Observations on the conversion of pteroylmonoglutamate into polyglutamate in human system.
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Folic Acid↗
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A rapid colorimetric assay for gamma-glutamyl hydrolase (conjugase).
A simple procedure for the measurement of gamma-glutamyl hydrolase (conjugase) activity is described. Glutamic acid released from pteroylpenta-gamma-glutamate by hog kidney and chicken pancreas conjugases was quantitated using the dye 4,4'-bis(dimethylamino)benzophenone hydrazone. The procedure involves hydrolysis of the folylpoly-gamma-glutamate substrate by conjugase, conversion of glutamate to alpha-ketoglutarate by L-glutamate dehydrogenase and colorimetric measurement of the BDBH derivative of alpha-ketoglutarate. The release of as little as one nmol of glutamic acid from the substrate can be measured by this procedure, which is well suited for the assay of a variety of conjugase preparations. In addition, the method should provide a general assay for the enzymatic hydrolysis of various folate and antifolate polyglutamates.
Animals↗
Methotrexate analogues. 34. Replacement of the glutamate moiety in methotrexate and aminopterin by long-chain 2-aminoalkanedioic acids.
Eight previously unreported methotrexate (MTX) and aminopterin (AMT) analogues with the L-glutamate moiety replaced by DL-2-aminoalkanedioic acids containing up to 10 CH2 groups were synthesized from 4-amino-4-deoxy-N10-methylpteroic or 4-amino-4-deoxy-N10-formylpteroic acid. All the compounds were potent inhibitors of purified L1210 mouse leukemia dihydrofolate reductase (DHFR), with IC50's of 0.023-0.034 microM for the MTX analogues and 0.054-0.067 microM for the AMT analogues. The compounds were not substrates for, but were inhibitors of, partially purified mouse liver folylpolyglutamate synthetase (FPGS). Activity was correlated with the number of CH2 groups in the side chain. The IC50's for inhibition of cell growth in culture by the chain-extended MTX analogues were 0.016-0.64 microM against CEM human leukemic lymphoblasts and 0.0012-0.026 microM against L1210 mouse leukemia cells. However, the optimal chain length for growth-inhibitory activity was species-dependent. Our results suggested that CEM cells were inhibited most actively by the analogue with nine CH2 groups, while L1210 cells were most sensitive to the analogue with six CH2 groups. Among the AMT analogues, on the other hand, the most active compound against L1210 cells was the one with nine CH2 groups, which had an IC50 of 0.000 65 microM as compared with 0.0046 microM for MTX and 0.002 microM for AMT. A high degree of cross-resistance was observed between MTX and the chain-extended compounds in two MTX-resistant cell lines, CEM/MTX and L1210/R81. All the MTX analogues were active against L1210 leukemia in mice on a qd X 9 schedule, with optimal increases in lifespan (ILS) of 75-140%. Notwithstanding their high in vitro activity, the AMT analogues were more toxic and less therapeutically effective than MTX analogues of the same chain length even though neither series of compounds possessed FPGS substrate activity. These MTX and AMT analogues are an unusual group of compounds in that they retain the dicarboxylic acid structure of classical antifolates yet are more lipophilic than the parent compounds because they have more CH2 groups and are almost equivalent in vivo to MTX on the same schedule even though they do not form polyglutamates.
Amino Acids, Dicarboxylic↗
[Folic acid metabolism in rapidly growing tissues: synthesis of pteroylpolyglutamates].
Pteroylpolyglutamates are regarded as the functional coenzyme form of folate: hence their synthesis may represent important regulatory step in the folate-dependent processes. To verify this hypothesis the pteroylpolyglutamate content was studied in rapidly growing tissues, which require a large availability of folate coenzymes, essential as carbon atom donors in the synthesis of purine and pyrimidine nucleotides and in the metabolism of some aminoacids. In the developing chick embryo, the higher polyglutamates are the only folate forms present and their content increases progressively during the development of the embryo. In rat liver regenerating after partial hepatectomy the higher polyglutamate forms decrease below the normal values from 12 to 72 h after the operation; the normal values are restored after 180 h. The initial drop is not related to a lower synthesis, but to a greater utilization of these coenzymes during the early periods of regeneration, when biosynthetic processes are markedly increased. In fact the content of polyglutamates in regenerating liver can be maintained at normal levels by increasing the availability of precursors via folic acid administration to hepatectomized rats.
Animals↗
5-fluorouracil: a pharmacological paradigm in the use of cytotoxics.
1. Painstaking progress in drug development is well illustrated by 5-fluorouracil (5FU), originally designed 40 years ago as a fluorinated analogue of the naturally occurring base uracil. Innovative pharmacokinetic and pharmacodynamic strategies have seen significant clinical improvements for cancer patients over the past decade. 2. 5-Fluorouracil acts by three main mechanisms. Principally, the intermediate metabolite fluorodeoxyuridine monophosphate inhibits a key enzyme in pyrimidine biosynthesis, namely thymidylate synthase (TS). Additionally, 5FU is metabolized to ribo- and deoxy-ribonucleotides, which act as false bases for incorporation into RNA and DNA. 3. Biomodulation of 5FU has been attempted with methotrexate (MTX), folinic acid, interferons, cisplatin and radiotherapy. Methotrexate augments the actions of 5FU by inhibiting dihydrofolate reductase and decreasing the folate pool required for pyrimidine biosynthesis, inhibiting TS via MTX-polyglutamate and directly inhibiting purine biosynthesis. Interferons increase steady state concentrations of 5FU. 5-Fluorouracil enhances the cytotoxicity of cisplatin and radiotherapy by inhibiting DNA repair. Folinic acid enhances TS inhibition by increasing the intracellular pool of folates that stabilize the 5FU-TS complex. 4. 5-Fluorouracil has a short plasma half-life. Thymidylate synthase inhibition is limited to the S-phase of the cell cycle and only a small fraction of most cancer cells are in S-phase at any one time. Increased response rates seen with infusional protocols may reflect the effective recruitment of additional mechanisms of cytotoxicity, not dependent on cell cycle, including effects on RNA synthesis. 5. Patients with localized metastatic disease may benefit from locoregional treatments. These include hepatic intra-arterial therapy with related compounds, such as floxuridine, which reach high concentrations at sites of tumour, while systemic toxicities are minimized by efficient hepatic clearance. 6. Recent developments include orally bioavailable formulations, such as ftorafur, capecitabine and the combination of 5FU with the dihydropyrimidine phosphate dehydrogenase inhibitor ethynyluracil. Recognition of diurnal variation in the activity of such key enzymes as DPD has led to the administration of 5FU at regulated, variable infusion rates (chronomodulation). These promising pharmacological approaches may further improve clinical outcomes in common cancers.
Antimetabolites, Antineoplastic↗
Folates as inhibitors of glutamate dehydrogenase.
Folates and tetrahydrofolates inhibit beef liver glutamate dehydrogenase (EC 1.4.1.2). Double reciprocal plats indicate a competitive inhibition for alpha-ketoglutarate-glutamate by folic acid and methotrexate and a complex or mixed type for NAD-NADH site. Pteroic acid is not inhibitory at the concentrations studied. The addition of up to four gamma-linked glutamyl residues to folic and tetrahydrofolic acids increases the inhibition. Further chain elongation of the gamma-peptide had no effect on the inhibitory activity. The p-aminobenzoate poly-gamma-glutamates were less inhibitory than the corresponding folyl polyglutamates.
Aminobenzoates↗
Identification of a novel gene, dep, associated with depolymerization of the capsular polymer in Bacillus anthracis.
Bacillus anthracis produces a gamma-linked poly-D-glutamic acid capsule that is essential for virulence. A 6.2 kb fragment of B. anthracis DNA (cap), when present in Escherichia coli, produces a capsular polymer that is immunologically identical to that produced by B. anthracis. By immunodiffusion analysis of E. coli strains carrying varying portions of the cap region, we identified a novel gene (dep) responsible for degradation of the capsular polymer of B. anthracis. The simultaneous presence of the cap region and the dep gene caused production of low-molecular-weight, degraded capsular polymer both in E. coli and in B. anthracis, whereas the cap region alone caused production of a high-molecular-weight capsule. The dep gene mapped immediately downstream of the cap region within a 1.8 kb fragment and was transcribed in the same direction. This fragment was sequenced and a 1401 bp open reading frame (ORF) was found that is predicted to encode a peptide with molecular weight of 51,460. By in vitro transcription-translation analysis, this ORF was shown to be the dep gene product. The deduced amino acid sequence of the dep product has sequence similarity to E. coli and mammalian gamma-glutamyltranspeptidase (GGT). However, the Dep protein did not have GGT activity. The Dep protein appears to be an enzyme that catalyses the hydrolysis of the poly-D-glutamic acid capsule. Although the biological functions of the dep gene are unknown, it is possible that low-molecular-weight, diffusible polyglutamates produced through the action of the dep gene may act to inhibit host defence mechanisms.
Amino Acid Sequence↗
Mouse folylpoly-gamma-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors.
Folylpoly-gamma-glutamate synthetase (FPGS) is the enzyme responsible for metabolic trapping of reduced folate cofactors in cells for use in nucleotide and amino acid biosynthesis. There are two isoforms of FPGS expressed in mouse tissues, one is expressed in differentiated tissue, principally liver and kidney, and the other in all rapidly proliferating cell types. The present study sought the functional difference that would explain the evolution of two mouse FPGS species. Recombinant cytosolic mouse isozymes were compared with respect to steady state kinetics, chain length of polyglutamate derivatives formed, and end-product inhibition by the major reduced folylpentaglutamate cofactors. Both isoforms were equally effective in catalyzing the addition of a mole of glutamic acid to reduced folate monoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of the model folate, 5,10-dideazatetrahydrofolate. In contrast, the FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH(2)-H(4)PteGlu(5) and (6S)-H(4)PteGlu(5) than the isoform expressed in differentiated tissues, as demonstrated by 13- and 6-fold lower inhibition constants (K(i)), respectively. Interestingly, each isozyme was equally sensitive to inhibition by (6R)-10-CHO-H(4)PteGlu(5). We drew the conclusion that the decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by 5,10-CH(2)-H(4)PteGlu(5-6) and H(4)PteGlu(5-6) may have evolved to permit accumulation of a larger folate cofactor pool than that found within rapidly proliferating tissue.
Amino Acid Sequence↗
Observations on the presence of pteroyl polyglutamates in human serum.
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Animals↗
Human thymidylate synthetase. II. Derivatives of pteroylmono- and -polyglutamates as substrates and inhibitors.
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Folic Acid↗
Bacteriophage tail components. IV. Pteroyl polyglutamate synthesis in T4D-infected Escherichia coli B.
The nature of pteroyl polyglutamates in uninfected and T4D bacteriophage-infected Escherichia coli B has been examined. (3)H-p-aminobenzoic acid has been used to label the folate compounds and gel permeation chromatography on glass beads to separate the folate compound by molecular size. It has been found that, although the major folate compound in uninfected bacteria is pteroyl triglutamate, E. coli B cells also contain folate compounds having as many as six glutamate residues. Infection with T4D stimulated the addition of glutamate residues to the lower-molecular-weight host pteroyl compounds, resulting in the conversion of the host compounds into the hexaglutamate form. This viral-induced conversion is chloramphenicol sensitive and appears to be due to a late phage gene product. The phage gene responsible for this conversion has not been identified. In cells infected with a T4D mutant defective in gene 28, there was an apparent production of the large pteroyl polyglutamates equivalent in size to pte(glu)(9-12). These high-molecular-weight forms were converted into pte(glu)(6) by incubation with bacterial extracts made after infection with T4D 28(+). Apparently, the product of T4D gene 28(+) is capable of specifically cleaving the high-molecular-weight polyglutamates to the form necessary for phage tail assembly.
Aminobenzoates↗
Effects of gamma-glutamyl hydrolase on folyl and antifolylpolyglutamates in cultured H35 hepatoma cells.
A subline of H35 hepatoma cells (H35D cells) that have been made resistant to 5,10-dideazatetrahydrofolate exhibits an increase in gamma-glutamyl hydrolase (GH) activity. GH is a lysosomal enzyme in H35 and H35D cells on the basis of comparison of the distribution of enzyme activity with other known lysosomal enzymes. The hydrolysis rate of methotrexate polyglutamate with isolated, intact lysosomes is 4-5-fold greater in H35D cells than in H35 cells. GH activity in isolated lysosomes is in part dependent on the presence of a reducing agent such as mercaptoethanol. Permeabilization of lysosomal preparations from both cell types by Triton X-100 causes a 10-fold enhancement in GH activity. The result of the enhanced activity of GH in H35D cells is a marked reduction in antifolylpolyglutamate concentration, with the parent antifolate being the predominant intracellular species found under all conditions tested. Unlike antifolates, the total intracellular folate concentration is nearly identical in both cells under standard culture conditions up to 10 microns folic acid. However, the chain length of folylpolyglutamates consists of predominantly triglutamates and tetraglutamates in H35D cells with increased GH, whereas it consists of pentaglutamates and hexaglutamates in the parental cells. At 50 and 100 microns folic acid, the folate accumulation in H35D cells is less than half that of H35 cells, and the predominant polyglutamate species in the H35D cells are the diglutamates through the tetraglutamates. The results demonstrate that the two H35 cell lines having equal folylpolyglutamate synthetase but that one with enhanced lysosomal GH activity exhibits a marked reduction in the amount and gamma-glutamyl chain length of folylpolyglutamates and antifolylpolyglutamates.
Animals↗
Antitumor activity of antifolate inhibitors of thymidylate and purine synthesis in human soft tissue sarcoma cell lines with intrinsic resistance to methotrexate.
We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.
Antimetabolites, Antineoplastic↗
Purification and partial characterization of rat liver folate binding protein: cytosol I.
The high molecular weight folate binding protein of rat liver cytosol has been purified to apparent homogeneity. Purification was achieved by using a combination of gel filtration, O-(diethylaminoethyl)cellulose chromatography, and affinity chromatography. This folate binding protein was initially identified during purification by an in vivo labeling procedure involving intraperitoneal injection of [3H]folic acid prior to sacrifice and subsequently by its ability to bind naturally reduced [3H]folate polyglutamates in vitro. A molecular weight of 210 000 was estimated by gel chromatography. This is distinct from the trifunctional formyl-methenyl-methylene synthetase of rat liver which has a molecular weight of 225 000. Sodium dodecyl sulfate electrophoresis revealed a single band with a molecular weight of about 100 000 which suggests the native protein is composed of two identical subunits. The partially purified protein contains bound tetrahydropteroylpentaglutamate.
Animals↗
Novel antifolate drugs.
Antimetabolites are active chemotherapeutic agents for many solid tumor and hematologic malignancies. Folate antagonists, purine analogues, and pyrimidine analogues are the three main categories of antimetabolites. Methotrexate, the most studied folate antagonist, is effective in many malignancies. Methotrexate inhibits dihydrofolate reductase, which leads to accumulation of polyglutamated folates, causing further inhibition of thymidylate synthase and glycinamide ribonucleotide formyltransferase. Subsequently, the lack of reduced folate substrates impairs synthesis of purine nucleotides, thymidylate, and certain amino acids, which can lead to cell death. However, methotrexate resistance develops through several mechanisms, including decreased folate carrier-mediated membrane transport, dihydrofolate reductase gene amplification, specific transcription-translational modifications, and downregulation of intracellular methotrexate polyglutamation. Antifolate drug development has focused on agents designed to overcome different aspects of methotrexate resistance. This article reviews the enzymatic targets for antifolates, describes the known mechanisms of antifolate resistance, and summarizes the current development of novel antifolate agents. Discussed specifically are trimetrexate, edatrexate, raltitrexed, pemetrexed, ZD9331, lometrexol, LY309887, GW1843, OSI-7904(L), and nolatrexed, all of which have unique clinical pharmacology and are in various stages of development. The toxicity of antifolates has been sporadic and difficult to predict clinically. Supplementation with folic acid and vitamin B(12) has been shown to reduce the toxicity of pemetrexed without affecting efficacy and has increased the therapeutic index for this novel agent.
Antimetabolites, Antineoplastic↗
Structural basis for recognition of polyglutamyl folates by thymidylate synthase.
Thymidylate synthase (TS) catalyzes the final step in the de novo synthesis of thymidine. In vivo TS binds a polyglutamyl cofactor, polyglutamyl methylenetetrahydrofolate (CH2-H4folate), which serves as a carbon donor. Glutamate residues on the cofactor contribute as much as 3.7 kcal to the interaction between the cofactor, substrate, and enzyme. Because many ligand/receptor interactions appear to be driven largely by hydrophobic forces, it is surprising that the addition of hydrophilic, soluble groups such as glutamates increases the affinity of the cofactor for TS. The structure of a polyglutamyl cofactor analog bound in ternary complex with deoxyuridine monophosphate (dUMP) and Escherichia coli TS reveals how the polyglutamyl moiety is positioned in TS and accounts in a qualitative way for the binding contributions of the different individual glutamate residues. The polyglutamyl moiety is not rigidly fixed by its interaction with the protein except for the first glutamate residue nearest the p-aminobenzoic acid ring of folate. Each additional glutamate is progressively more disordered than the previous one in the chain. The position of the second and third glutamate residues on the protein surface suggests that the polyglutamyl binding site could be utilized by a new family of inhibitors that might fill the binding area more effectively than polyglutamate.
Amino Acid Sequence↗
5,10-Dideazatetrahydrofolic acid (DDATHF) transport in CCRF-CEM and MA104 cell lines.
5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly converted to high chain length polyglutamates. Transport via this system was inhibited in folate-depleted cells by 10 nM folic acid.(ABSTRACT TRUNCATED AT 400 WORDS)
Biological Transport↗
5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase.
Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.
Cell Division↗