Semax prevents learning and memory inhibition by heavy metals

Separate and joint effect of Semax, ascorbic acid, lead diacetate, and ammonium molybdate on avoidance conditioning in rats was studied. It was established that the heavy metal salts inhibited the avoidance response, and the peptide counteracted this inhibition as strongly as ascorbic acid or to a comparable degree. These findings confirm the antioxidant properties of Semax.     

Nootropic and analgesic effects of Semax following different routes of administration

Heptapeptide Semax (MEHFPGP) is the fragment of ACTH(4-10) analogue with prolonged neurotropic activity. The aim of the present work was to study the Semax effects on learning capability and pain sensitivity in white rats following intraperitoneal and intranasal administration in different doses. Semax nootropic effects were studied in the test of acquisition of passive avoidance task. Pain sensitivity was estimated in Randall-Selitto paw-withdrawal test. It was shown that Semax exerts nootropic and analgesic activities following intraperitoneal administration. Analysis of dependence of these effects on dose resulted in different dose-response curves. Following intranasal administration, Semax was more potent in learning improvement compared to intraperitoneal administration. The peptide failed to affect the animal pain sensitivity following intranasal administration as opposed to intraperitoneal administration. The data obtained suggest different mechanisms and brain structures involved in realization of the nootropic and analgesic effects of Semax.     

The Binding of Semax, ACTH 4–10 Heptapeptide, to Plasma Membranes of the Rat Forebrain Basal Nuclei and Its Biodegradation

The binding characteristics of the peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) to plasma membranes of basal nuclei of the rat forebrain and the dynamics of its degradation during its incubation with these membranes were studied. Binding of the homogeneously labeled [G-³H]Semax was shown to be time-dependent, specific, and reversible. Specific binding of the heptapeptide depended on calcium ions and was characterized by the dissociation constant of the ligand–receptor complex K _d 2.41 ± 1.02 × 10^–9 M and by the concentration of binding sites B _max 33.5 ± 7.9 × 10^–15 mol/mg of protein. A method of studying Semax biodegradation in the presence of plasma membranes of rat brain was developed. It is based on the use of the peptide homogeneously labeled with tritium and on an HPLC analysis with UV detection at 220 and 254 nm of the peptide fragments formed. The half-life of Semax in the presence of the plasma membranes was demonstrated to be longer than 1 h. Dipeptidylaminopeptidases are considered to be the main enzymes responsible for its biodegradation; they successively cleave Semax to the HFPGP pentapeptide and the PGP tripeptide.     

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd²⁺, Ni²⁺, and Co²⁺ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested. Higher concentration (0.1 mM) of Cu²⁺ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx. Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu²⁺ salts (20–30% inhibition), 0.5 mM ZnSO₄ (9.7%), 0.5 and 0.05 mM ZnCl₂ (34.3% and 20.8%), 0.1 mM salts of Cd²⁺, Ni²⁺, Co²⁺ (35–78%). 1 mM FeS_O4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl₃ (14%), 0.05 mM ZnS_O4 (9.6%), 0.005 mM ZnCla and ZnS_O4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdS_O4 (20.8 and 12.4%). All other tested salts-salts of Mn²⁺ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co²⁺ and Ni²⁺, 0.001 mM salts of Cu²⁺, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.     

Induction of metabolic shocks in source and sink of two cucurbits by molybdenum stress

Different concentrations of ammonium molybdate (10(-7) to 10(-4)M) affected the levels of metabolites in the source and sink organs of the seedlings of C. melo and C. vulgaris and data were recorded at 7, 14 and 21 days after treatment (DAT) of molybdenum (Mo). Reducing and non reducing sugars declined with an increase in concentration of ammonium molybdate from 10(-7) to 10(-4) M. Soluble protein and dry weight of seedlings increased in source at lower concentration (10(-7) M) and gradually decreased in all other concentrations (10(-6), 10(-5) and 10(-4) M). Starch was slightly accumulated in hypocotyl and fresh weight constantly declined with an increase in ammonium molybdate concentration from 10(-7) to 10(-4) M in all the parts of seedlings viz. cotyledon, hypocotyl and roots. Thus molybdenum at higher concentration induced decline in the metabolite levels in source and sink as well as in transporting organs.     

Molybdate reduction by Pseudomonas sp. strain DRY2

Aims: To isolate and characterize a potent molybdenum‐reducing bacterium. Methods and Results: A minimal salt medium supplemented with 10 mmol l^?1 molybdate, glucose (1·0%, w/v) as a carbon source and ammonium sulfate (0·3%, w/v) as a nitrogen source was used in the screening process. A molybdenum‐reducing bacterium was isolated and tentatively identified as Pseudomonas sp. strain DRY2 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny. Strain DRY2 produced 2·4, 3·2 and 6·2 times more molybdenum blue compared to Serratia marcescens strain DRY6, Enterobacter cloacae strain 48 and Eschericia coli K12, respectively. Molybdate reduction was optimum at 5 mmol l^?1 phosphate. The optimum molybdate concentration that supported molybdate reduction at 5 mmol l^?1 phosphate was between 15 and 25 mmol l^?1. Molybdate reduction was optimum at 40°C and at pH 6·0. Phosphate concentrations higher than 5 mmol l^?1 strongly inhibited molybdate reduction. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide and cyanide did not inhibit the molybdenum‐reducing enzyme activity. Chromium, copper, mercury and lead inhibited the molybdenum‐reducing activity. Conclusions: A novel molybdenum‐reducing bacterium with high molybdenum reduction capacity has been isolated. Significance and Impact of the Study: Molybdenum is an emerging global pollutant that is very toxic to ruminants. The characteristics of this bacterium suggest that it would be useful in the bioremediation of molybdenum pollutant.     

Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport

Exposure to Cd²⁺ and Pb²⁺ has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd²⁺ and Pb²⁺. It was shown that the preincubation of rat brain nerve terminals with Cd²⁺ (200 μM) or Pb²⁺ (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in l-[¹⁴C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered l-[¹⁴C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd²⁺ or Pb²⁺ caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn²⁺ did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd²⁺ and Pb²⁺ may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca²⁺ channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na⁺-dependent glutamate uptake.     

A comparison of growth rate of algae as influenced by variation in nitrogen nutrition inChlorella pyrenoidosa andScenedesmus obliquus

Algae were cultivated in nutrient solutions containing nitrates or ammonium salts as the nitrogen source. We measured the culture density in the case of autotrophic and mixotrophic cultivation at different pH values, in the presence of nitrate also at different concentrations and in the absence of molybdenum. The dry weight and amount of nitrogen in the cells under these conditions were determined. According to results obtained we conclude thatChlorella, in contrast toScenedesmus, prefers ammonia as the nitrogen source in the first growth period, later both prefer nitrogen from nitrates. Nitrogen from ammonium salts has a positive effect only during short-time cultivation. If glucose was used as a mixotrophic energy source (or perhaps H-donor), the unsuitability of ammonia as a nitrogen source was more marked. We suppose that the lower growth intensity of older cultures (i.e., after seven days) in ammonia medium is the result of the use of endogenous energy sources. Both algae have adaptive systems making it possible to use nitrate nitrogen even in the absence of molybdate.     

The mechanism of action of molybdenum and tungsten upon collagen structures in vivo

The effect of in vivo administration of molybdenum (as sodium molybdate) and tungsten (as sodium tungstate) was investigated in the skin of laboratory rats. It was proved that the amount of both bound molybdenum and tungsten in collagen is relatively small being 0.05 and 0.06 moles per mole respectively. Besides the fraction of firmly bound molybdenum and tungsten a much higher extractable pool of both these metals was found. It was also demonstrated that in vivo shadowing of collagen is caused by the fraction of loosely bound metals. On the other hand pronounced changes were shown in the mechanical properties of connective tissue after molybdate and tungstate administration. Surprisingly, the change in mechanical properties indicated a lower level of cross-linking after the administration of the investigated metals. It is therefore concluded that bitopical binding of molybdenum and tungsten in the collagen structure is unlikely. It also appears that the biological effect of these metals is due to the competition with copper and the interference with the physiological cross-linking reactions based on the partial blockade of lysyloxidase.     

Blockade of heavy metals accumulation in Chlorella vulgaris cells by 24-epibrassinolide

This study was conducted on the influence of 24-epibrassinolide (24-epiBL) mixed with varying concentrations of heavy metals (copper, lead, cadmium, zinc) upon the growth and accumulation of these heavy metals in the cell of the alga Chlorella vulgaris Beijerinck (Chlorophyceae). Heavy metals at the concentration of 10^–3 M, alone or mixed with 24-epiBL, showed a lethal effect on C. vulgaris. At metal concentrations of 10^–6–10^–4 M, a combination with 24-epiBL appeared to have a stronger stimulatory effect on a number of cells than a single metal (a stronger inhibitory effect). 24-EpiBL at the concentration of 10^–8 M in combination with heavy metals (in the range 10^–6–10^–4 M) blocked metal accumulation in algal cells. 24-EpiBL has an anti-stress effect on C. vulgaris contaminated by heavy metals. The inhibitory effect on metal accumulation of 24-epiBL mixed with different heavy metals was arranged in the following order: zinc > cadmium > lead > copper. This process is correlated with the stimulation of growth of C. vulgaris. The stimulatory effect of 24-epiBL mixed with heavy metals leading to an increased pH in the medium (5.28–6.20) was significantly higher than the impact due to the increased acidity in the medium due to metals alone (pH 3.10–5.85). Lower pH increased the toxicity of heavy metals in C. vulgaris cells.     

Dependence of long-lasting effects of the ACTH(4-10) analogue semax on the time of its neonatal administration

Long-lasting behavioural effects of chronic administration of synthetic ACT(4-10) analogue Semax (MEHFPGP) during early neonatal life were studied. The peptide was injected daily intraperitoneally in dose 0.05 mg/kg during the first, second or second-third weeks of postnatal development. It was shown that the peptide injections during the first week lead to a decrease and during second or second-third weeks--to an increase of exploratory activity in 4-8-week aged rats. Furthermore, the peptide adminictration at all times diminished anxiety and improved learning ability of adult rats. The data obtained show that Semax neonatal administration during the first three weeks of life modulates development of brain structures involved in regulation of exploration, anxiety and learning.     

Electron crystallography of the scrapie prion protein complexed with heavy metals

The insolubility of the disease-causing isoform of the prion protein (PrP^Sc) has prevented studies of its three-dimensional structure at atomic resolution. Electron crystallography of two-dimensional crystals of N-terminally truncated PrP^Sc (PrP 27-30) and a miniprion (PrP^Sc106) provided the first insights at intermediate resolution on the molecular architecture of the prion. Here, we report on the structure of PrP 27-30 and PrP^Sc106 negatively stained with heavy metals. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the β-helix that was proposed for the structure of PrP^Sc. Differential staining also confirmed the location of the internal deletion of PrP^Sc106 at or near these densities.     

Effect of sodium molybdate on the binding of androgen-receptor complexes to germ cell and sertoli cell chromatin

This study explores the effect of molybdate on androgen receptors in relation to chromatin binding. Two fractions of testicular and prostatic cytosol were obtained by ammonium sulfate precipitation at 15–37% (I) and 37–47% (II) saturation. Both fractions from either tissue exhibited specific binding of [³H]-androgens which was thermolabile. With the exception of testicular fraction II, all fractions bound to Sertoli cell and germ cell chromatin.Prostatic [³H]-androgen-receptor complexes isolated from cytosols in the presence of 10 mM sodium molybdate showed a decreased ability (only 20–40%) to bind to either Sertoli cell or germ cell chromatin. Similarly, testicular [³H]-androgen-receptor complexes isolated in the presence of molybdate bound less well (60–70%) to Sertoli cell chromatin. On the other hand, inclusion of molybdate subsequent to ammonium sulfate precipitation did not significantly alter the binding ability of the receptor complexes to either chromatin. Also, the presence of molybdate during the receptor-chromatin interaction did not significantly decrease the ability of either prostatic or testicular androgen-receptor complexes to bind to Sertoli cell chromatin. These results indicate that the addition of molybdate prior to ammonium sulfate precipitation may impede the activation of androgen-receptor complexes by salt, resulting in a decreased ability of the steroid-receptor complexes to bind to chromatin acceptor sites. The data also indicate that molybdate exerts its inhibitory effect directly on the nonactivated receptor complexes and not on the chromatin acceptor sites.     

The copper-molybdenum antagonism in ruminants. I. The formation of thiomolybdates in animal rumen

The formation of thiomolybdates, MoO_xS_4?x^2?(x = 0, 1, 2, or 3), from molybdate and sulphide salts in aqueous media has been studied under conditions which simulate the fluid phase in the rumen. The influences of the sulphide:molybdenum ratio, pH and phosphate levels on the nature of the species formed were investigated. The thiomolybdates, in particular the MoS_4^2? ion, have been implicated as the active intermediates in the widespread molybdenum induced copper deficiency that affects ruminants. The results presented here show that, under physiological conditions, di- and trithiomolybdates will form more readily than tetrathiomolybdate.     

The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii

 Azotobacter vinelandii produces five siderophores with different metal binding properties, depending on the concentrations of Fe(III) and molybdate in the growth medium. The three lower protonation constants of the unusual bis(catecholamide) siderophore azotochelin (L) were determined by a simultaneous spectrophotometric and potentiometric titration as log K ₅=3.65(5), log K ₄=7.41(3) and log K ₃=8.54(4). The metal-ligand equilibrium constant for [MoO₂(L)]^3– was obtained from analysis of the absorbance concentration data: at 20  °C and pH 6.6, log K _eq=4(1). Based on an average log K _a value of 12.1 for the two basic phenolic oxygens of azotochelin, the equilibrium formation constant was converted into the conventional formation constant K _f(MoL) = [MoO₂L^{3 –}]/[MoO₂ ²⁺][L^{5 –}] = 10³⁵ M^–1. To assess the influence of molybdenum-siderophore interactions on metal uptake in A. vinelandii, the dose-response effect of molybdate in the growth medium on siderophore biosynthesis was followed by UV-vis spectroscopy and HPLC. It could be shown that the formation of molybdenum siderophore complexes clearly reduces the concentration of free siderophores available for iron solubilization. Furthermore, in media with initial molybdate concentrations up to 100 μM, the molybdenum azotochelin complex is the predominant molybdenum species, suggesting that azotochelin might also possess sequestering activity towards molybdenum. Even higher molybdate levels result in a complete repression of the synthesis of the tetradentate siderophore azotochelin, while they initiate the alternative release of the more efficient iron chelator, the hexadentate siderophore protochelin. Received: 20 April 1998 / Accepted: 29 June 1998     

蚯蚓作用下污泥重金属形态变化及其与化学生物学性质变化的关系

城市污泥处理是一项世界性难题,污泥农业利用是其最简单有效的资源化利用方式之一,但污泥中较高的重金属含量限制了其实际推广应用,利用蚯蚓-超富集植物联合修复污泥重金属的方法已引起国内外研究者的关注。以新鲜城市脱水污泥为研究对象,接种赤子爱胜蚓(Eisenia fetida)进行室内培养试验,系统研究蚯蚓作用下污泥重金属形态的变化,及其与污泥氧化还原条件、化学和微生物性质变化的关系,以期为蚯蚓-超富集植物联合修复技术在污泥重金属处理中的应用提供理论依据。结果表明,试验前期蚯蚓在污泥中能正常生长和存活,前20 d总生物量增加了52%。蚯蚓可以显著促进污泥中的Cu、Zn、Cd、Ni等重金属从残渣态和铁锰态等稳定形态向交换态和水溶态等有效形态转化。还可以显著降低污泥中还原性物质的含量,减缓p H值下降速度,降低总有机碳含量,促进铵态氮向硝态氮转化,减少污泥微生物的数量并增加其种群活性。蚯蚓作用下,污泥中重金属的活化程度与还原性物质的含量呈显著负相关,而与微生物种群的活性呈显著正相关(P0.05)。综上所述,蚯蚓可以促进污泥重金属的活化,并改善污泥的肥力条件,为修复植物在污泥中的正常生长和对重金属离子的快速吸收提供有利条件。     

Specific binding of aluminium and iron ions to a cation‐selective cell wall channel of Microthrix parvicella

Heavy metal salts containing aluminium or iron are used in wastewater treatment to control excessive growth of the Gram‐positive bacterium Microthrix parvicella, frequently observed in wastewater plants suffering from bulking, foaming and scum. Microthrix parvicella belongs to the class Actinobacteria but not to mycolata, although its taxonomic position in this class is not identified. Investigations using the microspheres adhesion to cells method (MAC) suggested that M. parvicella is as strongly hydrophobic as the mycolic acid containing actinomycetes. The cell wall of M. parvicella was investigated for the presence of water‐filled channels using the lipid bilayer assay. An ion‐permeable channel called MppA with a conductance of 600 pS in 1 M KCl was identified in cell wall extracts and purified to homogeneity. The cation‐selective channel showed no voltage‐dependent closure at higher voltages. Interestingly, MPPA could be blocked by heavy metal ions. Binding of polyvalent cations such as iron and aluminium was studied in titration experiments and revealed stability constants for their binding to MppA up to 700 M^?1. The cell wall channel of M. parvicella contains a binding site for polyvalent cations which may play a crucial role for the effect of heavy metals salts on M. parvicella‐dominated activated sludge.     

Effect of a cyclic analog of enkephalin on learning and memory in the mouse

In experiments on mice, the effect of cyclic analogue of enkephalin (CAE) on the processes of learning and memory was studied in control animals and in animals with changed functional state of monoaminergic brain systems. Administration of the peptide to intact animals significantly accelerated the acquisition of conditioned reflexes of two-way avoidance and did not significantly affect the retention of these reflexes and their subsequent reproduction. Retention and reproduction of conditioned reflexes elaborated in one combination, was disturbed. Administration of iprazid did not eliminate the "accelerating effect" of CAE on the formation of conditioned reflexes of the two-way avoidance but sharply disturbed their retention. In such conditions, the amnesing iprazid effect increased still more. Besides, under CAE effect, the activity of acetylcholinesterase in the motor and especially in the visual cortex of the mice increased. The obtained data testify to an important role of the monoaminergic and cholinergic brain mechanisms in realization of CAE effects on the processes of learning and memory.     

Microwave-assisted pretreatment of woody biomass with ammonium molybdate activated by H2O2

Pretreatments for enzymatic saccharification are crucial for the establishment of lignocellulosic biorefineries. In this study, we focused on ammonium ions and peroxometal complexes as potential delignifying agents. We first examined the pretreatment of beech wood with nine different ammonium salts in the presence of H₂O₂. Significant pretreatment effects were found only for ammonium molybdate, which is transformed to a peroxometal complex on reacting with H₂O₂. Since microwave sensitizer effects are expected for (peroxo)molybdate, beech wood was pretreated using external heating and microwave irradiation. As a result, a maximum sugar yield of 59.5% was obtained by microwave irradiation at 140 °C for 30 min, while external heating in an autoclave gave a sugar yield of 41.8%. We also found that an ammonium ion is the key counterion accelerating the pretreatment with molybdate. These results highlight the powerful selective delignifying capability of the H₂O₂-activated ammonium molybdate system energised by microwave radiation.