Influence of cadmium on the distribution of the essential trace elements zinc and copper in the liver and kidneys of rats

Cd induced changes of Zn and Cd distribution in the liver and kidneys were studied in relation to Cd metallothionein (MT) synthesis. Wistar male rats were given CdCl₂ by sc injection of .8, 1.5, and 3.0 mg Cd/kg three times a week for three weeks. Cd levels of liver and kidneys increased with the increment of Cd dosage and 80–90% of Cd was found in the cytosol. The MT fractions contained 80–89% cytosolic Cd in the liver and 55–75% Cd in the kidneys. Zn concentrations in the liver increased following Cd administration, But Zn in the kidneys showed only slight increase. There was a distinct decrease of Cu concentration in the liver of the 3.0 mg group. In contrast, Cu concentrations in the kidneys increased about three times in the .8 and 1.5 mg Cd groups, but Cu in the 3.0 mg group showed only 1.5 times increase. The changes of these metal concentrations were observed mainly in the cytosol. Non-MT-Cd in the kidneys was maximum in the 1.5 mg group, but the 3.0 mg group showed significant decrease. In parallel with this decrease of Cd, Cu and Zn in the kidneys showed similar decrease. When the kidneys are injured, Zn and Cu appear to leak from this organ.     

Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sar-coplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca2 uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca2 -ATPase activity in SR and changing of character of Ca2 release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca2 transport of SR.     

Preliminary investigation of sequence-independent DNA binding proteins in rat skeletal muscle sarcoplasmic reticulum and their function

To observe the binding of plasmid DNA to non-nuclear DNA binding proteins in sarcoplasmic reticulum (SR) and the effects of this binding on SR function, sarcoplasmic reticulum proteins in rat skeletal muscle were isolated by differential centrifuge and sucrose density-gradient centrifuge. The results showed that there are two sequence-independent DNA binding proteins in SR proteins, the molecular weights of which are 83 and 58 ku, respectively. Ca²⁺ uptake and release of SR were remarkably promoted by the binding of plasmid DNA to DNA binding proteins in SR, the mechanism is probably through increasing of Ca²⁺-ATPase activity in SR and changing of character of Ca²⁺ release channel ryanodine receptors induced by the binding. These results suggest that there exist DNA binding proteins in SR and its binding to DNA may affect Ca²⁺ transport of SR.     

Protective effects of selenium against mercury toxicity as studied in the rat liver and kidney by nuclear analytical techniques

Mercuric chloride and sodium selenite were separately administered to male rats in the drinking water or in a combination (2.5 mmol Hg/L and 0.1 mmol Se/L). The mercuric chloride group showed histopathological lesions, as evidenced by cell necrosis in the liver and tubular necrosis in the kidney. The sodium selenite group showed some depression in growth, but pathological changes were found neither in the liver nor in the kidney. Simultaneous administration of both compounds produced a protective effect on weight loss and histopathology. These effects were associated with some small structures in the kidney proximal tubules and to some structure in the extracellular space in the liver. Thin, unstained cryosections were freeze-dried and examined in the Studsvik Nuclear Microprobe. The structures observed in the liver and the kidney were shown to contain both selenium and mercury.     

Variation of the level of mercury and metallothionein in the kidneys and liver of rats with time of exposure to sodium selenite

Sodium selenite was administered to rats before, after, and simultaneously with mercuric chloride. In all animal groups, mercury was administered intravenously in doses of 0.5 mg/kg every other day for two weeks. Selenium was given intragastrically either in a single dose of 7.0 mg Se/kg or in repeated doses of 0.1 mg Se/kg every day for weeks. It was demonstrated that, depending on the administration schedule, selenium induced significant changes in the binding of mercury by soluble fraction proteins both in the kidneys and in the liver. In every exposure, the mercury content decreased mainly in the low-molecular weight proteins, and the level of metallothionein-like proteins was diminished in the both organs. In the kidneys, the mercury content showed a correlation with the level of metallothionein (r=0.78). Amounts of mercury below 10 μg/g kidney do not stimulate metallothionein biosynthesis in this organ. A distinct interaction effect was observed in the case of a simultaneous administration of equimolar amounts of both the metals in question.     

Structural state of the Na+ /d-glucose cotransporter in calf kidney brush-border membranes Target size analysis of Na+-dependent phlorizin binding and Na+-dependent d-glucose transport

Target sizes of the renal sodium-d-glucose cotransport system in brush-border membranes of calf kidney cortex were estimated by radiation inactivation. In brush-border vesicles irradiated at ?50°C with 1.5 MeV electron beams, sodium-dependent phlorizin binding, and Na⁺-dependent d-glucose tracer exchange decreased exponentially with increasing doses of radiation (0.4–4.4 Mrad). Inactivation of phlorizin binding was due to a reduction in the number of high-affinity phlorizin binding sites but not in their affinity. The molecular weight of the Na⁺-dependent phlorizin binding unit was estimated to be 230 000 ± 38 000. From the tracer exchange experiments a molecular weight of 345 000 ± 24 500 was calculated for the d-glucose transport unit. The validity of these target size measurements was established by concomitant measurements of two brush-border enzymes, alkaline phosphatase and γ-glutamyltransferase, whose target sizes were found to be 68 570 ± 2670 and 73 500 ± 2270, respectively. These findings provide further evidence for the assumption that the sodium-d-glucose cotransport system is a multimeric structure, in which distinct complexes are responsible for phlorizin binding and d-glucose translocation.     

Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites

Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K_d = 0.15 μM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1″ phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.     

Cadmium,copper, and zinc levels in the rice and rice field soil of Houston,Texas

Fifty-one pairs of hulled rice samples and the soil from which each rice sample was grown were analyzed for heavy metals in August, 1979, in order to estimate the background contamination of cadmium (Cd), copper (Cu), and zinc (Zn) in rice grown in the Houston, Texas area. Both samples were divided into three groups by soil types and colors. The cadmium concentration in Texas rice was only one-half to one-quarter lower than that of Asian rice. However, the levels of Cu and Zn in rice in Texas were similar to those reported. Soil heavy metals were lower than ever reported, but these values were consistent with the geochemical characteristics of the Texas Houston area. No particular relationship was found between the three metals in rice and the metals in soil where the sampled rice was grown.     

Identification of the critical structural determinants of the EF-hand domain arrangements in calcium binding proteins

EF-hand calcium binding proteins (CaBPs) share strong sequence homology, but exhibit great diversity in structure and function. Thus although calmodulin (CaM) and calcineurin B (CNB) both consist of four EF hands, their domain arrangements are quite distinct. CaM and the CaM-like proteins are characterized by an extended architecture, whereas CNB and the CNB-like proteins have a more compact form. In this study, we performed structural alignments and molecular dynamics (MD) simulations on 3 CaM-like proteins and 6 CNB-like proteins, and quantified their distinct structural and dynamical features in an effort to establish how their sequences specify their structures and dynamics. Alignments of the EF2-EF3 region of these proteins revealed that several residues (not restricted to the linker between the EF2 and EF3 motifs) differed between the two groups of proteins. A customized inverse folding approach followed by structural assessments and MD simulations established the critical role of these residues in determining the structure of the proteins. Identification of the critical determinants of the two different EF-hand domain arrangements and the distinct dynamical features relevant to their respective functions provides insight into the relationships between sequence, structure, dynamics and function among these EF-hand CaBPs.     

Characteristics of high-affinity [3H]adenosine binding to rat brain synaptosomes and turkey erythrocyte membranes

High affinity binding sites for [³H]adenosine in rat brain and in turkey erythrocytes can be identified by binding experiments. Displacement experiments using a number of adenosine analogs indicate that these high affinity sites do not represent the R-type adenosine receptors which mediate activation of adenylate cyclase, although the binding is theophylline sensitive. Similarly, the binding of [³H]adenosine is not to the P-site, which mediates inhibition of adenylate cyclase, since the high affinity binding persists in the presence of 2′,5′-dideoxyadenosine. Furthermore, these results remain qualitatively similar also in the presence of dipyridamole which blocks adenosine transport sites. We conclude that theophylline sensitivity does not indicate that [³H]adenosine binding sites correspond to adenosine receptors coupled to adenylate cyclase.     

Ethanol increases mitochondrial cytochrome P450 2E1 in mouse liver and rat hepatocytes

Enhanced hepatic levels of cytochrome P450 2E1 (CYP2E1) may play a key role in the pathogenesis of some liver diseases because CYP2E1 represents a significant source of reactive oxygen species. Although a large fraction of CYP2E1 is located in the endoplasmic reticulum, CYP2E1 is also present in mitochondria. In this study, we asked whether ethanol, a known inducer of microsomal CYP2E1, could also increase CYP2E1 within mitochondria. Our findings indicated that ethanol increased microsomal and mitochondrial CYP2E1 in cultured rat hepatocytes and in the liver of lean mice. This was associated with decreased levels of glutathione, possibly reflecting increased oxidative stress. In contrast, in leptin-deficient obese mice, ethanol administration did not increase mitochondrial CYP2E1, nor it depleted mitochondrial glutathione, suggesting that leptin deficiency hampers mitochondrial targeting of CYP2E1. Thus, ethanol intoxication increases CYP2E1 not only in the endoplasmic reticulum but also in mitochondria, thus favouring oxidative stress in these compartments.     

25-Hydroxylation of 1-α-hydroxyvitamin D-3 in rat and human liver

1α-Hydroxyvitamin D-3 25-hydroxylase activity was measured in subcellular fractions of rat and human liver. The formation of 1,25-dihydroxyvitamin D-3 was determined by high pressure liquid chromatography. In rat liver 1α-hydroxyvitamin D-3 25-hydroxylase activities were found in the purified nuclei, the heavy mitochondrial fraction and the microsomal fraction. The enrichment of 25-hydroxylase activity was highest in the heavy mitochondrial fraction. With this fraction a minimum amount (about 0.5 mg) of protein was required before formation of 1,25-dihydroxyvitamin D-3 could be detected. Above this amount the reaction was linear with amount of protein up to at least 2 mg/ml. The reaction was also linear with time up to 60 min. An apparent K_m value of 2·10^?5 M was found. The mitochondrial 25-hydroxylase was stimulated by addition of cytosolic protein or bovine serum albumin. The degree of stimulation was dependent on the amount of mitochondrial protein present in the incubation mixture. Maximal stimulation was seen with 0.2 mg/ml of either protein in the presence of 0.5 mg mitochondrial protein. The stimulating effect remained after heating the protein for 5 min at 100°C. The cytosolic protein did not stimulate a reconstituted mitochondrial 1α-hydroxyvitamin D-3 25-hydroxylase. The mitochondrial vitamin D-3 25-hydroxylase was inhibited both by cytosolic protein and by bovine serum albumin. Human liver revealed only one 1α-hydroxyvitamin D-3 25-hydroxylase activity located to the heavy mitochondrial fraction. The results are in agreement with previous studies on the localization of vitamin D-3 25-hydroxylase in rat and human liver. The difference in localization of the 25-hydroxylase between rat and human liver implies that studies on the regulation of the microsomal 25-hydroxylase in rat liver may not be relevant to the situation in human liver.     

Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is locted Important variables that were studied included adding NADH, adding, induction of enzymed by intake of phenobarbital and the effects of oxygen. Reduction of nonparamagnetic derivatives and oxidation to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADh or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b₅ and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylmines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells.     

The role of aryl hydrocarbon receptor and the reactive oxygen species in the modulation of glutathione transferase by heavy metals in murine hepatoma cell lines

Glutathione transferase (GST) is a phase II detoxifying enzyme that plays a protective mechanism against oxidizing substances and toxic contaminants. Among these contaminants, heavy metals and polycyclic and halogenated aromatic hydrocarbons (PHAHs) have been shown to exert their toxic effects through the modulation of detoxifying enzymes, including the GSTs. Recently, we showed that heavy metals particularly Hg2+, Pb2+, and Cu2+ modulate the expression of phase II detoxifying enzymes such as NAD(P)H:quinone oxidoreductase 1 and Gsta1 in a concentration- and time-dependent manner. However, the effect of heavy metals and their potential interactions with aryl hydrocarbon receptor (AhR) ligands, PHAHs, on total Gst activity is still unknown. In the current study, we have investigated the effects of Hg2+, Pb2+, and Cu2+ in the absence and presence of four AhR ligands on the total Gst activity and reactive oxygen species (ROS) production in wild-type and AhR-deficient Hepa 1c1c7 cells. Our results showed that Hg2+ and Cu2+, but not Pb2+, significantly induced Gst activity in wild-type cells, whereas all metals induced the Gst activity in AhR-deficient cells. The induction of Gst activity by heavy metals was strongly correlated with an increase in the ROS production in wild-type, but not in AhR-deficient cells. Co-administration of heavy metals with AhR ligands differentially modulated Gst activity, in that co-exposure to Hg2+ plus AhR ligands could be beneficial in protecting against cytotoxicity as demonstrated by the increase in Gst activity with a proportional decrease in ROS production. Whereas co-exposure to Cu2+ plus AhR ligands was more toxic in that a decrease in Gst activity and an increase in oxidative stress of the cell were observed. We concluded that heavy metals differentially modulate the Gst activity through oxidative stress- and AhR-mediated mechanisms.     

Synthesis and binding potencies of cyclohexapeptide somatostatin analogs containing naphthylalanine and arylalkyl peptoid residues

We report the synthesis, binding affinities to the recombinant human somatostatin receptors, and structure‐activity relationship studies of compounds related to the cyclic hexapeptide, c‐[Pro⁶‐Phe⁷‐D‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹], L‐363,301 (the numbering in the sequence refers to the position of the residues in native somatostatin). The Pro residue in this compound is replaced with the arylalkyl peptoid residues Nphe (N‐benzylglycine), (S)βMeNphe [(S)‐N‐[(α‐methyl)benzyl]glycine] or (R)βMeNphe [(R)‐N‐[(α‐methyl)benzyl]glycine] and l ‐1‐naphthylalanine is incorporated into either position 7 or 11 of the parent compound. The synthesis and binding data of the Nnal⁶ ([N‐naphthylmethyl]glycine) analog of L‐363,301 is also reported. The incorporation of the Nnal residue into position 6 of L‐363,301 resulted in an analog with weaker binding affinities to all hsst receptors but enhanced selectivity towards the hsst2 receptor compared with the parent compound. The other compounds bind effectively to the hsst2 receptor but show some variations in the binding to the hsst3 and hsst5 receptors resulting in different ratios of binding affinities to the hsst5 and hsst2 or hsst3 and hsst2, respectively. The incorporation of the Nphe residue into position 6 and the Nal residue into position 7 of L‐363,301 led to a compound which binds potently to the hsst2 and has increased selectivity towards this receptor (weaker binding to hsst3 and hsst5 receptors) compared with the parent compound. The analogs with β‐methyl chiral substitutions in the aromatic peptoid side chain and Nal in position 7 or 11 bind effectively to the hsst2 and hsst5 receptors. They exhibit similar ratios of binding affinities to the hsst5 and hsst2 receptors as observed for L‐363,301. There are however minor differences in binding to the hsst3 receptor among these analogs. These studies allow us to investigate the influence of additional hydrophobic groups on the binding activity to the isolated human somatostatin receptors and the results are important for the design of other somatostatin analogs. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Specificity of covalently stabilized complexes of 125I-labeled human somatotropin and components of the lactogenic binding sites of rat liver

¹²⁵I-labeled human somatotropin specifically bound to the lactogenic sites of microsomal membranes from pregnant rat liver, originated a radioactive covalent complex of M_r 63,000 upon reaction with dimethyl suberimidate, disuccinimidyl suberate (DSS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The amoun of this species was closely parallel with the preexisting amount of the ligand-receptor complex. Photoactivation of a hormone derivative bound to the receptor also gave rise to the 63 K species. A ternary complex of receptor, hormone and Triton X-100 cross-linked with DSS yielded the 63 K species and a new one of 96 K. The data indicate that the 63 K complex involves the radioactive hormone and a constituent of the binding site. The 96 K species could comprise a second component of the receptor.     

Autoradiographic reevaluation of the binding properties of 125I-[Leu31,Pro34]peptide YY and 125I-peptide YY3-36 to neuropeptide Y receptor subtypes in rat forebrain

125I-[Leu31,Pro34]peptide YY (PYY) and 125I-PYY3-36, initially described as selective neuropeptide Y Y1 and Y2 receptor ligands, respectively, were recently shown to label also Y4 and Y5 receptors. We used receptor autoradiography to assess whether these ligands can be reliably used to investigate the various neuropeptide Y receptors in rat forebrain. In most of the brain regions examined (in coronal sections at the level of dorsal hippocampus), specific 125I-[Leu31,Pro34]PYY binding was completely inhibited by 1 microM BIBP-3226, a selective Y1 receptor ligand, but unaffected by 10 nM rat pancreatic polypeptide, selectively inhibiting Y4 receptors, suggesting that Y4 receptors are present in negligible numbers compared with Y1 receptors in the areas examined. Significant numbers of BIBP-3226-insensitive 125I-[Leu31,Pro34]PYY binding sites were measured in the CA3 subfield of the hippocampus only, possibly representing Y5 receptors. 125I-PYY3-36 binding was unchanged by 1 microM BIBP-3226, whereas a population of 125I-PYY3-36 binding sites was sensitive to 100 nM [Leu31,Pro34]neuropeptide Y, likely representing Y5 receptors. The possibility of distinguishing between Y2 and Y5 receptors using 125I-PYY3-36 as radioligand was validated by their different regional distribution and their distinct changes 24 h after kainate seizures, i.e., binding to Y5 receptors was selectively decreased in the outer cortex, whereas binding to Y2 receptors was enhanced in the hippocampus. Thus, the use of selective unlabeled compounds is required for distinguishing the various receptor subtypes labeled by 125I-[Leu31,Pro34]PYY and 125I-PYY3-36 in rat brain tissue.