Induction of a new ribonucleotide reductase after infection of mouse L cells with pseudorabies virus.

The mammalian ribonucleotide reductase consists of two nonidentical subunits, protein M1 and M2. M1 binds nucleoside triphosphate allosteric effectors, whereas M2 contains a tyrosine free radical essential for activity. The activity of ribonucleotide reductase increased 10-fold in extracts of mouse L cells 6 h after infection with pseudorabies virus. The new activity was not influenced by antibodies against subunit M1 of calf thymus ribonucleotide reductase, whereas the reductase activity in uninfected cells was completely neutralized. Furthermore, packed infected cells (but not mock-infected cells) showed an electron paramagnetic resonance spectrum of the tyrosine free radical of subunit M2 of the cellular ribonucleotide reductase. These data given conclusive evidence that on infection, herpesvirus induces a new or modified ribonucleotide reductase. The virus-induced enzyme showed the same sensitivity to inhibition by hydroxyurea as the cellular reductase. The allosteric regulation of the virus enzyme was completely different from the regulation of the cellular reductase. Thus, CDP reduction catalyzed by the virus enzyme showed no requirement for ATP as a positive effector, and no feedback inhibition was observed by dTTP or dATP. The virus reductase did not even bind to a dATP-Sepharose column which bound the cellular enzyme with high affinity.     

Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

Stereoselective, strong inhibition of ribonucleotide reductase from E. coli by cisplatin

Using ribonucleotide reductase (EC 1.17.4.1) purified from E. coli clones with overproducing plasmids for the B1 and B2 subunits, respectively, studies have been carried out of the inhibition of this enzyme by cisplatin. Under anaerobic conditions, using the dithiol, reduced form of the enzyme, it was found that ribonucleotide reductase is extremely sensitive to cisplatin: greater than 90% inhibition was achieved with 2-fold molar excess of platinum reagent even at 10(-8)M enzyme. Inhibition was essentially instantaneous and irreversible to G-25 gel filtration. The site of inhibition was found to be the B1 subunit. Transplatin was much less effective. Inhibition of the enzyme by cisplatin (molar ratio cisplatin:B1 = 4.3) led to a decrease in thiol titre corresponding to approximately 1 thiol group per dimer of B1 subunits under conditions leading to 94% inactivation of the ribonucleotide reductase activity.     

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis

Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast. The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms. In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP. On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity. Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP. The relative molecular mass of the holoenzyme is about 250 000. The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy. GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonucleotide and ATP. Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents. Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (I50) is reached only at 2-6 mM hydroxyurea concentration. Up to 50% reactivation occurs spontaneously after removal of the inhibitor. In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure. Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.     

Enhancement of ribonucleotide reductase activity at low enzyme concentrations by polyethylene glycol

The estimation of ribonucleotide reductase in cell extracts has been problematical on account of abnormally low activities at low enzyme concentrations, presumably due to subunit dissociation. This problem can be alleviated by assaying the enzyme in the presence of polyethylene glycol. The presence of 15% polyethylene glycol during the assay greatly stimulated ribonucleotide reductase activity at low enzyme concentrations and allowed measurement of enzyme activity in as little as 10(5) mouse L929 cells, a 30-fold enhancement of assay sensitivity. Enzyme activity measured in the presence of 15% polyethylene glycol was proportional to enzyme concentration, thus making possible the accurate measurement of very low levels of ribonucleotide reductase.     

Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region.

Recombinant plasmids containing all or part of the genetic region of Escherichia coli coding for the two subunits of ribonucleoside diphosphate reductase (proteins B1 and B2) were constructed with the aid of the multicopy plasmid pBR322. Two of these plasmids (pPS1 and pPS2) appeared to carry both a regulator and the complete structural information for the enzyme and, after transformation of E. coli, directed a 10- to 20-fold overproduction of both proteins B1 and B2. The other plasmids (pPS101 and pPS201) carried structural information for only protein B2. Cells carrying pPS1 and pPS2 showed a 5- to 500-fold increased resistance against the drug hydroxyurea. This establishes that in E. coli the inhibition of deoxyribonucleic acid synthesis by hydroxyurea is fully explained by its action on ribonucleotide reductase.     

Isolation and characterization of an Escherichia coli K-12 mutant deficient in glutaredoxin.

Mutants of Escherichia coli K-12 deficient in glutaredoxin were isolated and partially characterized. The mutants have detectable but significantly reduced glutaredoxin activity in assays of whole cells made permeable with ether as well as in assays of crude extracts coupled to ribonucleotide reductase. In vivo, the mutants appear to be deficient in both sulfate and ribonucleotide reduction, suggesting that in vivo glutaredoxin is the preferred cofactor for ribonucleotide reductase and adenosine 3'-phosphate 5'-phosphosulfate reductase. Complementation of the mutant phenotype by transformants was used to clone the wild-type glutaredoxin allele. The transformants had a high level of glutaredoxin activity and contained a plasmid with an insert that had a restriction endonuclease pattern identical to that predicted by the DNA sequence for glutaredoxin determined by Hoog et al. (J.-O. Hoog, H. von Bahr-Lindstrom, H. Jornvall, and A. Holmgren, Gene 43:13-21, 1986).     

Investigation of an octapeptide inhibitor of Escherichia coli ribonucleotide reductase by transferred nuclear Overhauser effect spectroscopy

Several peptides contained within the C-terminal sequence of the B2 subunit of Escherichia coli ribonucleotide reductase (RNR) were investigated for their ability to inhibit the enzyme, presumably by interfering with association of the B1 and B2 subunits. AcYLVGQIDSE, corresponding by sequence homology to a nonapeptide that inhibits herpes simplex RNR [Gaudreau et al. (1987) J. Biol. Chem. 262, 12413] shows no inhibition of the E. coli enzyme (IC50 greater than 3 mM), whereas AcDDLSNFQL, the C-terminal octapeptide of the E. coli B2 subunit, is a noncompetitive inhibitor (Ki = 160 microM). Neither bradykinin (RPPGFSPFR) nor the pentapeptide AcSNFQL inhibits the E. coli enzyme. Transferred nuclear Overhauser enhancement spectroscopy was used to probe the conformation of AcDDLSNFQL when it is bound to the B1 subunit. These experiments suggest that the peptide adopts a turn in the region of Asn5 and Phe6 and that a hydrophobic cluster of the phenylalanine and leucine side chains is involved in the interaction surface.     

Thioredoxin reductase-mediated hydrogen transfer from Escherichia coli thioredoxin-(SH)2 to phage T4 thioredoxin-S2.

Thioredoxin from Escherichia coli B and phage T4-infected E. coli B are small hydrogen carrier proteins which in their reduced forms are specific hydrogen donors to E. coli and T4-induced ribonucleotide reductase, respectively. The oxidation-reduction active group of both thioredoxins consists of a single cystine residue which is reduced to sulfhydryl form by NADPH in the presence of E. coli thioredoxin reductase. Reduction of T4 thioredoxin-S2 to thioredoxin-(SH)2 led to a 3-fold increase in the quantum yield of tyrosine fluorescence. By using the spectrofluorimetric properties of T4 thioredoxin and E. coli thioredoxin as markers for their oxidized and reduced forms we have shown that E. coli thioredoxin reductase catalyzed the reaction: (see article) whose equilibrium constant favors formation of E. coli thioredoxin-S2 and T4 thioredoxin-(SH)2. This finding suggests that in the T4-infected cell most of the deoxyribonucleotides required for the viral DNA might be synthesized by the T4-induced ribonucleotide reductase while the host ribonucleotide reductase is inactive due to the shortage of reduced E. coli thioredoxin.     

The small subunit of ribonucleotide reductase is encoded by one of the most abundant translationally regulated maternal RNAs in clam and sea urchin eggs

In both clam oocytes and sea urchin eggs, fertilization triggers the synthesis of a set of proteins specified by stored maternal mRNAs. One of the most abundant of these (p41) has a molecular weight of 41,000. This paper describes the identification of p41 as the small subunit of ribonucleotide reductase, the enzyme that provides the precursors necessary for DNA synthesis. This identification is based mainly on the amino acid sequence deduced from cDNA clones corresponding to p41, which shows homology with a gene in Herpes Simplex virus that is thought to encode the small subunit of viral ribonucleotide reductase. Comparison with the B2 (small) subunit of Escherichia coli ribonucleotide reductase also shows striking homology in certain conserved regions of the molecule. However, our attention was originally drawn to protein p41 because it was specifically retained by an affinity column bearing the monoclonal antibody YL 1/2, which reacts with alpha-tubulin (Kilmartin, J. V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582). The finding that this antibody inhibits the activity of sea urchin embryo ribonucleotide reductase confirmed the identity of p41 as the small subunit. The unexpected binding of the small subunit of ribonucleotide reductase can be accounted for by its carboxy-terminal sequence, which matches the specificity requirements of YL 1/2 as determined by Wehland et al. (Wehland, J., H. C. Schroeder, and K. Weber, 1984, EMBO [Eur. Mol. Biol. Organ.] J., 3:1295-1300). Unlike the small subunit, there is no sign of synthesis of a corresponding large subunit of ribonucleotide reductase after fertilization. Since most enzymes of this type require two subunits for activity, we suspect that the unfertilized oocytes contain a stockpile of large subunits ready for combination with newly made small subunits. Thus, synthesis of the small subunit of ribonucleotide reductase represents a very clear example of the developmental regulation of enzyme activity by control of gene expression at the level of translation.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure

The quaternary structure of ribonucleotide reductase of Escherichia coli was investigated, with the use of purified B1 and B2 proteins and bifunctional cross-linking agents. The holoenzyme is known to be an alpha 2 beta 2 tetramer consisting of two dimeric proteins: B1 (alpha 2) and B2 (beta 2). The cross-linking data support a model in which both of the beta subunits interact closely with only one of the two alpha subunits. Some of the interactions involving B2 were localized to the C terminus of the protein by use of truncated B2 protein (beta',beta'), a proteolytic cleavage product of B2 in which the 30 carboxy-terminal residues are missing from each of the beta subunits. Other interactions were indicated by the ability of glutaredoxin, but not thioredoxin, to inhibit some of the cross-linking reactions. We also asked whether ribonucleotide reductase interacts closely with other proteins inside the cell, by adding cross-linkers directly to suspensions of whole bacteria. Proteins in extracts of these cross-linked bacteria were resolved electrophoretically and probed with a monoclonal antibody to the B1 protein. High-molecular-mass products were detected, supporting the utility of this method for identifying intracellular interactions among enzymes of DNA precursor biosynthesis.     

Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Vaccinia virus-induced ribonucleotide reductase can be distinguished from host cell activity

Increased ribonucleotide reductase activity has been detected in vaccinia virus-infected BSC-40 cells. We have studied certain biochemical and kinetic properties of CDP reduction in extracts from infected and uninfected cells. ATP inhibited reductase activity in crude extracts by rapid and extensive substrate phosphorylation. Substitution of adenylylimido-diphosphate (AMP-PNP), a noncleavable analog that functions as positive activator for reductase, but inhibits phosphorylation and cleavage of substrate, allowed us to reliably measure reductase activity. In the presence of AMP-PNP, CDP reduction by extracts from infected or uninfected cells was linear with time for 60 min and with enzyme concentration, except at very low enzyme levels. Activities from both sources were optimally active at pH 8.1. Variation of AMP-PNP and Mg2+ concentrations revealed, however, that in the absence of exogenous Mg2+, AMP-PNP strongly stimulated virus-induced CDP reduction, but inhibited endogenous CDP reduction. In the presence of the activator, increasing Mg2+ concentrations progressively inhibited the induced activity, but stimulated the endogenous activity up to a 1:2 Mg2+/activator molar ratio. The vaccinia virus-induced activity was highly dependent on AMP-PNP and was not detectable over underlying cellular activity in its absence. Determination of substrate kinetics with respect to CDP revealed a threefold-lower Km for the virus-induced enzyme as compared with the cellular enzyme. These data suggest, but do not prove, that a novel ribonucleotide reductase is expressed on infection by vaccinia virus.     

An active ribonucleotide reductase from Arabidopsis thaliana cloning, expression and characterization of the large subunit.

In all living organisms, deoxyribonucleotides, the DNA precursors, are produced by reduction of the corresponding ribonucleotides catalyzed by ribonucleotide reductase. In mammals as in Escherichia coli, the enzyme consists of two proteins. Protein R1 is the proper reductase as it contains, in the substrate binding site, the reducing active cysteine pair. Protein R2 provides a catalytically essential organic radical. Here we report the cloning, expression, purification and characterization of protein R1 from Arabidopsis thaliana. Expression in E. coli was made possible by coexpression of tRNAArg4 which is required for the utilization of AGA and AGG as codons for arginines. Protein R1 shows extensive similarities with protein R1 from mammals: (a) it shows 69% amino-acid sequence identity to human and mouse R1 protein; (b) it is active during CDP reduction by dithiothreitol, in the presence of protein R2 [Sauge-Merle, S., Laulhère, J.-P., Coves, J., Ménage, S., Le Pape, L. & Fontecave, M. (1997) J. Biol. Inorg. Chem. 2, 586-594]; (c) activity is stimulated by thioredoxin and ATP and is inhibited by dATP, showing that as in the mammalian enzyme, the plant ribonucleotide reductase seems to be allosterically regulated by positive (ATP) and negative (dATP) effectors.     

B12 Coenzyme-dependent Ribonucleotide Reductase in Rhizobium Species and the Effects of Cobalt Deficiency on the Activity of the Enzyme

This investigation revealed that the ribonucleotide reductases in extracts of Rhizobium leguminosarum, R. trifolii, R. phaseoli, R. japonicum, and R. meliloti 3DOal (ineffective in nitrogen fixation) are dependent upon B(12) coenzyme for activity. Rhizobium and certain Lactobacillus species are the only two groups of organisms known to contain B(12) coenzyme-dependent ribonucleotide reductases. Extracts of cobalt-deficient R. meliloti cells assayed in the presence of optimum B(12) coenzyme showed a 5- to 10-fold greater ribonucleotide reductase activity than comparable extracts from cells grown on a complete medium. Furthermore, cobalt-deficient cells were abnormally elongated and contained reduced contents of deoxyribonucleic acid. The addition of purified deoxyribonucleosides to cobalt-deficient cultures of R. meliloti failed to alleviate deficiency symptoms.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase.

The small subunit of iron-dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine-122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide-directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron-related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3-kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25-35 times higher level as compared to the host strain.     

Affinity chromatography on anti-B1 monoclonal gels for purification and characterization of protein B1 from Escherichia coli ribonucleotide reductase

Affinity gels were prepared from four monoclonal antibodies against the B1 protein of ribonucleotide reductase of Escherichia coli. The gels were used to purify protein B1 and also to study some of its properties. Gels from the nonneutralizing monoclonal anti-B1-k bound as much as 2 mg of B1/mL and were employed to prepare essentially pure B1 protein in a single step from extracts of wild-type E. coli and strains overproducing the subunit. However, B1 prepared from wild-type extracts had a lowered specific activity, suggesting some denaturation during elution of the protein from the column. Addition of the allosteric effector dATP during affinity chromatography changed the chromatographic pattern. Some protein B2, the second subunit of the reductase, remained in all cases bound to the gels together with B1. The gel prepared from anti-B1-c retained two additional proteins. In other experiments involving binding of proteolytic fragments of B1 to various antibodies, we also found a striking effect of dATP, suggesting that dATP made protein B1 less accessible to proteolysis. In these experiments fragments around 15K still had the ability to bind monoclonals, making possible more detailed investigations of the structural contacts between B1 and the monoclonals.     

Hydroxyurea-resistant mouse L cells with elevated levels of drug-resistant ribonucleotide reductase activity

We describe the isolation and partial characterization of a mouse L-cell line which is resistant to normally highly cytotoxic concentrations of hydroxyurea. A detailed analysis of the target enzyme ribonucleotide reductase in both wild-type and hydroxyurea-resistant enzyme preparations suggests that the drug-resistant cells form a ribonucleotide reductase enzyme which contains a structural alteration, rendering it less sensitive to inhibition by hydroxyurea. K₁ values for hydroxyurea inhibition of ribonucleotide reduction in enzyme preparations from hydroxyurea-resistant cells were significantly higher than corresponding values from preparations from wild-type cells. The K_m for CDP reduction in enzyme preparations of drug-resistant cells was approximately threefold higher than the corresponding parental wild-type value. In addition, in vivo enzyme assays detected a major difference between the temperature profiles of ribonucleotide reduction in nucleotide-permeable drug-resistant and wild-type cells. When levels of ribonucleotide reductase activity were measured in vivo, it was found that the drug-resistant cells contained approximately 3 times the wild-type level of CDP reductase activity and twice wild-type level of GDP reductase activity. This combination of enhanced enzyme levels plus an altered sensitivity to drug inhibition can easily account for the drug-resistance phenotype. The properties of these hydroxyurea-resistant cells indicate that they will be useful for genetic and biochemical studies.This work was supported by the N.S.E.R.C. of Canada and the Muscular Dystrophy Association of Canada through research funds (J. A. W.) and by the N.R.C. of Canada through a graduate scholarship (B. A. K.).     

Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(II).

Ribonucleotide reductase is a highly regulated, rate-limiting activity in the synthesis of DNA. A previous study has shown that the Escherichia coli enzyme is inhibited by the clinically important antitumor agent cis-diamminedichloroplatinum(II) (DDP), and this has led to the hypothesis that ribonucleotide reductase is an important site of action for this chemotherapeutic agent. This hypothesis has been directly tested in this investigation. We observed that DDP inhibits the mammalian ribonucleotide reductase, with 50% inhibition occurring at 0.3 mM. Unlike the E. coli enzyme where only one of the two protein components is targeted by DDP, we observed that both of the mammalian proteins (R1 and R2) were sites for the inhibitory activity of the drug. Colony-forming experiments, enzyme activity studies, and analyses of R1 and R2 message levels in mutant cell lines containing either high levels of ribonucleotide reductase activity or exhibiting resistance to the cytotoxic effects of DDP were used to further investigate the potential role of ribonucleotide reductase in DDP cytotoxic action and drug resistance. These studies did not support a hypothesis formulated in the earlier investigation that inhibition of ribonucleotide reductase is an important component of DDP cytotoxic activity or that it is a major participant in DDP resistance mechanisms. From a biological point of view, DDP is a very active drug, and in addition to its cytotoxic effects it is capable of inducing a variety of cellular changes. Whether or not the inhibition of mammalian ribonucleotide reductase activity that we have described in this study plays a role in mediating any of these other effects remains to be determined.