Synthesis and possible applications of biotin-linked copper clusters

The trifunctional aziridine XAMA-7 (CAS 57116-45-7) has been used to form crosslinks between a deep red-violet copper cluster of the type Cu(I)8Cu(II)6pen12Cl5- (pen=penicillamine) and molecules with biological activity such as d-biotin and proteins. A complex containing biotin, bovine serum albumin and the copper cluster displayed activity toward affinity columns of avidin on Agarose, and the red-violet pigment was immobilized on the gel. This interaction was completely blocked in gels which had been pretreated with d-biotin carboxylic acid. The free and biologically active versions of the cluster have some potential for biomedical applications. For example, the short-lived positron emitter 64Cu (suitable for positron tomography) may be carried in the cluster's structure. The cluster is paramagnetic, but it is a relatively weak effector of water proton spin-lattice relaxation. Other members of this structural group of inorganic compounds may have better magnetic properties, and the crosslinking reaction with aziridines appears to be generally applicable to the group.     

Density functional study of bare gold clusters: the ten-vertex neutral system

Four novel Au₁₀ structures have been located by means of density functional methods and their geometry and electronic structure are discussed. Furthermore, the behavior of less extensive basis sets in conjunction with the B3PW91 functional is compared to a highly accurate and more extensive energy-consistent scalar-relativistic pseudopotential and basis set for neutral ten-vertex gold clusters. The values obtained for several structural parameters for known and novel optimized Au₁₀ systems are discussed.     

Release of copper from yeast copper-thionein after S-alkylation of copper-thiolate clusters.

Our knowledge on the release of copper from Cu-thionein in biological systems is limited. Other than oxidative cleavage or direct transfer, the possibility of an alkylation mechanism seemed attractive. Iodoacetamide and methyl methanesulphonate were successfully employed to alkylate the Cu-thiolate sulphur atom of homogeneous Cu(I)-thionein from yeast. The alkylation caused a weakening of the Cu-S bonding, which led to the release of copper. After equilibrium dialysis a proportion of the released copper was found in the dialysis buffer. When iodoacetamide was used carboxymethylcysteine was detected in the protein hydrolysate. A 10-fold molar excess over cysteine was sufficient for complete alkylation, which could be conveniently monitored by c.d. at 328 and 359 nm. The reaction proceeded under both aerobic and anaerobic conditions. E.p.r. measurements of Cu2+ revealed unequivocally the complete cleavage of the Cu-thiolate bonding in less than 5 h. It is possible that this mode of copper release might be of relevance to the molecular transport of this biochemically important transition metal.     

Mechanism of copper mediated triple helix formation at neutral pH in Drosophila satellite repeats

The highly repeated Drosophila melanogaster AAGAGAG satellite sequence is present at each chromosome centromere of the fly. We demonstrate here how, under nearly physiological pH conditions, these sequences can form a pyrimidine triple helix containing T.A-T and CCu.G-C base triplets, stabilized by Cu2+ metal ions in amounts mirroring in vivo concentrations. Ultraviolet experiments were used to monitor the triple helix formation at pH 7.2 in presence of Cu2+ ions. Triplex melting is observed at 23 degrees C. Furthermore, a characteristic signature of triple helix formation was obtained by Fourier transform infrared spectroscopy. The stabilization of the C.G-C base triplets at pH 7.2 is shown to occur via interactions of Cu2+ ions on the third strand cytosine N3 atom and on the guanine N7 atom of the polypurine target strand forming CCu.G-C triplets. Under the same neutral pH conditions in absence of Cu2+ ions, the triple helix fails to form. Possible biological implications are discussed.     

Identification of methionine-rich clusters that regulate copper-stimulated endocytosis of the human Ctr1 copper transporter

Copper uptake and subsequent delivery to copper-dependent enzymes are essential for many cellular processes. However, the intracellular levels of this nutrient must be controlled because of its potential toxicity. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. Recent studies have shown that elevated copper stimulates the endocytosis and degradation of the hCtr1 protein, and this response is likely an important homeostatic mechanism that prevents the overaccumulation of copper. The domains of hCtr1 involved in copper-stimulated endocytosis and degradation are unknown. In this study we examined the importance of potential copper-binding sequences in the extracellular domain and a conserved transmembrane (150)MXXXM(154) motif for copper-stimulated endocytosis and degradation of hCtr1. The endocytic response of hCtr1 to low copper concentrations required an amino-terminal methionine cluster ((40)MMMMPM(45)) closest to the transmembrane region. However, this cluster was not required for the endocytic response to higher copper levels, suggesting this motif may function as a high affinity copper-sensing domain. Moreover, the transmembrane (150)MXXXM(154) motif was absolutely required for copper-stimulated endocytosis and degradation of hCtr1 even under high copper concentrations. Together with previous studies demonstrating a role for these motifs in high affinity copper transport activity, our findings suggest common biochemical mechanisms regulate both transport and trafficking functions of hCtr1.     

Calculation of the Second-Order Polarizability of a Warm Magnetized Plasma

An analysis is made of the general expression for the density of a nonlinear charge induced in a magnetized plasma in the interaction between two arbitrary waves. Asymptotic expressions for the nonlinear induced charge density are derived for the first time in the case where both of the interacting waves are short-scale.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Polarizability of the ionic cloud around a charged membrane sheet

The polarizability of the ionic cloud around a charged membrane sheet parallel to the surface is calculated. The membrane is modeled as an infinitely thin and long strip of width 2d'. The Poisson-Boltzmann equation is solved with an external electric field as a perturbation. The polarizability is given in closed integral form, with numerical results. At higher salt concentration the polarizability varies approximately as kappa(-3), where kappa is the Debye-Hückel constant. The given theoretical result can account for the a.c. electrodichroism of purple membrane suspensions.     

Cisplatin stabilizes a multimeric complex of the human Ctr1 copper transporter: requirement for the extracellular methionine-rich clusters

Cisplatin is a highly effective cancer chemotherapy agent. However, acquired resistance currently limits the clinical utility of this drug. The human high affinity copper importer, hCtr1, and its yeast and murine orthologues have been shown to mediate the uptake of cisplatin. This transporter is located at the plasma membrane under low copper conditions, and excess copper concentrations stimulate its endocytosis and degradation. In this study we further examined the link between cisplatin and hCtr1 by examining whether cisplatin can also stimulate the endocytosis and degradation of hCtr1. The steady-state location of hCtr1 and its endocytosis from the plasma membrane were not altered by cisplatin treatment. Unexpectedly, cisplatin treatment of a cell line expressing hCtr1 revealed the time- and concentration-dependent appearance of a stable hCtr1 multimeric complex, consistent with a homotrimer, which was not observed following copper treatment of these same cells. Mutagenesis studies identified two methionine-rich clusters in the extracellular amino-terminal region of hCtr1 that were required for stabilization of the hCtr1 multimer by cisplatin, suggesting that these sequences bind cisplatin and form crosslinks between hCtr1 polypeptides. Treatment with the metal chelator dimethyldithiocarbamate disassembled the hCtr1 multimer following cisplatin exposure, suggesting that platinum was an integral component of this complex. These studies provide the first evidence for a direct interaction between cisplatin and the hCtr1 protein and establish that cisplatin and copper have distinct biochemical consequences on this transporter.     

The effect of copper and zinc at neutral and acidic pH on the general haematology and osmoregulation of Oreochromis mossambicus

The objective of this study was to determine the effect of sublethal copper and zinc concentrations at a neutral and an acidic pH, on selected haematological parameters as well as on the total osmolality and electrolyte concentrations of Oreochromis mossambicus. In general, at neutral pH copper and zinc caused blood acidosis, increases in circulating white blood cell numbers, causing stimulation of the immune system, and a rapid release of red blood cells from haemopoietic tissue, as reflected in the decreases in mean corpuscular volume. Increases in red blood cells are attributed to an increase in the oxygen-carrying haemoglobin as an adaptation to altered respiratory homeostasis caused by copper and zinc. These increases are therefore a secondary reaction to the metals and not the result of direct stimulation of the haemopoietic tissue. In contrast, at an acidic pH copper and zinc concentrations usually caused blood alkalosis and decreases in white blood cell numbers, due to the bioconcentration of metals which blocks and suppresses the leucopoietic tissue. Decreases were also recorded in red blood cell counts, haemoglobin, haematocrit and mean corpuscular volume, which can be ascribed to anaemic and hypoxic conditions, gill damage and impaired osmoregulation.     

Simultaneous Detection of Local Polarizability and Viscosity by a Single Fluorescent Probe in Cells

Many intracellular reactions are dependent on the dielectric (“polarity”) and viscosity properties of their milieu. Fluorescence imaging offers a convenient strategy to report on such environmental properties. Yet, concomitant and independent monitoring of polarity and viscosity in cells at submicron scale is currently hampered by the lack of fluorescence probes characterized by unmixed responses to both parameters. Here, the peculiar photophysics of a green fluorescent protein chromophore analog is exploited for quantifying and imaging polarity and viscosity independently in living cells. We show that the polarity and viscosity profile around a novel hybrid drug-delivery peptide changes dramatically upon cell internalization via endosomes, shedding light on the spatiotemporal features of the release mechanism. Accordingly, our fluorescent probe opens the way to monitor the environmental effects on several processes relevant to cell biochemistry and nanomedicine.     

Probing the geometries,relative stabilities,and electronic properties of neutral and anionic AgnSm (n + m ≤ 7) clusters

The vertical excitation energies of 3,4-dicyano-6-methoxy and 3,4-dicyano-6,7-dimethoxy carbostyril have been computed with different approximations for the time-dependent density functional theory (TD-DFT) procedure and with different implementations of the continuum solvation model COSMO. Different DFT functionals were tested in TD-DFT and Tamm-Dancoff approximations (TDA) for the excitation energies in the gas phase. TDA-B3LYP showed the best agreement with the experimental data. Then TDA-B3LYP computations were performed combined with the COSMO model of solvation comparing a linear response (LR) and a post-configuration interaction (CI) implementation of the fast solvent reorganization. The post-CI solvent model overestimates the π→π* transitions and strongly underestimates the n→π* transition. The TDA approximation in combination with the linear response implementation of the COSMO solvation model perfectly computes the experimental results. TDA-LR is the most reliable method for the computation of the vertical excitation energies in a solvent. Comparison with explicit solvent calculations shows there is only a minor effect on the energies of the electronic interaction of the solute with the solvent.     

The neutral theory is dead. The current significance and standing of neutral and nearly neutral theories

Comparative studies of DNA sequences provide opportunities for testing the neutral and the selection theories of molecular evolution. In particular, the separate estimation of the numbers of synonymous and nonsynonymous substitutions is a powerful tool for detecting selection of the latter. The difference in the patterns of these two types of substitutions of mammalian genes turned out to be in accord with the slightly deleterious or nearly neutral mutation theory for nonsynonymous changes. Interaction systems at the amino acid level were suggested to be responsible for such nearly neutral, or very weak, selection. Synonymous substitutions are not strictly neutral, but because of their minute effect, random drift predominates such that the rate of substitution is only slightly less than the completely neutral prediction. It was concluded that the strictly neutral theory has not held up as well as the nearly neutral theory, yet remains invaluable as a null hypothesis for detecting selection. On the other hand, the main difference between the nearly neutral and the traditional selection theories is that the former predicts rapid evolution in small populations, whereas the latter predicts rapid evolution in large populations.     

Influence of substituents on the structures of hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates

By changing the substituents on 1,2,4-triazole ring, six novel organic-inorganic hybrid complexes constructed from tetranuclear copper(I) 1,2,4-triazolate clusters and octamolybdates, [{Cu₄(L)_x}Mo₈O₂₆] (L = 3,5-diamino-1,2,4-triazole (datrz) and x = 4 for 1; L = 3-amino-1,2,4-triazole (3atrz) and x = 4 for 2; L = 3,5-dimethyl-1,2,4-triazole (dmtrz) and x = 4 for 3; L = 3,5-dimethyl-4-amino-1,2,4-triazole (dmatrz) and x = 6 for 4; L = 3,5-diethyl-4-amino-1,2,4-triazole (deatrz) and x = 4 for 5; L = 3,5-di(n-propyl)-4-amino-1,2,4-triazole (dpatrz) and x = 3 for 6), were obtained. The tetranuclear Cu(I) cluster in compound 1 acts as charge-compensating unit, which is the first polynuclear metal 1,2,4-triazole structure only with N1, N2 bridging mode. Compounds 2, 4, 5 and 6 are of polymeric 1D chains and 3 is of a 2D layer structure. In 2, three distinct Cu(I)-coordination geometries, distorted tetrahedral, T-shaped and V-shaped linear Cu(I), are observed in the same structure. The first extended hybrid structure constructed by δ-octamolybdates is founded in 4. A novel [Mo₈O₂₆]⁴⁻ anion is found in 5, which contains only three crystallographically independent Mo atoms. In compounds 5 and 6, terminal oxo groups of octamolybdate cluster act as μ₃-oxo bridges to link the copper(I) coordination complexes; such an unusual linking manner is unique in the coordination chemistry of octamolybdates with transition metal fragments. The influences of substituent on the structures of the tetranuclear units are also discussed in details.     

Mechanisms for solubilization of cobalt,copper and nickel from Indian Ocean nodules at near neutral pH by a marine isolate

Polymetallic ocean nodules offer an alternative source for extracting valuable strategic metals like Cu, Co and Ni. A novel biodissolution process was carried out, employing the cell-free spent growth medium from a marine organism (Bacillus M1) isolated from nodules; and Cu, Co and Ni solubilization from the nodules was observed to be beyond the theoretical solubility limits at near neutral pH. Different characterization techniques revealed the presence of phenolic substances in the spent growth medium, which might have formed soluble complexes with the transition metals. The low prevailing E_h redox value in the medium suggested a strong reducing environment, favoring the reductive dissolution of the oxides. A correlation study of dissolution of Cu, Co and Ni with that of Mn and Fe in the nodules was made to investigate the mechanisms of metal solubilization by the marine isolate. Under the influence of a strong reducing environment coupled with complexation by a phenolic substance present in the spent growth medium, Mn and Fe oxides were solubilized from the nodules, resulting in concomitant dissolution of Cu, Co and Ni associated with them in the nodules.     

Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters

Carrot (Daucus carota L.) embryogenic callus (EC) loses its embryogenic competence and becomes nonembryogenic callus (NC) during long-term culture. With the loss of embryogenic competence, the cell clusters become smaller and the extent of intercellular attachments is reduced. Pectic fractions prepared from EC and NC were separated into two subfractions by gel filtration. A difference in sugar composition between EC and NC was found only in the high-molecular-mass (ca. 1300 kDa) subfraction, and the ratio of the amount of arabinose to that of galactose (Ara/Gal) was strongly and positively correlated with the size of cell clusters in several different cultures. From the results of sugar-composition and methylation analyses, and the results of treatment with exo-arabinanase, models of the neutral sugar chains of pectins from EC and NC are proposed. Both neutral sugar chains are composed of three regions. The basal region is composed of linearly linked arabinan 5-Ara_f> moieties in both types of callus. The middle galactan region is composed of 6-linked galactose, some of which branches at the 3 and 4 positions, and this region is larger and more frequently branched in NC than in EC. Finally, the terminal arabinan region is composed of 5-linked arabinose, branched at the 3 position, and the size of the terminal arabinan is larger in EC than in NC. The significance of the neutral sugar chains of pectins in the interaction of cell wall components and intercellular attachment is discussed.Abbreviations Ara/Gal ratio (w/w) of the amount of arabinose to that of galactose - EC embryogenic callus - NC non-embryogenic callus - T-Ara_f terminal arabinose The authors are grateful to Dr. Naoto Shibuya of the National Institute of Agrobiological Resources for his gift of exo-arabinanase.     

Dynamics of neutral biodiversity

Hubbell's neutral model has become a major paradigm in ecology. Whereas the steady-state structure is well understood, results about the dynamical aspects of the model are scarce. Here we derive dynamical equations for the Simpson diversity index. Both mean and variance of the diversity are proven to satisfy stable linear system dynamics. We show that in the stationary limit we indeed recover previous results, and we supplement this with numerical simulations to validate the dynamical part of our analytical computations. These findings are especially relevant for experiments in microbial ecology, where the Simpson diversity index can be accurately measured as a function of time.     

Invasibility of neutral communities

Comparative data in invasion ecology show that (i) disturbance enhances community invasibility, (ii) there is a positive relationship between residence time of an invader and its success, (iii) there are broadly constant proportions of newly arrived species to those that become established and dominant (“tens rule”), and (iv) invasive species have higher growth rates in comparison with non-invasive species. I use a simple neutral model to test whether these patterns occur in communities with all species identical and no species-specific interactions. In the model, local communities are grouped into continents with immigration rates smaller between than within the continents. Species coming from the other continent are considered to be alien and their fates are recorded. In the model, disturbance predictably increases species numbers and numbers of individuals of aliens. However, the model makes different predictions on effects of disturbance on three processes involved in alien species spreading: establishment (positive effect of disturbance), naturalization (negative effect) and dominance (positive effect). The predictions do not change if variation of growth rates is incorporated into the model. The model also predicts positive relationship between residence time and abundance. Total community size had little effect on success of alien species. The broad agreement of the predictions of the neutral model with the patterns from the field suggests that some of these general patterns of community invasibility are to some degree fully independent of any specific biological assumptions and by themselves may not provide many insights on underlying biological processes. Aggregate data should therefore be used with great caution and statistical patterns must be removed by means of generating null model predictions.