Oxidative modification of human ceruloplasmin by methylglyoxal: an in vitro study

Methylglyoxal (MG) is an endogenous physiological metabolite which is present in increased concentrations in diabetics. MG reacts with the amino acids of proteins to form advanced glycation end products. In this in vitro study, we investigated the effect of MG on the structure and function of ceruloplasmin (CP) a serum oxidase carrier of copper ions in the human. When CP was incubated with MG, the protein showed increased electrophoretic mobility which represented the aggregates at a high concentration of MG (100 mM). MG-mediated CP aggregation led to the loss of enzymatic activity and the release of copper ions from the protein. Radical scavengers and copper ion chelators significantly prevented CP aggregation. CP is an important protein that circulates in plasma as a major copper transport protein. It is suggested that oxidative damage of CP by MG may induce perturbations of the copper transport system and subsequently lead to harmful intracellular condition. The proposed mechanism, in part, may provide an explanation for the deterioration of organs in the diabetic patient.     

Recommendations for testing new chelating agents for removal of incorporated actinides from the body

Summary The body content of certain radionuclides and the consequences of accidental incorporation may be reduced by treatment with chelating agents. Such agents have been widely studied and have proved to be useful in man. Chelation therapy may also be advantageous in certain cases of heavy metal poisoning. There is still a need to develop new, more efficient and less toxic agents and better therapeutic schedules for using the existing agents. So far as possible the testing of potential new compounds should be carried out using standardized methods.Supported by the Radiation Protection Programme of the Commission of the European Communities-publication no 1889     

Toxicology of vanadium compounds in diabetic rats: The action of chelating agents on vanadium accumulation

The possible use of vanadium compounds in the treatment of diabetic patients is now being evaluated. However, previously to establish the optimal maximum dose for diabetes therapy, it should be taken into account that vanadium is a highly toxic element to man and animals. The toxic effects of vanadium are here reviewed. The tissue vanadium accumulation, which would mean an additional risk of toxicity following prolonged vanadium administration is also discussed. Recently, it has been shown that coadministration of vanadate and TIRON, an effective chelator in the treatment of vanadium intoxication, reduced the tissue accumulation of this element, decreasing the possibility of toxic side effects derived from chronic vanadium administration without diminishing the hypoglycemic effect of vanadium. However, previously to assess the effectiveness of this treatment in diabetic patients, a critical reevaluation of the antidiabetic action of vanadium and its potential toxicity is clearly needed.     

Cadmium inhibits delta-aminolevulinate dehydratase from rat lung in vitro: interaction with chelating and antioxidant agents

The effect of cadmium (Cd(2+)) on delta-aminolevulinate dehydratase (delta-ALA-D) activity from rat lung in vitro was investigated. delta-ALA-D activity, a parameter for metal intoxication, has been reported as a target of Cd(2+) in different tissues. The protective effect of monotherapies with dithiol chelating (meso-2,3-dimercaptosuccinic acid (DMSA) and 2,3-dimercaptopropane-1-sulfonic acid (DMPS)) or antioxidant agents (ascorbic acid, diphenyl diselenide (PhSe)(2), and N-acetylcysteine (NAC)) was evaluated. The effect of a combined therapy (dithiol chelatingxantioxidant agent) was also studied. Zinc chloride (ZnCl(2)) and dithiothreitol (DTT) were used to investigate the mechanisms involved in cadmium, chelating and antioxidant effects on delta-ALA-D activity. Cadmium inhibited rat lung delta-ALA-D activity at low concentrations. DTT (3mM), but not ZnCl(2) (100microM), protected the inhibition of enzyme activity caused by Cd(2+). Chelating agents were not effective in restoring the enzyme activity. DMPS and DMSA presented inhibitory effect on enzyme activity. DTT restored the inhibition caused by both chelating agents, but ZnCl(2) restored only the inhibitory effect induced by DMSA. These compounds caused a marked potentiation of delta-ALA-D inhibition induced by Cd(2+). ZnCl(2) did not restore inhibition of enzyme activity caused by Cd(2+) plus chelating agents. Conversely, DTT restored the inhibition induced by Cd(2+)/DMSA, but not by Cd(2+)/DMPS. Antioxidants were not effective in ameliorating delta-ALA-D inhibition induced by Cd(2+), whereas ascorbic acid potentiated the enzyme inhibition induced by this metal. A combined effect of Cd(2+)xDMPSx(PhSe)(2) and Cd(2+)xDMPSxNAC was observed. There was no combined effect of Cd(2+)xchelatorxantioxidants when DMSA was used. This study demonstrated that Cd(2+)inhibited delta-ALA-D activity and chelating and antioxidant agents, alone or combined, did not restore the enzyme activity. In contrast, these compounds potentiated the inhibition induced by Cd(2+) in rat lung.     

A study of the electron transfer properties of the heme undecapeptide from cytochrome c by 1H nmr spectroscopy

Nuclear magnetic resonance (nmr) spectroscopy has been used to investigate the heme undecapeptide from cytochrome c. Assignments of resonances to specific residues have been made based on spin decoupling, redox titration, and the pH and temperature dependence of resonance lines. An outline structure is presented based on the assignments, secondary shift data, and the x-ray crystal structure of cytochrome c. An equation is derived to relate the width of an nmr line during a redox titration to the percentage of each oxidation state. Using this equation the self-exchange rate constant for electron transfer for the heme peptide is 1.3 x 10(7) M-1 sec-1 at 330 degrees K. Discussion of the self-exchange rate constants of cytochrome c, cytochrome c3, and cytochrome c551 is related to this constant for the heme undecapeptide.     

The discovery of the nature of ferredoxin in photosystems: A recollection

I describe here my recollection of the story of the discovery of the nature of ferredoxin in photosystems that began in 1965: this story involved the EPR measurements by a young physicist J.H.M. Thornley, using samples provided by J.F. Gibson and D. Hall, and in collaboration with F.R. Whatley.     

Opioid receptor agonists in the rabbit colon: comparison of in vivo and in vitro studies

Myoelectrical activity was recorded in the proximal and distal colon of rabbits using chronically implanted electrodes. The motility in both the proximal and distal colon was inhibited by the intravenous (IV) administration of the following opioid agonists for mu receptors: morphine and fentanyl, kappa receptors: ethylketazocine (EKC) and U 50 488 H, and delta receptors: D-Ala2 D-Leu5-enkephalin (DADLE) and D-Ser2 Leu-enkephalin-Thr6 (DSLET). In contrast, the myoelectric activity in the distal colon was increased during the infusion of an endogenous kappa opioid agonist, dynorphin (DYN). All of these effects were prevented by naloxone pretreatment. During in vitro studies using extraluminal force transducers, fentanyl, U 50 488 H and DSLET inhibited spontaneous contractions of the proximal colon, but U 50 488 H and DSLET caused a substantial increase in the motility of the distal colon. The observed motor responses in the proximal and distal colon following opioid agonist administration indicate that the control of these two intestinal segments may be different. It is suggested that the stimulatory effect of dynorphin on the distal colon is peripherally-mediated while inhibition of the whole colon by opioid agonists regardless of subtypes seems to be centrally-mediated.     

Determination of free Ca ion concentrations with an ion-selective electrode in the presence of chelating agents in comparison with calculated values.

Experimentally determined free Ca ion concentrations, measured with a Ca-selective electrode, were compared with values calculated with a computer program utilizing stability constants of the chelating agents: NTA, EDTA, and EGTA used to set the free ion concentration in the range of 10^?3 to 10^?6m. In the presence of 0.1 m KCl, 2 mm MgCl₂, 20 mm Hepes (pH 7.4), 2 mm ATP, 0.1 mm CaCl₂ (total concentration), and various ligand concentrations the measured free Ca²⁺ levels were found to be approximately six to seven times greater than the computer-derived values. Apparent stability constants for Ca-ATP, Ca-EDTA, and Ca-EGTA were determined under these experimental conditions.     

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.     

Amino acid sequencing by gas chromatography-mass spectrometry using trifluoro-dideuteroalkylated peptide derivatives: C. The primary structure of the carboxypeptidase inhibitor from potatoes

The carboxypeptidase inhibitor from potatoes has been used to demonstrate the utility of gas chromatography-mass spectrometry for the determination of the primary structure of such large polypeptides. Two mixtures of oligopeptide fragments, obtained by limited acid hydrolysis and enzymatic digestion of this polypeptide, were transformed into the corresponding mixtures of O-trimethyl-silylated trifluoro-dideuteroethyl polyamino alcohols which were then analyzed by gas chromatography-mass spectrometry. The resulting mass spectral and retention index data allowed the identification of 61 oligopeptide fragments which were assembled by the computer by positioning all 39 amino acid residues in a unique sequence (with the exception of the assignment of the primary amide groups of Asn and Gln).     

The postnatal development of serum zinc, copper and ceruloplasmin in the horse

1. Serum samples were collected from ten foals at predetermined times during the first 12 months following birth and zinc and copper concentrations and ceruloplasmin activity were evaluated. 2. Serum zinc concentrations were found to be quite variable with respect to age (range = 67-95 micrograms/dl). 3. Serum copper concentrations increased in a linear fashion from day 0 to day 28 before levelling off at 190-247 micrograms/dl. 4. Ceruloplasmin activity was found to correlate with the concentration of serum copper (r = 0.92) and reached a plateau at an activity of 30-38 IU by day 28.     

Regulatory cells in human bone marrow: Suppression of an in vitro primary antibody response

Human bone marrow (BMC) contains regulatory cells that can suppress the in vitro primary PFC response of normal allogeneic spleen or tonsillar cells and autologous peripheral blood cells. Suppression is dependent upon the dose of BMC added, but is not due to cell crowding nor to excessive cytotoxicity, and requires the presence of viable, metabolically active BMC. BMC are maximally inhibitory when added during the first 24 hr of culture and do not cause an induced shift in the kinetics of the response. Thus, suppression reflects inhibition of early inductive events in the antibody response. The target of suppression is the non-T cell, with either polyclonal activator or Ag being required for maximal suppression. DNA synthesis of normal tonsillar cells is not inhibited by BMC. Characterization of the human bone marrow-suppressor cell has shown it to be radiosensitive, E-rosette negative, Fc receptor positive, and to reside in the large, weakly adherent cell population after velocity sedimentation and in the lymphocyte-depleted fraction after sucrose density gradient separation. Pretreatment of the bone marrow-suppressor cell with anti-human thymocyte serum does not abrogate suppression. We speculate on a possible physiologic role for this cell.     

Auto and heterotransplantation of the ovary to the uterus or parotid region in beef cows and prepuberal heifers

Eighteen mature estrous cycling beef cows and 9 prepuberal heifers were stratified by breed, age and weight to determine the effect of ovary-transplantation to a proximal site (right uterine horn) (U) and distal site (parotid region) (P) upon ovarian activity. Active ovaries (AO), ovaries with the corpus luteum (CL), were autotransplanted to the myometrium of the U in 3 cows and to the muscles of the P in 2 cows and their inactive ovaries (IO), ovaries without a CL, remained. Active ovaries of 6 cows were removed and heterotransplanted to 6 prepuberal heifers and their 10 were heterotransplanted to the U or the parotid (3). Six heifers received either a mature AO in the U or in the parotid. Three heifers were ovariectomized and their ovaries were heterotransplanted to 6 cows, 3 per site. Cows and heifers were slaughtered randomly 2 months after surgery and their ovaries were collected for microscopic and histological analysis. The transplants were successfully accomplished in 94% of the cows and in 83% of the heifers. Both of the unsuccessful heterotransplantations were located in the uterus. More estrous activity was found (P<.025) in cows than in heifers with their own ovaries in situ . All prepuberal ovaries in situ showed follicular development when mature AO were transplanted to either the U or parotid. The same trend was found in prepuberal ovaries transplanted to mature cycling cows. Cows with an IO in situ and AO transplanted to either site had more estrous activity than did ovariectomized cows with an IO transplanted to either site. Pregnancy rates in mature cycling cows with an least one ovary in situ were higher (P<.005) in cows with an ovary in the parotid region than cows with an ovary transplanted to the uterus.     

Rat enterocyte Na+ transport in vitro action of parathyroid hormone and calcitonin

Parathyroid hormone (PTH) and calcitonin exert well known effects on the renal tubule which are thought to involve specific hormone receptors and adenyl cyclase. In the intestine, it is not clear whether the action of PTH and calcitonin is only indirect or also direct, and their mechanisms of action are much less well established. In the present study, possibly direct effects of PTH and calcitonin on Na⁺ transport in isolated intestinal epithelial cells of rats were investigated. In the presence of bovine PTH (1.2 I.U./ml) in the incubation medium, the Na⁺ efflux rate constant (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$) of isolated enterocytes was significantly reduced when compared to that in control experiments with the hormone vehicle only. The mean depression of ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ induced by bovine PTH was 26% as compared to the control (100%) and to that induced by ouabain (4.0mM) which was 44%. No depressant effect of bovine PTH on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ was observed when the isolated enterocytes were incubated with ouabain (4.0 mM). Thus, bovine PTH appeared to inhibit the ouabain-sensitive Na⁺ pump. When incubating the isolated epithelial cells in an EGTA-containing Ca²⁺-free medium, bovine PTH lost its capacity to inhibit (${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$). Thus, the presence of extracellular Ca²⁺ appeared necessary for the inhibitory effect of bovine PTH. In contrast to its effect on ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$, bovine PTH induced no change in net Na⁺ uptake by isolated enterocytes. Moreover, no significant effect on enterocyte Na⁺ transport could be demostrated for salmon or porcine calcitonin at two different concentrations in the incubation medium. Neither bovine PTH nor salmon calcitonin induced significant changes in enterocyte cyclic AMP or cyclic GMP concentrations. It was concluded that bovine PTH, but not calcitonin, exerted a direct inhibitory effect on the ouabain-sensitive ${}^{\mathrm{o}}\text{K}{}_{\mathrm{Na}}$ of isolated rat enterocytes. The effect of bovine PTH occured without measurable activation of the cyclic nucleotide system but needed the presence of Ca²⁺ in the incubation medium to be operative.     

Kinetics of the removal of ferric ion from transferrin by aminoalkylphosphonic acids

The kinetics of ion removal at 25 degrees C in 0.1 M Tris, pH 7.4 by a series of phosphonic acids have been evaluated. The initial rate of iron removal is first order in ferric-transferrin, but shows a hyperbolic dependence on the concentration of the phosphonate ligand. At high ligand concentrations the reaction is clearly biphasic, and the data are interpreted in terms of nonequivalent rate constants for iron removal from the two transferrin iron-binding sites. Rate constants for three phosphonic acid ligands are approximately 0.025 min-1 and approximately 0.007 min-1 for the faster and slower binding sites. The results are discussed in relation to the conformational change mechanism for iron removal from transferrin proposed by Coward et al. [21].