal funiculus, and obviously consist of nonprimary projections to the brainstem. These results suggest that nitric oxide represents a widely used messenger molecule in the frog spinal cord, in particular with respect to the processing of sensory information.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

Prevention by copper of cadmium sequestration by metallothionein in liver.

Following chronic CdCl2 administration to rats, more than 98% of the metal in liver supernatant is bound to the low molecular weight binding protein, metallothionein. Simultaneous administration of high doses of Cd and copper salts result in an increase in toxicity which is accompanied by a failure of Cd sequestration by metallothionein in vivo. This may be due to an aggregation of metallothionein which has been observed in the presence of copper in vitro.     

Stockpiling of DNA polymerases during oogenesis and embryogenesis in the frog, Xenopus laevis

The amounts of the various forms of DNA polymerase (alpha 1, alpha 2, beta, and gamma) have been determined in oocytes, eggs, and embryos of the frog, Xenopus laevis. During oogenesis the relative proportions and absolute levels of all forms changed dramatically. In stage I (early) oocytes, DNA polymerase-gamma, the "mitochondrial" polymerase, was the predominant form. During oocyte growth, DNA polymerase-alpha 1 and -alpha 2 increased by more than 100-fold, DNA polymerase-beta by 15-fold, and DNA polymerase-gamma by only 8-fold. During oocyte maturation and ovulation, the levels of all forms of DNA polymerase roughly doubled. The mature stage VI oocyte contained 5 orders of magnitude more DNA polymerase activity than is found in an individual somatic cell. DNA polymerase-alpha 1 and -alpha 2, the "replicative" polymerases, were the predominant forms in mature oocytes and ovulated unfertilized eggs. During fertilization, the relative proportions and absolute levels of the four forms remained constant. During subsequent stages of embryogenesis, the total amounts of DNA polymerase-alpha 1 and -alpha 2 declined slightly from cleavage through gastrulation, the stages of most rapid chromosomal DNA replication. The rapid increase in cell number during early embryogenesis establishes the same levels of DNA polymerase/cell as are present in adult somatic cells. After neurulation, the absolute levels of DNA polymerase-alpha 1 and -alpha 2 increased in proportion to increases in cell number. The absolute levels of DNA polymerase-beta remained constant, and the levels of DNA polymerase-gamma increased 2-fold throughout embryogenesis.     

Accelerated gene evolution and subfunctionalization in the pseudotetraploid frog Xenopus laevis

Background  

Ancient whole genome duplications have been implicated in the vertebrate and teleost radiations, and in the emergence of diverse angiosperm lineages, but the evolutionary response to such a perturbation is still poorly understood. The African clawed frog Xenopus laevis experienced a relatively recent tetraploidization ~40 million years ago. Analysis of the considerable amount of EST sequence available for this species together with the genome sequence of the related diploid Xenopus tropicalis provides a unique opportunity to study the genomic response to whole genome duplication.     

Classification of DNA polymerase activities from ovaries of the frog,Xenopus laevis

Four distinct DNA polymerase activities were isolated from ovaries of the frog Xenopus laevis. Specific assays for each activity were established. The isolated activities were characterized by molecular weight, template-primer preferences, and sensitivity to specific inhibitors as Xenopus laevis ovarian DNA polymerases-α₁, -α₂, -β, and -γ. All previously described Xenopus laevis DNA polymerases were classified using these properties.     

Global realized niche divergence in the African clawed frog Xenopus laevis

Although of crucial importance for invasion biology and impact assessments of climate change, it remains widely unknown how species cope with and adapt to environmental conditions beyond their currently realized climatic niches (i.e., those climatic conditions existing populations are exposed to). The African clawed frog Xenopus laevis, native to southern Africa, has established numerous invasive populations on multiple continents making it a pertinent model organism to study environmental niche dynamics. In this study, we assess whether the realized niches of the invasive populations in Europe, South, and North America represent subsets of the species’ realized niche in its native distributional range or if niche shifts are traceable. If shifts are traceable, we ask whether the realized niches of invasive populations still contain signatures of the niche of source populations what could indicate local adaptations. Univariate comparisons among bioclimatic conditions at native and invaded ranges revealed the invasive populations to be nested within the variable range of the native population. However, at the same time, invasive populations are well differentiated in multidimensional niche space as quantified via n‐dimensional hypervolumes. The most deviant invasive population are those from Europe. Our results suggest varying degrees of realized niche shifts, which are mainly driven by temperature related variables. The crosswise projection of the hypervolumes that were trained in invaded ranges revealed the south‐western Cape region as likely area of origin for all invasive populations, which is largely congruent with DNA sequence data and suggests a gradual exploration of novel climate space in invasive populations.