Growth-modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes

The tripeptide H-Gly-His-Lys-OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.     

Evaluation of metals in a defined medium for Pichia pastoris expressing recombinant beta-galactosidase

Culture growth and recombinant protein yield of the Pichia pastoris GS115 methanol utilization positive system were studied in response to the types and levels of metals present in the growth medium and the supplemental salts typically used for these fermentations. Magnesium and zinc were both required to support cell growth but at significantly reduced levels compared to the control. However, supplementation with calcium, cobalt, iron, manganese, iodine, boron, and molybdenum were not required to sustain cell mass. When the medium was reformulated with only zinc and magnesium, the cells grew to 12-15 generations, which are expected for high cell density fed-batch fermentations. Product yields of the recombinant protein beta-galactosidase were significantly influenced by the trace metal concentrations. By using response surface and full factorial designs, maximum protein yield occurred when the concentration of zinc salt was limited to the level necessary only to support cell mass while protein yield positively correlated to increasing levels of the remaining trace metal salts. These studies are the first to show that excess trace metals must be optimized when developing P. pastoris based fed-batch fermentations.     

Trace element uptake by L-cells as a function of trace elements in a synthetic growth medium

The concentration of trace elements in L-cells has been studied as a function of the trace metal content of the growth medium. Cells were cultured in synthetic media which contained varying trace amounts of the elements manganese, iron, cobalt, copper, zinc and molybdenum. The cellular concentration of the elements potassium, iron, copper and zinc were then determined. It was found that the cell accumulates trace metals at a different rate than they are made available. Deficiencies in zinc could be “induced” in the cell by increasing the concentration of iron, manganese and cobalt; cellular iron deficiencies were observed at larger medium concentrations of zinc, manganese, copper and cobalt. Trace metal uptake by the cell was seen to parallel the utilization by multicellular organisms.     

Effect of divalent cations of the amphetamine-induced stimulation of [3H]catechol synthesis in the striatum.

Abstract— The effects of divalent cations on the stimulation of [³H]catechol formation in striatal slices induced by d-amphetamine was studied in order to determine the role of calcium in this action of amphetamine. In the absence of any divalent cations in the medium, amphetamine did not significantly stimulate [³H]catechol synthesis in striatal slices, but it produced a marked stimulation of synthesis when calcium (1.25 mm ) was added to the medium. In the presence of calcium (1.25 mm ), high concentrations of magnesium (15mm ), other divalent cations (2.5 mm ) such as barium, strontium, manganese and cobalt, as well as verapamil, inhibited the amphetamine-induced stimulation. When the slices were incubated in medium containing no divalent cations, the addition to the medium of either strontium, cobalt, zinc, or magnesium (2.5 mm ) could not support the amphetamine-induced stimulation of [³H]catechol synthesis, while the addition of barium resulted in a significant stimulation of synthesis. In contrast, the stimulation produced by amphetamine in the presence of manganese was comparable to that observed when calcium had been added to the medium. Since amphetamine did not alter the specific activity of [³H]tyrosine in the tissue in the presence of any of the divalent cations tested, the amphetamine-induced stimulation of [³H]catechol synthesis was probably due to an increase in tyrosine hydroxylase activity. Calcium and manganese were also able to support the stimulation of [³H]catechol synthesis in striatal slices induced by high potassium concentration. However, compared to the effects with amphetamine, manganese was much less effective than calcium in supporting the stimulation induced by high potassium concentration. These results show that specific divalent cations can support the stimulation of catechol synthesis induced by amphetamine in striatal slices, and suggest that the entry of these specific ions into cells, presumably dopamine neurons, is involved in this action.     

Heavy metals in some Chinese herbal plants

The concentrations of nine heavy metals, cadmium, cobalt, copper, iron, manganese, nickel, lead, zinc and mercury in 42 Chinese herbal medicinal plants were determined. Generally, all the samples studied had, relative to the other trace metals, higher concentrations of iron, manganese, and zinc. The concentration range of the metals determined was comparable to that in many of the East Asian vegetables and fruits. A few samples were found to contain relatively higher concentrations of the toxic metals such as cadmium, lead, and mercury. This was probably caused by contamination during air-drying and preservation.     

Effects of carbon source, phosphorus concentration, and several micronutrients on biomass and geosmin production by Streptomyces halstedii

The effects of various carbon sources, phosphorus concentration, and different concentrations of the micronutrients calcium, cobalt, copper, iron, manganese, potassium, and zinc were determined on biomass dry weight production, geosmin production, and geosmin/biomass (G/B) values for Streptomyces halstedii, a geosmin-producing actinomycete isolated from the sediment of an aquaculture pond. Of the substrates tested, maltose as a sole carbon source promoted maximal growth by S. halstedii while mannitol promoted maximal geosmin production, and galactose yielded the highest G/B values. Fish-food pellets and galactose were poor substrates for growth. Increasing phosphorus concentrations enhanced geosmin production and G/B values. Of the seven micronutrients tested, zinc, iron, and copper had the most profound effects on biomass and geosmin production. Increasing zinc concentrations promoted biomass production while inhibiting geosmin production and G/B values; increasing concentrations of copper and iron inhibited biomass and geosmin production. Increased copper concentrations had the greatest effect in preventing growth and geosmin production by S. halstedii. Journal of Industrial Microbiology & Biotechnology (2001) 26, 241–247. Received 20 September 2000/ Accepted in revised form 17 January 2001     

Quantitation of seven elements in Mycobacterium phlei and Mycobacterium bovis by neutron activation analysis.

Quantitative determination of the elements potassium, sodium, manganese, magnesium, iron, cobalt and zinc was performed in mycobacteria by neutron activation analysis. Mycobacterium phlei ATCC 19 249 at different phase of growth (4, 8, 13, 23 and 37 days old cultures), and 14 days old Mycobacterium bovis BCG cultures and uninoculated semi-synthetic Sauton culture media were examined. The elements studied could be divided into three groups; sodium, potassium and magnesium could be regarded as major, iron as minor, and zinc, manganese and cobalt as trace elements. M. phlei contained, with the exception of zinc, higher amounts of elements than M. bovis. Other metals (aluminium, antimony, rubidium) could also be detected.     

Relationship of trace metals to the alcohol-effect in citric acid synthesis by Aspergillus fonsecaeus

Summary Synthesis of citric acid by Aspergillus fonsecaeus in a basal medium supplemented with trace metals (iron, zinc, copper, and manganese) and methanol has been investigated. Methanol was found to be stimulatory to acid synthesis when manganese or manganese and iron were present. No other mineral tested could be substituted for manganese in the presence of the alcohol. However, comparable yields of citric acid could be obtained if methanol were deleted, the concentration of iron increased and zinc substituted for manganese.This investigation was conducted under a contract with the United States Department of Agriculture, authorized by the Research and Marketing Act, and administered through the Northern Regional Research Laboratory.     

The effects of 17beta-estradiol and ethanol on zinc- or manganese-induced toxicity in SK-N-SH cells

Serious neurodegenerative disorders are increasingly prevalent in our society and excessive oxidative stress may be a key mediator of neuronal cell death in many of these conditions. A variety of metals, such as manganese and zinc, are essential trace elements but can reach localized toxic concentrations through various disease processes or environmental exposures and have been implicated as having a role in neurodegeneration. Both manganese and zinc exist as bivalent cations and are essential cofactors/activators for numerous enzymes. Evidence suggests one action of these metals, when concentrated beyond physiological levels, may be to inhibit cellular energy production, ultimately leading to increased radical formation. Our studies were undertaken to directly investigate the toxic effects of manganese and zinc in an immortalized neuronal-like cell line (SK-N-SH) by testing interactions with the antioxidant, 17beta-estradiol, and the neurotoxin, ethanol. Employing undifferentiated SK-N-SH cells, we found that these metals caused biphasic effects, enhancing cell proliferation at low doses and inducing cell death at higher doses. Zinc was both more efficacious and more potent than manganese in enhancing growth and in causing cell death. 17beta-Estradiol and ethanol enhanced the proliferative actions of zinc and manganese across a wide concentration range. Furthermore, co-treatment with either 17beta-estradiol or ethanol afforded protection against manganese-, but not zinc-induced toxicity. Finally, combined administration of 17beta-estradiol and ethanol to SK-N-SH cells resulted in both a loss of growth enhancement and protective properties that were observed when these substances were administered individually. We also noted that the toxic effects occurred more rapidly from zinc than manganese exposure. Taken together, these data suggest that oxidative stress likely has a role in cell death resulting from toxic exposure to either zinc or manganese, but there is a difference in the precise mechanism of their effects.     

Long-Term Sorption of Metals Is Similar among Plastic Types: Implications for Plastic Debris in Aquatic Environments

Concerns regarding plastic debris and its ability to accumulate large concentrations of priority pollutants in the aquatic environment led us to quantify relationships between different types of mass-produced plastic and metals in seawater. At three locations in San Diego Bay, we measured the accumulation of nine targeted metals (aluminum, chromium, manganese, iron, cobalt, nickel, zinc, cadmium and lead) sampling at 1, 3, 6, 9 and 12 months, to five plastic types: polyethylene terephthalate (PET), high-density polyethylene (HDPE), polyvinyl chloride (PVC), low-density polyethylene (LDPE), and polypropylene (PP). Accumulation patterns were not consistent over space and time, and in general all types of plastic tended to accumulate similar concentrations of metals. When we did observe significant differences among concentrations of metals at a single sampling period or location in San Diego Bay, we found that HDPE typically accumulated lesser concentrations of metals than the other four polymers. Furthermore, over the 12-month study period, concentrations of all metals increased over time, and chromium, manganese, cobalt, nickel, zinc and lead did not reach saturation on at least one plastic type during the entire 12-month exposure. This suggests that plastic debris may accumulate greater concentrations of metals the longer it remains at sea. Overall, our work shows that a complex mixture of metals, including those listed as priority pollutants by the US EPA (Cd, Ni, Zn and Pb), can be found on plastic debris composed of various plastic types.     

Biomethanation of spent wash: Heavy metal inhibition of methanogenesis in synthetic medium

Five heavy metals detected in distillery waste were lead (1.0–8.8 μg/ml), copper (1.7–15.7 μg/ml), zinc (3.1–11.8 μg/ml), iron (36.0–43.5 μg/ml), and manganese (3.0–5.1 μg/ml). Their toxicity to biomethanogenesis in a synthetic medium containing 1% sodium acetate, propionate, or butyrate was measured by batch fermentation, after cultivating the bacterial biomass semicontinuously. Lead, copper, and zinc in decreasing order were found to be toxic to biomethanogenesis. Lead at the concentration of 10 μg/ml completely stopped methane production. Iron did not produce any notable change in the process while manganese stimulated the rate of methane production. The toxicity of lead, copper, and zinc to methanogenic bacteria and methane production was dose-dependent but the growth of acetogenic bacteria was impaired at higher concentrations (2.5–10.0 μg/ml) of lead, copper, and zinc. Manganese stimulated the growth of only methanogenic bacteria, but not that of non-methanogenic bacteria or acetic acid production. The reduction in the synthesis of acetic acid via butyrate was more in the presence of these three metals than the synthesis of this acid via propionate.     

Hydrogen cyanide, a secondary metabolite of Pseudomonas aeruginosa.

Seventy-four of 110 strains of Pseudomonas aeruginosa tested produced detectable amounts of HCN from growth in 2% peptone or nutrient agar. Of the 25 species of12 bacterial and fungal genera tested, other than P. aeruginosa, only P. fluorescens and P. polycolor gave positive HCN tests. Cyanide is produced after cessation of active growth. Iron was stimulatory to cyanogenesis in concentration above 1 muM, while copper, zinc, cobalt, and manganese at concentrations of 20 muM had no effect. Cyanogenesis id dependent on the temperature of incubation within ranges which allow complete growth. Inorganic phosphate in concentrations between 90 and 300 mM allows growth but inhibits HCN production. Growth of cells anaerobically, using nitrate as the electron acceptor, results in low cyanide yields, which can be partially reversed by subsequent aerobic incubation. These results indicate that HCN is a secondary metabolite of P. aeruginosa.     

Metal Concentrations in Cerebrospinal Fluid and Blood Plasma from Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal degenerative disorder of motor neurons. The cause of this degeneration is unknown, and different causal hypotheses include genetic, viral, traumatic and environmental mechanisms. In this study, we have analyzed metal concentrations in cerebrospinal fluid (CSF) and blood plasma in a well-defined cohort (n?=?17) of ALS patients diagnosed with quantitative electromyography. Metal analyses were performed with high-resolution inductively coupled plasma mass spectrometry. Statistically significant higher concentrations of manganese, aluminium, cadmium, cobalt, copper, zinc, lead, vanadium and uranium were found in ALS CSF compared to control CSF. We also report higher concentrations of these metals in ALS CSF than in ALS blood plasma, which indicate mechanisms of accumulation, e.g. inward directed transport. A pattern of multiple toxic metals is seen in ALS CSF. The results support the hypothesis that metals with neurotoxic effects are involved in the pathogenesis of ALS.     

EDTA chelation effects on urinary losses of cadmium, calcium, chromium, cobalt, copper, lead, magnesium, and zinc

The efficacy of a chelating agent in binding a given metal in a biological system depends on the binding constants of the chelator for the particular metals in the system, the concentration of the metals, and the presence and concentrations of other ligands competing for the metals in question. In this study, we make a comparison of the in vitro binding constants for the chelator, ethylenediaminetetraacetic acid, with the quantitative urinary excretion of the metals measured before and after EDTA infusion in 16 patients. There were significant increases in lead, zinc, cadmium, and calcium, and these increases roughly corresponded to the expected relative increases predicted by the EDTA-metal-binding constants as measured in vitro. There were no significant increases in urinary cobalt, chromium, or copper as a result of EDTA infusion. The actual increase in cobalt could be entirely attributed to the cobalt content of the cyanocobalamin that was added to the infusion. Although copper did increase in the post-EDTA specimens, the increase was not statistically significant. In the case of magnesium, there was a net retention of approximately 85% following chelation. These data demonstrate that EDTA chelation therapy results in significantly increased urinary losses of lead, zinc, cadmium, and calcium following EDTA chelation therapy. There were no significant changes in cobalt, chromium, or copper and a retention of magnesium. These effects are likely to have significant effects on nutrient concentrations and interactions and partially explain the clinical improvements seen in patients undergoing EDTA chelation therapy.     

Seasonal changes in the concentrations of Ca,Fe, K,Mg, Mn and Na in eelgrass (Zostera marina L.) in the Limfjord,Denmark

The seasonal changes in concentrations of calcium, iron, potassium, magnesium, manganese and sodium in above- and below-ground parts of eelgrass (Zostera marina L.) were studied at three locations in the Limfjord, Denmark.The concentrations of calcium, iron and manganese in eelgrass differed significantly at the three stations. Above-ground parts of eelgrass contained significantly higher concentrations of potassium and manganese than below-ground parts, whereas the concentrations of iron and sodium were highest in the below-ground parts. Calcium and mangnesium concentrations in the two fractions were not significantly different.Significant seasonal variations were observed in the concentrations of calcium, potassium, manganese and sodium in above-ground parts of eelgrass and in the concentrations of iron, magnesium and sodium in below-ground parts. Iron, potassium, manganese and, to an extent, sodium showed a similar seasonal pattern with maximum concentrations in the summer and minimum concentrations in the spring or fall, whereas calcium concentrations in the above-ground parts showed the opposite pattern.The observed seasonal variation patterns are discussed in relation to the respective metals and changes in environmental factors.     

Inhibitory effect of molybdenum and vanadium salts on aflatoxin B1 synthesis by Aspergillus flavus

The effects of the elements zinc, manganese, iron, copper, molybdenum, and vanadium, added in various salt forms, on mycelial weights and aflatoxin B1 accumulation in the mycelium of Aspergillus flavus were investigated in liquid shake cultures. Ammonium heptamolybdate, when added to a complete medium at concentrations of 50-100 mg/L, appreciably reduced aflatoxin B1 accumulation without affecting growth of the fungus. Sodium molybdate and sodium monovanadate also reduced aflatoxin B1 yields without affecting mycelial growth but to a lesser extent. The addition of zinc sulphate stimulated aflatoxin B1 production in all media used. The influence of the other trace elements on aflatoxin production depended on the level of trace elements present in the basal medium. In general, manganese chloride had a stimulatory effect, whereas copper sulphate depressed yields. Mycelial levels of aflatoxin had peaked and then declined before mycelial dry weights had reached maximum. High yields of aflatoxin B1 were obtained in media having a final pH as low as pH 2.8.     

Comparisons of nine heavy metals in salt gland and liver of greater scaup (Aythya marila), black duck (Anas rubripes) and mallard (A. platyrhynchos)

Levels of nine heavy metals were measured in the livers and salt glands of greater scaup (Aythya marila), black duck (Anas rubripes) and mallard (A. platyrhynchos) from Raritan Bay, New Jersey to determine if the functioning avian salt gland concentrates heavy metals. Heavy metals examined were cadmium, cobalt, chromium, copper, lead, mercury, manganese, nickel and zinc. Heavy metal levels varied significantly by species and tissue for chromium, copper, lead, and manganese, and by tissue for cobalt, mercury, nickel and zinc. In comparing tissues cobalt was higher in the salt glands than in livers of all three species; chromium and nickel were higher in the salt gland than liver for mallard and black duck; and lead, manganese and zinc were higher in the liver than the salt gland in greater scaup. Generally metal levels were higher in the salt gland for mallard and black duck, and in the liver for greater scaup.     

Interactive effects of cadmium,lead and zinc on root growth of two metal tolerant genotypes ofHolcus lanatus L.

Lead and zinc tolerant genotypes ofHolcus lanatus L. were grown in culture solution at different cadmium, lead and zinc concentrations, and combinations. In all treatments, an increased inhibition of root length with increasing concentrations of heavy metals was observed. Growth of genotype 1 was better than that of genotype 2 in all treatments, suggesting that genotype 1 is more tolerant. The better root growth of genotype 1, at different cadmium concentrations, than that of genotype 2, indicated the existence of a co-tolerance or greater tolerance of genotype 1 to cadmium. Heavy metal combinations resulted in increased lead or zinc uptake by plants, while cadmium was decreased. In a lead-zinc combination, decreased lead and increased zinc uptake were detected. The different interactive effects of heavy metals on root growth of genotype 1 (additive or synergistic) and genotype 2 (additive or antagonistic) may suggest their differential susceptibility to the above metals.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Advances in biotreatment of acid mine drainage and biorecovery of metals: 1. Metal precipitation for recovery and recycle

Acid mine drainage (AMD), an acidic metal-bearingwastewater, poses a severe pollution problem attributedto post mining activities. The metals usuallyencountered in AMD and considered of concern for riskassessment are arsenic, cadmium, iron, lead, manganese,zinc, copper and sulfate. The pollution generated byabandoned mining activities in the area of Butte, Montanahas resulted in the designation of the Silver Bow Creek–ButteArea as the largest Superfund (National Priorities List) sitein the U.S. This paper reports the results of bench-scalestudies conducted to develop a resource recovery basedremediation process for the clean up of the Berkeley Pit.The process utilizes selective, sequential precipitation (SSP)of metals as hydroxides and sulfides, such as copper, zinc,aluminum, iron and manganese, from the Berkeley Pit AMDfor their removal from the water in a form suitable foradditional processing into marketable precipitates and pigments.The metal biorecovery and recycle process is based on completeseparation of the biological sulfate reduction step and themetal precipitation step. Hydrogen sulfide produced in the SRBbioreactor systems is used in the precipitation step to forminsoluble metal sulfides. The average metal recoveries usingthe SSP process were as follows: aluminum (as hydroxide) 99.8%,cadmium (as sulfide) 99.7%, cobalt (as sulfide) 99.1% copper(as sulfide) 99.8%, ferrous iron (sulfide) 97.1%, manganese(as sulfide) 87.4%, nickel (as sulfide) 47.8%, and zinc (as sulfide)100%. The average precipitate purity for metals, copper sulfide,ferric hydroxide, zinc sulfide, aluminum hydroxide and manganesesulfide were: 92.4, 81.5, 97.8, 95.6 , 92.1 and 75.0%, respectively.The final produced water contained only calcium and magnesiumand both sulfate and sulfide concentrations were below usablewater limits. Water quality of this agriculturally usable watermet the EPA's gold standard criterion.