35S-cysteine Incorporation Research Articles

Functional integrity of metallothionein genes in testicular cell lines

The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose- and time-dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S-cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells.

成人歯肉由来線維芽細胞のＤＮＡ合成とタンパク質合成におよぼすカドミウムとフッ素の影響

Cadmium (Cd) and fluoride (F) affect the metabolism of hard tissues, but their effect on human beings is not yet known. The purpose of this study was to investigate the effect of Cd and/or F on DNA and protein syntheses of human cultured cells. Four human gingival fibroblast primary cultures (GF-5, 6, 12 and 13 cells) and two human malignant cell lines (HeLa S3cells and KU-2 cells) were used. All cells were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum. After 24hr incubation, cells were cultured in media containing CdCl2 and/or NaF in order to study their growth, DNA and protein synthesis, and intracellular Cd level. The growth and DNA synthesis of cells were inhibited by 2.0mM NaF or 10-4mM CdCl2. The incorporation of 14C-leucine in cells was increased by 0.5mM NaF or 10-5mM-10-4mM CdCl2. The enhancement of incorporation of 14C-leucine was greater in cells exposed to NaF plus CdCl2 than in cells exposed to CdCl2 alone. Increased incorporation of 35S-cysteine in cells was observed with 10-5mM-10-4mM CdCl2 or NaF plus CdCl2. However, F had no effect on the incorporation of 35S-cysteine. Gel filtration was performed with Sephadex G75. 35S peak was found in an metallothionein (MT) fraction. The intracellular Cd level was lower in gingival fibroblasts than in malignant cells. These results indicate that Cd induced the synthesis of MT in both human gingival fibroblasts and malignant cells. It also seems that F has the ability to stimulate the inducement of MT synthesis by Cd.

Comparative study on metallothionein induction in whole testicular tissue and isolated Leydig cells

The present study aimed at investigating whether previously reported interstrain differences of mice in testicular susceptibility to cadmium toxicity can be attributed to different metallothionein expression in the testes and other organs. Furthermore, purified Leydig cell systems were employed to identify a testicular compartment capable to respond specifically to exogenous cadmium by increased metallothionein expression. It was shown that species differences in testicular damage after cadmium administration can not be explained by differences in cadmium-induced stimulation of hepatic and renal metallothionein. In contrast to these organs, no metallothionein response to cadmium was observed in testicular homogenates. However, both freshly isolated and purified rat Leydig cells and a murine tumor Leydig cell line are not only able to synthesize metallothionein under basal conditions, but also respond to exogenous cadmium by increased synthesis of metallothionein (35S-cysteine incorporation) and an increased Mt-mRNA content. Extracts from freshly isolated and purified rat Leydig cells contained a cadmium-binding component which eluted in gel chromatography with the same properties as metallothionein. It was concluded that the use of purified cell systems is a valuable tool to investigate possible targets of cadmium toxicity.

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

The incorporation of 35S-cysteine and 3H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of 35S-cysteine over 3H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.

Induction of metal binding proteins in striped bass, Morone saxatilis, following cadmium treatment

1. 1. The time course of induction of cytosolic metal binding proteins (MBP) was observed following up to three daily intramuscular injections of cadmium chloride (0.2 mg cadmium/injection). 2. 2. Low molecular weight binding proteins were resolved by gel permeation chromatography on Sephadex G-75. Based on total metal binding capacity, the concentration of crude MBP increased 2.6 fold. This level of induction of MBP was confirmed by polarographic analysis. 3. 3. Initial binding of cadmium to MBP resulted in displacement of zinc, while at later times, zinc associated with MBP increased above control levels. 4. 4. Using 35S-cysteine incorporation, it was shown that the rate of hepatic MBP synthesis was significantly greater than controls and sham injected fish 18 hr after the third cadmium injection. 5. 5. Due to interfering proteins of molecular weights similar to the metal binding proteins one dimensional PAGE was not capable of verifying induction. However, the metal binding proteins were resolved using two dimensional gel electrophoresis.

Inhibition of LPS toxicity for macrophages by metallothionein-inducing agents.

Parenteral administration of adrenal corticosteroids or particular transition metal salts are known to protect mice from the lethal effects of bacterial lipopolysaccharides (LPS). To determine if both groups of substances act through similar biologic mechanisms, their capacity to protect macrophages from the direct toxic effects of LPS was examined in vitro. When added simultaneously with LPS at culture initiation, 10 to 100 microM cortisone increased the viability of normal peritoneal macrophages as determined by trypan blue exclusion. Prednisolone and corticosterone protected LPS-treated macrophages at even lower concentrations (0.1 to 1 microM); estradiol and testosterone failed to alter cell viability at any concentration tested. Protection was dependent on de novo synthesis because inclusion of 20 nM actinomycin C1 or 1 microM cycloheximide with 10 microM corticosterone during a 4-hr pretreatment period blocked induction of the protective effect. Murine macrophages were also protected by micromolar concentrations of zinc, cadmium, mercury, and manganese, but not by calcium or lead. As was obtained with corticosteroids, heavy metal-induced protection depended on de novo RNA and protein synthesis. Because all substances that protected against LPS are known inducers of metallothionein in somatic cells, peritoneal macrophages were assayed for the presence of this unique, cytoplasmic protein. Within 2 to 8 hr, 10 microM cadmium caused three to fivefold increases in the incorporation of 35S-cysteine and in the binding of 203Hg into the TCA-soluble fraction of cell lysates that was excluded on centrifugally accelerated Sephadex G-10 columns. These results suggest macrophages may be protected from LPS-mediated cytotoxicity through synthesis of a sulfhydryl-rich, metal-binding protein. Although its mechanism of action remains unknown, it is proposed that metallothionein may function homeostatically by altering intracellular concentrations of zinc or may play a regulatory role by facilitating transfer of heavy metals among metal-requiring apoproteins.

Effects of methylmercury on zinc-thionein levels of rat liver.

The induction of metallothionein synthesis by administration of methylmercury chloride as studied. A repetitive sc dose of methylmercury chloride to rats in amounts of 10 mg/kg/day for 3 days produced profound hypozincemia and 3-fold increase in hepatic zinc bound to metallothionein by the initial 24 h. The incorporation of 35S-cysteine into hepatic metallothionein was about 4 times larger for rats treated with methylmercury, which was comparable with it for rats treated with cadmium, than for control rats. These results suggest the induction of de novo synthesis of metallothionein by administration of methylmercury, although methylmercury was found to be unable to bind to produced metallothionein both in vivo and in vitro. A stress produced by methylmercury administration appears to be involved in the induction of hepatic zinc-thionein by methylmercury, through its mechanism is not clear.

736 EFFECT OF EXTRACELLULAR COPPER (Cu) ON METALLOTHIONEIN METABOLISM IN NORMAL AND MENKES FIBROBLASTS

Previously, we demonstrated that the abnormal accumulation of Cu in Menkes disease (MD) fibroblasts (Fb) is associated with an increased amount of metallothionein (MT) (2-3 times normal) (Pediat. Res., in press). To define the basic defect in MD, we investigated the effect of varying concentrations of extracellular Cu (exCu) on the regulation of CuMT metabolism informal (N) and MD Fb. Relative levels of MT were measured by 35S-cysteine incorporation into the 10,000 MW Cu-binding cytosolic protein. Total intracellular Cu was measured by atomic absorption spectrometry. After exposure of N and MD Fb to 2 ug/ml Cu (as CuCl2) maximal levels of MT synthesis were attained within 8 h and remained elevated for at least 56 h. Additional Cu (up to 20 ug/ml) did not increase the rate of MT synthesis above that observed with 2 ug/ml. Pulse-chase experiments demonstrated no difference in the half-life (T1/2=48-60 h) of MTs in N or MD Fb in the presence or absence of exCu. These results demonstrated that MT was an inducible protein in both N and MD Fb. Also, the rate of degradation of the MTs was the same in both the N and MD Fb. Although the mechanism of MT induction by exCu appears normal in MD Fb, the suppression of MT synthesis in the absence of excess Cu may be defective. This regulatory defect results in abnormally high constitutive levels of MT in MD Fb. Whether this defect in regulation of MT synthesis is transcriptional or translational is currently under investigation.

Rapid induction of copper-binding proteins in the gills of metal exposed mussels

The exposure of the mussel Mytilus galloprovincialis Lam. to sublethal concentrations of Cu 2+ (0.08 ppm) for 24–48 hr results in the appearance of low molecular weight (about 12,000), heat stable copper-binding proteins in the soluble extracts obtained from the gills. 2. The soluble low molecular weight copper-binding proteins do not occur in the gill extracts from the controls and from the mussels submitted to a short intoxication (4 hr). 3. The incorporation of 35S cysteine into the 12,000 molecular weight copper-rich fraction from the gills of mussels exposed for 48 hr to Cu 2+ is 7–10 fold greater than in controls. This result strongly supports the inducible nature of these proteins. 4. The low molecular weight proteins synthesized in response to the uptake of Cu 2+ show many of the characteristics of metallothioneins, having a high SH content, few aromatic amino acids and a high metal binding capacity.

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket, Teleogryllus commodus (walk.).

Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) MATERIAL IN THE PARAMEDIAL NEUROSECREtory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of 35S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after ovariectomy. A functional relationship between the PNC and the ovaries is suggested.

Mistranslation in E. coli

Flagellin, the protomeric subunit of bacterial flagella, contains no cysteine. We have detected the incorporation of trace quantities of 35S-cysteine into flagellin, highly purified and then resolved by SDS polyacrylamide gel electrophoresis, to measure mistranslation in vivo. Under normal conditions, this value is about 6 × 10 −4 pmoles cysteine per pmole flagellin. This value is greatly increased during growth in low concentrations of streptomycin and neomycin, antibiotics which are known to stimulate misreading in vitro. Of the specific types of misreading which streptomycin stimulates in vitro, only misreading of the CGU and CGC arginine codons could give rise to illegitimate incorporation of cysteine. In agreement, partial arginine starvation increases the incorporation of 35S-cysteine into flagellin in a relA mutant, with or without streptomycin, but has no such effect in its isogenic relA + partner. Assuming from these results that 35S-cysteine incorporation into flagellin reflects misreading of CGU/C coda, we deduce a misreading probability per codon in the range of 10 −4.

A possible functional relationship between the subcommissural organ and the pineal complex and lateral eyes in Rana esculenta and Rana temporaria.

In previous publications (Diederen, 1972, 1973) evidence was presented that light and darkness hardly influence the secretory activity of the subcommissural organ (SCO) in intact frogs, i.e. frogs in which all photo-receptive organs are present. In the present study the influence of long daily photoperiods and of continuous darkness on the SCO of frogs in which the frontal organ, the pineal organ and the eyes were eliminated separately or in combination was studied. The parameters used to measure the secretory activity of the SCO were the amount of aldehyde-fuchsin(AF)-positive material in the SCO cells, the incorporation of 35S-cysteine by the SCO cells and the growth rate of Reissner's fibre (RF). Only in frogs in which the eyes as well as the entire pineal complex were eliminated was an unequivocal influence of light and darkness on the secretory activity of the SCO demonstrable: in blinded frogs without a pineal complex, the growth rate of RF is clearly enhanced in animals exposed to light. If the eyes or the pineal complex or the pineal organ alone have not been eliminated, then the stimulatory influence of light on the growth rate of RF can no longer be detected. This indicates that light causes the pineal complex and the eyes to exert an inhibitory influence on the secretory activity of the SCO. The hypothesis is put forward that in intact frogs influences of light on the secretory activity of the SCO, at least so far as the production of RF is concerned, are prevented by the photo-receptive organs. In this way a functional relationship between the SCO on the one hand and the pineal complex and eyes on the other hand may be expressed. A mechanism by which the influences of light on the SCO may be prevented by the pineal complex and the eyes is suggested.

Incorporation of cysteine into the cerebral neurosecretory system of adult desert locusts

Liquid scintillation counting techniques were used as a means of measuring the incorporation of 35S-cysteine into the neurosecretory system of adult female Schistocerca gregaria in different physiological conditions. The methods described were unable to measure uptake of 35S label into the cerebral neurosecretory cells but were successful in measuring incorporation rates into the storage lobes of the corpora cardiaca. The corpora cardiaca become labeled soon after injection of the 35S-cysteine, and the initial amount of label is proportional to the amount of neurosecretion within the gland. Loss of label from the hemolymph occurs very rapidly, and the 35S=cysteine concentrations become limiting for incorporation within the cerebral neurosecretory cells within the first hours of the experiment. Label within the corpora cardiaca increases instead of following the decreasing hemolymph label. The rate and time of increase of label within the corpora cardiaca therefore indicates the influx of newly labeled neurosecretion and enables comparisons to be made of neurosecretory turn-over in different females. Ten-day-old females synthesize and release neurosecretory material at a rapid rate. Immature females also synthesize and release material rapidly although at a lower rate than maturing females. Mature females have much less active neurosecretory systems than either maturing or immature females. The results from this liquid scintillation method of measuring radioactive labeling of neurosecretory systems support and amplify the results gained from autoradiographic techniques.

Influence of light and darkness on the subcommissural organ of Rana temporaria L. A cytological and autoradiographical study.

The influence of long daily photoperiods and of continuous darkness on the subcommissural organ (SCO) of Rana temporaria was studied. The amount of aldehyde-fuchsin (AF) positive material in the apical cell parts of the SCO proves to be less in light-adapted than in darkness-adapted animals in winter. The nuclear volume in the SCO of light- and of darkness-adapted animals, however, does not show any clear differences. Distinct differences in incorporation rate of 35S-cysteine between the SCO of light- and of darkness-adapted animals cannot be observed either in winter or in summer. The conclusion must be that light/darkness conditions hardly influence the secretory activity of the SCO cells of Rana temporaria. The results mentioned above were compared with those of experiments on pineal gland and preoptic nucleus. Incorporation of 35S-cysteine, which proceeds rather slowly, firstly takes place in the apical, i.e. supranuclear and perinuclear cell parts. Then the AF positive material located subnuclearly becomes gradually labelled. This is in support of the assumption that the apical cell parts are primarily concerned with synthesis and the basal cell parts with storage of secretory material. A marked influence of temperature on localization, appearance and amount of AF positive material in SCO cells has been observed.