Spermidine Binding Sites Research Articles

Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288–P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.

Molecular Dynamics Reveal Binding Mode of Glutathionylspermidine by Trypanothione Synthetase

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg2+ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N1-glutathionylation of N8-glutathionylspermidine, classifying N8-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N8-glutathionylspermidine was characterised as druggable.

Modulation of Differentiation-related Gene 1 Expression by Cell Cycle Blocker Mimosine, Revealed by Proteomic Analysis

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Spermine cytotoxicity to human colon carcinoma-derived cells (CaCo-2).

Spermine is a constituent of all vertebrate cells. Nevertheless, it exerts toxic effects if it accumulates in cells. Spermine is a natural substrate of the FAD-dependent polyamine oxidase, a constitutive enzyme of many cell types. It has been reported that the toxicity of spermine was enhanced if polyamine oxidase was inhibited. We were interested to examine spermine toxicity to human colon carcinoma-derived CaCo-2 cells because, in contrast to most tumor cell lines, CaCo-2 cells undergo differentiation, which is paralleled by changes in polyamine metabolism. CaCo-2 cells were remarkably resistant to spermine accumulation, presumably because spermine is degraded by polyamine oxidase at a rate sufficient to provide spermidine for the maintenance of growth. Inactivation of polyamine oxidase increased the sensitivity to spermine. A major reason for the enhanced spermine cytotoxicity at low polyamine oxidase activity is presumably the profound depletion of spermidine, and the consequent occupation of spermidine binding sites by spermine. Hydrogen peroxide and the aldehydes 3-aminopropanal and 3-acetamidopropanal, the products of polyamine oxidase-catalyzed splitting of spermine and N1-acetylspermine, contribute little to spermine cytotoxicity. Activation of caspase by spermine was insignificant, and the formation of DNA ladders, another indicator of apoptotic cell death, could not be observed. Thus it appears that cell death due to excessive accumulation of spermine in CaCo-2 cells was mainly nonapoptotic. The content of brush border membranes did not change between days 6 and 8 after seeding, and it was not affected by exposure of the cells to spermine. However, the activities of alkaline phosphatase, sucrase, and aminopeptidase in nontreated cells were considerably enhanced during this period, but remained low if cells were exposed to spermine. These changes appear to indicate that differentiation is prevented by intoxication with spermine, although other explanations cannot be excluded.

Mechanism of Action of Acamprosate. Part I. Characterization of Spermidine-Sensitive Acamprosate Binding Site in Rat Brain

It has been suggested that the anticraving drug, acamprosate, acts via the glutamatergic system, but the exact mechanism of action is still unknown. The aim of this study was to characterize [3H]acam-prosate binding and establish whether this showed any relation to sites on the NMDA receptor complex. We found saturable specific binding of [3H]acamprosate to rat brain membranes with a KD of 120 μM and a Bmax of 450 pmol/mg of protein. This acamprosate binding site was sensitive to inhibition by spermidine (IC50: 13.32 ± 1.1 μM; Hill coefficient = 1.04), and arcaine and glutamate both potentiated the inhibitory effect of spermidine. Acamprosate binding to the acamprosate binding site was also sensitive to inhibition by divalent cations (Ca2+, Mg2+, and Sr2+). Conversely, acamprosate displaced [14C]spermidine binding from rat brain membranes with an IC50, of 645 μM and a Hill coefficient = 1.74. This inhibitory effect of acamprosate was not affected by arcaine, and was associated with a significant reduction in Bmax and binding affinity for spermidine, suggesting an allosteric interaction between acamprosate and a spermidine binding site. These data are consistent with an effect of acamprosate on the NMDA receptor protein complex, and acamprosate was also found to alter binding of [3H]dizocilpine to rat brain membranes. When no agonists were present in vitro (minimal NMDA receptor activation), acamprosate markedly potentiated [3H]dizocilpine binding at concentrations in the 5 to 200 μ range. However, under conditions of maximal receptor activation (100 μM glutamate, 30 μM glycine), acamprosate only inhibited [3H]dizocilpine binding (at concentrations concentrations > 100 μM). When these binding studies were performed in the presence of 1 μM spermidine, the enhancing effects of acamprosate on [3H]dirocilpine binding were inhibited. The results show that acamprosate binds to a specific spermidine-sensitive site that modulates the NMDA receptor in a complex way. Together, with data from al Quatari et al. (see next paper), this work suggests that acamprosate acts as “partial co-agonist“ at the NMDA receptor, so that low concentrations enhance activation when receptor activity is low, whereas higher concentrations are inhibitory to high levels of receptor activation. This may be relevant to the clinical effects of acamprosate in alcohol-dependent patients during abstinence.

Mechanism of Action of Acamprosate. Part I. Characterization of Spermidine‐Sensitive Acamprosate Binding Site in Rat Brain

It has been suggested that the anticraving drug, acamprosate, acts via the glutamatergic system, but the exact mechanism of action is still unknown. The aim of this study was to characterize [3H]acamprosate binding and establish whether this showed any relation to sites on the NMDA receptor complex. We found saturable specific binding of [3H]acamprosate to rat brain membranes with a KD of 120 microM and a Bmax of 450 pmol/mg of protein. This acamprosate binding site was sensitive to inhibition by spermidine (IC50: 13.32 +/- 1.1 microM; Hill coefficient = 1.04), and arcaine and glutamate both potentiated the inhibitory effect of spermidine. Acamprosate binding to the acamprosate binding site was also sensitive to inhibition by divalent cations (Ca2+, Mg2+, and Sr2+). Conversely, acamprosate displaced [14C]spermidine binding from rat brain membranes with an IC50 of 645 microM and a Hill coefficient = 1.74. This inhibitory effect of acamprosate was not affected by arcaine, and was associated with a significant reduction in Bmax and binding affinity for spermidine, suggesting an allosteric interaction between acamprosate and a spermidine binding site. These data are consistent with an effect of acamprosate on the NMDA receptor protein complex, and acamprosate was also found to alter binding of [3H]dizocilpine to rat brain membranes. When no agonists were present in vitro (minimal NMDA receptor activation), acamprosate markedly potentiated [3H]dizocilpine binding at concentrations in the 5 to 200 microM range. However, under conditions of maximal receptor activation (100 microM glutamate, 30 microM glycine), acamprosate only inhibited [3H]dizocilpine binding (at concentrations concentrations >100 microM). When these binding studies were performed in the presence of 1 microM spermidine, the enhancing effects of acamprosate on [3H]dizocilpine binding were inhibited. The results show that acamprosate binds to a specific spermidine-sensitive site that modulates the NMDA receptor in a complex way. Together, with data from al Quatari et al. (see next paper), this work suggests that acamprosate acts as "partial co-agonist" at the NMDA receptor, so that low concentrations enhance activation when receptor activity is low, whereas higher concentrations are inhibitory to high levels of receptor activation. This may be relevant to the clinical effects of acamprosate in alcohol-dependent patients during abstinence.

Spermidine-preferential Uptake System in Escherichia coli: IDENTIFICATION OF AMINO ACIDS INVOLVED IN POLYAMINE BINDING IN PotD PROTEIN

Spermidine-binding sites on PotD protein, substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

Polyamine Binding to Plasma Membrane Vesicles Isolated from Zucchini Hypocotyls.

The general features of [14C]spermidine binding to plasmalemma vesicles isolated from zucchini (Cucurbita pepo L.) etiolated hypocotyls are reported in the present paper. The specific interaction of the polyamine with the plasma membranes was reversible and thermolabile, since it decreased by about 50% in the assay performed at 40[deg]C compared to that carried out on ice. On the contrary, nonspecific binding was unaffected by temperature. Specific spermidine binding showed a pH dependence with a maximum at pH 8.0 and it reached saturation between 0.75 and 1 mM external spermidine concentration. The value of the dissociation constant calculated from Scatchard analysis was 4.4 x 10-5 M. Specific spermidine interaction appeared to be sensitive to detergents and was markedly reduced by the presence of divalent cations, such as Mg2+ and Ca2+, whereas it was stimulated by monovalent cations. Polyamine binding sites were highly sensitive to pronase treatment. Competition experiments, performed using a series of compounds structurally related to spermidine, may provide some indication of the characteristics of spermidine binding sites. The results presented here suggest that specific spermidine binding occurs mainly with the protein component of the plasma membrane.

Glutathionylspermidine Metabolism in Escherichia coli.: PURIFICATION, CLONING, OVERPRODUCTION, AND CHARACTERIZATION OF A BIFUNCTIONAL GLUTATHIONYLSPERMIDINE SYNTHETASE/AMIDASE

Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W. (1975) J. Biol. Chem. 250, 2648-2654). Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.

Features of the spermidine-binding site of deoxyhypusine synthase as derived from inhibition studies. Effective inhibition by bis- and mono-guanylated diamines and polyamines

Several types of basic compounds structurally related to spermidine, one of the substrates for deoxyhypusine synthase, were tested as inhibitors of this enzyme. The results indicate that inhibitory compounds associate with the enzyme at the site of spermidine binding and must possess two charged primary amino or guanidino groups, or one of each. The efficiency of inhibition is related to the maximum possible distance between the primary amino groups and is adversely affected by substitutions on the secondary amino group or in the carbon chains of polyamines. The mono-guanyl derivatives are much more effective inhibitors than the parent amines or their bis-guanylated counterparts, N1-guanyl-1,7-diaminoheptane being the most effective compound with a Ki value of about 10 nM. Based on these observations we have proposed a model for the spermidine-binding site of deoxyhypusine synthase. Studies with Chinese hamster ovary cells reveal a direct correlation between prevention of hypusine formation by several guanyldiamines and their in vitro inhibition of deoxyhypusine synthase. This evidence for disruption of the initial step in the post-translational maturation of eukaryotic initiation factor 5A provides a basis for the potential control of protein biosynthesis and cell proliferation.

Identification and characterization of specific binding sites of [ 3H]spermidine in synaptic membranes of rat brain

Synaptic membranes of rat brain contained specific binding sites of [ 3H]spermidine (SPD) that exhibited an inverse temperature dependency, structure selectivity, reversibility, saturability, low affinity and high density with an uneven distribution profile. The affinities were not significantly different from each other in the rodent brain, while the highest density was found in the medulla-pons among the central structures examined with progressively lower densities in the midbrain, striatum, cerebellum, hypothalamus, hippocampus and cerebral cortex. The binding was insensitive to digestion by various proteases and glycosidases but sensitive to potentiation by phospholipases. A clear correlation was seen between the abilities of several natural and synthetic polyamines to displace [ 3H]SPD binding and to potentiate [ 3H] (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine binding to open cation channels associated with an N- methyl- d-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors. Treatment of brain membranes with deoxycholic acid resulted in a significant solubilization of [ 3H]SPD binding sites. Furthermore, [ 3H]SPD markedly associated with the acidic phospholipid phosphatidylserine irrespective of the presence of synaptic membranes in a manner sensitive to inhibition by a variety of calmodulin antagonists. These results suggest that endogenous polyamines may play a stimulatory role in neuronal responses mediated by the NMDA receptor ionophore complex through an interaction between their positive charges and negative charges of membranous phosphatidylserine in rat brain.

Polyamine-linked sepharoses: Preparation and application to mammalian spermine synthase

Seven different polyamine-linked Sepharose derivatives were prepared for the affinity chromatography of spermidine and spermine binding macromolecules: Spermine synthase from rat and hog brain was used as a model protein with a spermidine binding side. Comparative studies of the affinities of the enzymes for the seven matrixes suggested that two negative charges, three to four methylene groups apart, should be present at the decarboxylated S-adenosylmethionine binding site and should improve the binding of the enzyme to the Sepharose derivative. Two negative charges at the spermidine binding site would be expected to do the same. Three affinity matrixes linked with 1,17-diamino-4,9,14-triazaheptadecane, 1,21-diamino-4,9,13,18-tetraazaheneicosane, and 5-spermine carboxylic acid, respectively, had an affinity for spermine synthases higher than that of spermine-Sepharose, which has been used for the purification of spermine synthase. The first of these matrixes was used and proved to be effective for the purification.

Putrescine or spermidine binding site of aminopropyltransferases and competitive inhibitors

A model of the active site of aminopropyltransferases was proposed based on the study of a number of monoamino and diamino compounds as potential inhibitors and substrates, respectively, of spermidine synthase purified from pig liver. The active site seems to have a relatively large hydrophobic cavity adjacent to a negatively charged site, to which a protonated amino group of putrescine binds, with another amino group of putrescine being situated in the hydrophobic cavity as a free form to be aminopropylated by decarboxylated S-adenosylmethionine. On the basis of the above-mentioned model, another modified one was proposed for spermine synthase, and several compounds designed according to the modified model were found to potently inhibit spermine synthase, purified from rat brain, in competition with spermidine. The newly developed inhibitors were about two orders of magnitude more potent in vitro than a known inhibitor of spermine synthase, dimethyl(5′-adenosyl)sulfonium perchlorate.

A 13C NMR study of [5,8-13C2]spermidine binding to tRNA and to Escherichia coli macromolecules.

[5,8-13C2]Spermidine was prepared by synthesis, and its binding to macromolecular structures of Escherichia coli was studied. When added to E. coli cells, the two signals of [13C]spermidine (C-5, 47.8 ppm, and C-8, 39.6 ppm; JC-C = 5.8 Hz) were strongly broadened due to binding to macromolecules. When [13C]spermidine was added to E. coli tRNA, the C-5 resonance broadened to v1/2 = 4.7 Hz, whereas the C-8 resonance broadened to v1/2 = 2.7 Hz. tRNA-bound [13C]spermidine could be chased by [12C]spermidine or spermine, but not by putrescine or cadaverine. By using mixtures of [5-13C]- and [8-13C]spermidines (where 13C-13C coupling was avoided), it was possible to estimate a dissociation constant (Kd) of 3 x 10(-3) M using the C-5 v1/2obs values and a Kd of 2.10(-3) M using the C-8 v1/2obs values. The number of spermidine-binding sites (n) could also be estimated by fitting the bound spermidine molar fraction versus tRNA concentration. Values of n = 12 +/- 2 and 14 +/- 3 were obtained for C-5 and C-8, respectively. Measurements of line narrowing at increasing Mg2+ concentrations indicated that approximately 11 spermidines (of the 12-14 bound ones) could be displaced by the former, whereas 3 spermidines remain strongly bound to the tRNA backbone. Measurements of free and bound T1 allowed the determination of a correlation time of 10(-10)s for tRNA-bound spermidine.

3H]spermidine binding sites within the NMDA receptor complex in rat brain

3H]spermidine binding sites within the NMDA receptor complex in rat brain

Novel spermidine binding sites on the NMDA receptor complex : Yukio Yoneda and Kiyokazu Ogita, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-01, Japan

Novel spermidine binding sites on the NMDA receptor complex : Yukio Yoneda and Kiyokazu Ogita, Department of Pharmacology, Setsunan University, Hirakata, Osaka 573-01, Japan

Novel spermidine binding sites on the NMDA receptor complex

Novel spermidine binding sites on the NMDA receptor complex

Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata.

The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1,N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 microM and 379 microM, respectively. Since the disulfide form of N1-monoglutathionylspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.

Binding of spermidine to transfer ribonucleic acid.

The binding of spermidine to yeast tRNAPhe and Escherichia coli tRNAGlu2 at low and high ionic strength was studied by equilibrium dialysis. Once corrected for the expected Donnan effect, the binding at low ionic strength obeys the simple relationship of equivalent binding sites, and cooperative binding of spermidine to tRNA could not be detected. At low ionic strength (0.013 M Na+ ion), tRNAPhe (yeast) has 13.9 +/- 2.3 strong spermidine binding sites per molecule with Kd = 1.39 X 10(-6) M and a few weak spermidine binding sites which were inaccessible to experimentation; tRNAGlu2 (E. coli) has 14.8 +/- 1.6 strong spermidine binding sites and 4.0 +/- 0.1 weak spermidine binding sites with Kd = 1.4 X 10(-6) M and Kd = 1.23 X 10(-4) M, respectively. At high ionic strength (0.12 M monovalent cation) and 0.01 M Mg2+, tRNAPhe (yeast) has approximately 13 strong spermidine binding sites with an apparent Kd = 3.4 X 10(-3) M while the dimeric complex tRNAPhe X tRNAGlu2 has 10.4 +/- 1.2 strong spermidine binding sites per monomer with an apparent Kd = 2.0 X 10(-3) M. In the presence of increasing Na+ ion or K+ ion concentration, spermidine binding data do not fit a model for competitive binding to tRNA by monovalent cations. Rather, analysis of binding data by the Debye-Hückel approximation results in a good fit of experimental data, indicating that monovalent cations form a counterion atmosphere about tRNA, thus decreasing electrostatic interactions. On the basis of equilibrium binding analyses, it is proposed that the binding of spermidine to tRNA occurs predominantly by electrostatic forces.