Bioavailability of metalloporphyrin-based SOD mimics is greatly influenced by a single charge residing on a Mn site

Abstract

In the cell Mn porphyrins (MnPs) likely couple with cellular reductants which results in a drop of total charge from 5+ to 4+ and dramatically increases their lipophilicity by up to three orders of magnitude depending upon the length of alkylpyridyl chains and type of isomer. The effects result from the interplay of solvation, lipophilicit and stericity. Impact of ascorbate on accumulation of MnPs was measured in E. coli and in Balb/C mouse tumours and muscle; for the latter measurements, the LC/ESI-MS/MS method was developed. Accumulation was significantly enhanced when MnPs were co-administered with ascorbate in both prokaryotic and eukaryotic systems. Further, MnTnHex-2-PyP⁵⁺ accumulates 5-fold more in the tumour than in a muscle. Such data increase our understanding of MnPs cellular and sub-cellular accumulation and remarkable in vivo effects. The work is in progress to understand how coupling of MnPs with ascorbate affects their mechanism of action, in particular with respect to cancer therapy.     

Secondary succession is influenced by belowground insect herbivory on a productive site

We investigated the effects of insect herbivory on a plant community of a productive old-field community by applying foliar and soil insecticides in a full factorial design. During the first 3 years of succession, insecticide treatments had only minor effects on total cover abundance and species richness. However, species ranking within the plant community was strongly affected by soil insecticide but not by foliar insecticide. Creeping thistle, Cirsium arvense , dominated the experimental plots with reduced root herbivory, while square-stemmed willow-herb, Epilobium adnatum , dominated the control and the plots with foliar insecticide. When soil insecticide was applied, cover abundance of monocarpic forbs increased and cover abundance of polycarpic herbs decreased compared to the control. However, this effect was due to a few abundant plant species and is not based on a consistent difference between life history groups. Instead, application of soil insecticide promoted persistence of species that established at the start of succession, and suppressed species that established in the following years. We conclude that below-ground herbivory reduces competitive ability of resident species and, thus, facilitates colonization by late-successional species. Hence, soil insects can exert strong top-down effects on the vegetation of productive sites by affecting dominant plant species and altering competitive balances.     

Steroid-binding specificity of human sex hormone-binding globulin is influenced by occupancy of a zinc-binding site

One calcium-binding site (site I) and a second poorly defined metal-binding site (site II) have been observed previously within the amino-terminal laminin G-like domain (G domain) of human sex hormone-binding globulin (SHBG). By soaking crystals of this structure in 2.5 mm ZnCl(2), site II and a new metal-binding site (site III) were found to bind Zn(2+). Site II is located close to the steroid-binding site, and Zn(2+) is coordinated by the side chains of His(83) and His(136) and the carboxylate group of Asp(65). In this site, Zn(2+) prevents Asp(65) from interacting with the steroid 17beta-hydroxy group and alters the conformations of His(83) and His(136), as well as a disordered region over the steroid-binding site. Site III is formed by the side chains of His(101) and the carboxylate group of Asp(117), and the distance between them (2.7 A) is increased to 3.7 A in the presence of Zn(2+). The affinity of SHBG for estradiol is reduced in the presence of 0. 1-1 mm Zn(2+), whereas its affinity for androgens is unchanged, and chemically-related metal ions (Cd(2+) and Hg(2+)) have similar but less pronounced effects. This is not observed when Zn(2+) coordination at site II is modified by substituting Gln for His(136). An alteration in the steroid-binding specificity of human SHBG by Zn(2+) occupancy of site II may be relevant in male reproductive tissues where zinc concentrations are very high.     

New approach to the activation of anti-cancer pro-drugs by metalloporphyrin-based cytochrome P450 mimics in all-aqueous biologically relevant system

The low-molecular weight water-soluble Fe(III) and Mn(III) porphyrins--in biologically relevant phosphate-buffered saline medium with ascorbic acid as a source of electrons, under aerobic conditions but without co-oxidant - catalyze the hydroxylation of anti-cancer drug cyclophosphamide to active metabolite 4-hydroxycyclophosphamide in yields similar or higher than those typically obtained by the action of liver enzymes in vivo. The Fe(III) meso tetrakis(2,6-difluoro-3-sulfonatophenyl)porphyrin, highly electron-deficient at the metal site, was the most effective catalyst. If proven viable in vivo, this methodology could be expanded to localized or systemic activation of the entire family of oxazaphosphorine-based (and many other) anti-cancer drugs and become a powerful tool for an aggressive treatment of tumors with less toxic side effects to the patient.     

Light-induced charge separation in Photosystem I at low temperature is not influenced by vitamin K-1

The photoreduction of iron-sulfur centers was studied at low temperature in Photosystem I particles from spinach and the cyanobacterium Synechocystis 6803, which contain various amounts of vitamin K-1 (recently tentatively identified as the acceptor A1). The irreversible charge separation that was progressively induced at low temperature between P-700 and FA (or FB) by successive laser flashes was studied at 15 K. Its maximum amount after a large number of flashes was shown to be fairly independent of the number (0, 1 or 2) of vitamins K-1 per reaction center. Moreover, the first flash yield of this charge separation was diminished by only about 50% when vitamin K-1 was completely absent from the particles by comparison with particles containing one or two vitamin K-1 per reaction center. When FA and FB were prereduced, the iron-sulfur center FX was also reversibly photoreduced at 9 K in the absence of vitamin K-1. The implications of these results for the electron pathways of Photosystem I are discussed and it is proposed that a direct electron transfer from A0- to the iron-sulfur centers is highly efficient at low temperature.     

Effective size of a wild salmonid population is greatly reduced by hatchery supplementation

Many declining and commercially important populations are supplemented with captive-born individuals that are intentionally released into the wild. These supplementation programs often create large numbers of offspring from relatively few breeding adults, which can have substantial population-level effects. We examined the genetic effects of supplementation on a wild population of steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, by matching 12 run-years of hatchery steelhead back to their broodstock parents. We show that the effective number of breeders producing the hatchery fish (broodstock parents; N(b)) was quite small (harmonic mean N(b)=25 fish per brood-year vs 373 for wild fish), and was exacerbated by a high variance in broodstock reproductive success among individuals within years. The low N(b) caused hatchery fish to have decreased allelic richness, increased average relatedness, more loci in linkage disequilibrium and substantial levels of genetic drift in comparison with their wild-born counterparts. We also documented a substantial Ryman-Laikre effect whereby the additional hatchery fish doubled the total number of adult fish on the spawning grounds each year, but cut the effective population size of the total population (wild and hatchery fish combined) by nearly two-thirds. We further demonstrate that the Ryman-Laikre effect is most severe in this population when (1) >10% of fish allowed onto spawning grounds are from hatcheries and (2) the hatchery fish have high reproductive success in the wild. These results emphasize the trade-offs that arise when supplementation programs attempt to balance disparate goals (increasing production while maintaining genetic diversity and fitness).     

Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

In the vertebrate rod outer segment (ROS), the light-dependent activation of a GTP-binding protein (G-protein) and phosphodiesterase (PDE) is quenched by a process that requires ATP [Liebman, P.A., & Pugh, E.N. (1979) Vision Res. 19, 375-380]. The ATP-dependent quenching mechanism apparently requires the phosphorylation of photoactivated rhodopsin (Rho*); however, a 48-kilodalton protein (48K protein) has also been proposed to participate in the inactivation process. Purified species of phosphorylated rhodopsin containing 0, 2, or greater than or equal to 4 (high) phosphates per rhodopsin (PO4/Rho) were reconstituted into phosphatidylcholine (PC) vesicles and reassociated with a hypotonic extract from isotonically washed disk membranes that were depleted of 48K protein; PDE activation, in response to bleaching from 0.01% to 15% of the rhodopsin present, was measured. PDE activity was reduced by at least 30% at high fractional rhodopsin bleaches and by greater than 80% at low fractional rhodopsin bleaches in high PO4/Rho samples when compared to the activity measured in O PO4/Rho controls. A phosphorylation level of 2 PO4/Rho produced PDE activities that were intermediate between O PO4/Rho and high PO4/Rho samples at low bleaches, but were identical with the O PO4/Rho samples at high rhodopsin bleaches. Rhodopsin phosphorylation is thus capable of producing a graded inhibition of light-stimulated PDE activation over a limited range of (near physiological) bleach levels. This effect become less pronounced as the bleach levels approach those that saturate PDE activation. These results are consistent with increasing levels of phosphorylation, producing a reduction of the binding affinity of G-protein for Rho*.     

Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin

Endotoxin selectively induces monocyte Mn superoxide dismutase(SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. Inthis study we demonstrated that a mutant Escherichiacoli endotoxin lacking myristoyl fatty acid at the3' R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate comparedwith the wild-type E. coli endotoxinand markedly stimulated the activation of human monocyte nuclearfactor-B and the induction of Mn SOD mRNA and enzyme activity.However, in contrast to the wild-type endotoxin, it failed to inducesignificant production of tumor necrosis factor- and macrophageinflammatory protein-1 by monocytes and did not induce thephosphorylation and nuclear translocation of mitogen-activated proteinkinase. These results suggest that1) lipid A myristoyl fatty acid,although it is important for the induction of inflammatory cytokineproduction by human monocytes, is not necessary for the induction of MnSOD, 2) endotoxin-mediated inductionof Mn SOD and inflammatory cytokines are regulated, at least in part,through different signal transduction pathways, and3) failure of the mutant endotoxinto induce tumor necrosis factor- production is, at least in part,due to its inability to activate mitogen-activated protein kinase.

     

SfiI endonuclease activity is strongly influenced by the non-specific sequence in the middle of its recognition site

The SfiI endonuclease cleaves DNA at the sequence GGCCNNNN NGGCC, where N is any base and downward arrow is the point of cleavage. Proteins that recognise discontinuous sequences in DNA can be affected by the unspecified sequence between the specified base pairs of the target site. To examine whether this applies to SFII, a series of DNA duplexes were made with identical sequences apart from discrete variations in the 5 bp spacer. The rates at which SFII cleaved each duplex were measured under steady-state conditions: the steady-state rates were determined by the DNA cleavage step in the reaction pathway. SFII cleaved some of these substrates at faster rates than other substrates. For example, the change in spacer sequence from AACAA to AAACA caused a 70-fold increase in reaction rate. In general, the extrapolated values for k(cat) and K(m) were both higher on substrates with inflexible spacers than those with flexible structures. The dinucleotide at the site of cleavage was largely immaterial. SFII activity is thus highly dependent on conformational variations in the spacer DNA.     

Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate

A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa -subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.Abbreviations P_i phosphate - IEP inner envelope membrane protein - pIEP precursor form of IEP - SSU small subunit of ribulose-1,5-bisphosphate carboxylase oxygenase - IEP96_pep peptide specific antiserum to IEP96 - IEP96_pol polyspecific antiserum to IEP96     

Perception of loudness is influenced by emotion

Loudness perception is thought to be a modular system that is unaffected by other brain systems. We tested the hypothesis that loudness perception can be influenced by negative affect using a conditioning paradigm, where some auditory stimuli were paired with aversive experiences while others were not. We found that the same auditory stimulus was reported as being louder, more negative and fear-inducing when it was conditioned with an aversive experience, compared to when it was used as a control stimulus. This result provides support for an important role of emotion in auditory perception.     

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.     

Rouleau formation of erythrocytes is influenced by thrombocytes

Thrombocytes are activated by shearing and influence possibly the flow properties of blood. Erythrocytes resuspended in platelet poor plasma show a significantly increased deformability as well as reduced aggregability after addition of ADP as well as adrenaline in comparison with control. In platelet rich plasma the aggregation of erythrocytes becomes enhanced. This is discussed in respect of a release of macromolecules of platelets.     

Comprehensive pharmacokinetic studies and oral bioavailability of two Mn porphyrin-based SOD mimics,MnTE-2-PyP5+ and MnTnHex-2-PyP5+

The cationic, ortho Mn(III) N-alkylpyridylporphyrins (alkyl=ethyl, E, and n-hexyl, nHex) MnTE-2-PyP⁵⁺ (AEOL10113, FBC-007) and MnTnHex-2-PyP⁵⁺ have proven efficacious in numerous in vivo animal models of diseases having oxidative stress in common. The remarkable therapeutic efficacy observed is due to their: (1) ability to catalytically remove O₂^•− and ONOO⁻ and other reactive species; (2) ability to modulate redox-based signaling pathways; (3) accumulation within critical cellular compartments, i.e., mitochondria; and (4) ability to cross the blood–brain barrier. The similar redox activities of both compounds are related to the similar electronic and electrostatic environments around the metal active sites, whereas their different bioavailabilities are presumably influenced by the differences in lipophilicity, bulkiness, and shape. Both porphyrins are water soluble, but MnTnHex-2-PyP⁵⁺ is approximately 4 orders of magnitude more lipophilic than MnTE-2-PyP⁵⁺, which should positively affect its ability to pass through biological membranes, making it more efficacious in vivo at lower doses. To gain insight into the in vivo tissue distribution of Mn porphyrins and its impact upon their therapeutic efficacy and mechanistic aspects of action, as well as to provide data that would ensure proper dosing regimens, we conducted comprehensive pharmacokinetic (PK) studies for 24 h after single-dose drug administration. The porphyrins were administered intravenously (iv), intraperitoneally (ip), and via oral gavage at the following doses: 10 mg/kg MnTE-2-PyP⁵⁺ and 0.5 or 2 mg/kg MnTnHex-2-PyP⁵⁺. Drug levels in plasma and various organs (liver, kidney, spleen, heart, lung, brain) were determined and PK parameters calculated (C_max, C_24 h, t_max, and AUC). Regardless of high water solubility and pentacationic charge of these Mn porphyrins, they are orally available. The oral availability (based on plasma AUC_oral/AUC_iv) is 23% for MnTE-2-PyP⁵⁺ and 21% for MnTnHex-2-PyP⁵⁺. Despite the fivefold lower dose administered, the AUC values for liver, heart, and spleen are higher for MnTnHex-2-PyP⁵⁺ than for MnTE-2-PyP⁵⁺ (and comparable for other organs), clearly demonstrating the better tissue penetration and tissue retention of the more lipophilic MnTnHex-2-PyP⁵⁺.     

Dynamics in the Mn2+ binding site in single crystals of concanavalin A revealed by high-field EPR spectroscopy

EPR spectroscopy at 95 GHz was used to characterize the dynamics at the Mn(2+) binding site in single crystals of the saccharide-binding protein concanavalin A. The zero-field splitting (ZFS) tensor of the Mn(2+) was determined from rotation patterns in the a-c and a-b crystallographic planes, acquired at room temperature and 4.5 K. The analysis of the rotation patterns showed that while at room temperature there is only one type of Mn(2+) site, at low temperatures two types of Mn(2+) sites, not related by any symmetry, are distinguished. The sites differ in the ZFS parameters D and E and in the orientation of the ZFS tensor with respect to the crystallographic axes. Temperature-dependent EPR measurements on a crystal oriented with its crystallographic a axis parallel to the magnetic field showed that as the temperature increases, the two well-resolved Mn(2+) sextets gradually coalesce into a single sextet at room temperature. The line shape changes are characteristic of a two-site exchange. This was confirmed by simulations which gave rates in the range of 10(7)-10(8) s(-1) for the temperature range of 200-266 K and an activation energy of 23.8 kJ/mol. This dynamic process was attributed to a conformational equilibrium within the Mn(2+) binding site which freezes into two conformations at low temperatures.     

Potency of a tau fibrillization inhibitor is influenced by its aggregation state

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.