Biomedical subjects

M Muramatu

Publications and source records attributed to M Muramatu.

At least 19 recordsLinked to original sources

PubMed2000

Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells.

Extracellular stimuli trigger adipocyte differentiation by inducing the complex cascades of transcription. Transcription factors CCAAT enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor gamma (PPARgamma) play crucial roles in this process. Although ectopic expression of these factors in NIH-3T3 cells, a multipotential mesenchymal stem cell line, results in adipogenic conversion, little is known as to hormonal factors that regulate adipogenesis in these cells. In this report we demonstrate that PRL, a lactogenic hormone, enhances C/EBPbeta and PPARbeta mRNA expression and augments adipogenic conversion of NIH-3T3 cells. Moreover, we show that ectopic expression of the PRL receptor in NIH-3T3 cells results in efficient adipocyte conversion when stimulated with PRL and a PPARgamma ligand, as evidenced by expression of the adipocyte differentiation-specific genes as well as the presence of fat-laden cells. We further demonstrate that signal transducer and activator of transcription 5 (Stat5), a PRL signal transducer, activates aP2 promoter in a PRL-dependent manner. These results suggest that PRL acts as an adipogenesis-enhancing hormone in NIH-3T3 cells.

3T3 Cells↗

PubMed1998

Effects of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester, a specific trypsin inhibitor, on the growth of HL-60 cells.

Previous results showed that the synthetic compound amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester (APCA-OPhBut), a trypsin inhibitor, could specifically inhibit the activity of proteinase In and lead to growth arrest of Hela cells in early S phase. In this study, APCA-OPhBut exhibited inhibitory effects on the growth of HL-60 cells. Apoptotic cells were observed when the cells were cultured with APCA-OPhBut above 50 microM. Time course studies demonstrated that apoptotic cells were increased in a dose- and time-dependent manner. Flowcytometric assays demonstrated that HL-60 cells underwent slight G1 growth arrest after treatment with APCA-OPhBut. No change of Bcl-2 protein level was detected. The findings suggest that the intracellular trypsin-like protease inhibited by APCA-OPhBut not only plays a key role in DNA synthesis initiation but is also necessary for survival of certain cell lines.

Apoptosis↗

PubMed1998

Escherichia coli prlC gene encodes a trypsin-like proteinase regulating the cell cycle.

Proteinase In has previously been described as displaying a trypsin-like proteinase activity that momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication [Kato, M., Irisawa, T., Ohtani, M., and Muramatu, M. (1992) Eur. J. Biochem. 210, 1007-1014]. We purified the proteinase In from E. coli C600 and found that the 15 amino acid residues of its amino-terminal were identical with those of oligopeptidase A (OpdA), the product of the E. coli prlC gene. The purified proteinase had a molecular mass of approximately 67 kDa, which was also the same as that of oligopeptidase A. To further elucidate the relationship between proteinase In and oligopeptidase A, we assembled an expression vector to direct the synthesis of E. coli oligopeptidase A. The protein was expressed at a high level in E. coli BL21(DE3) and was produced mostly in the soluble, active form. Both the recombinant enzyme (rPrlC) and the purified proteinase In could hydrolyze trypsin substrates for proteinase In as well as benzyloxycarbonyl Ala- Ala-Leu p-nitroanilide (Z-AALpNA), described as a synthetic substrate for oligopeptidase A. The effects of various protease inhibitors on rPrlC were also very similar to those on proteinase In. The trypsin inhibitors 4-guanidino benzoic acid 4-tert-butylphenyl ester and antipain strongly inhibited the trypsin-like proteinase activity of the recombinant enzyme, but had no effect on its Z-AALpNA hydrolyzing activity. Cobalt ion, which greatly enhanced the OpdA activity, slightly inhibited the trypsin-like activity of the recombinant enzyme. These results strongly suggest that proteinase In is encoded by the E. coli prlC gene and is a multi-functional proteinase with two separate active sites.

Base Sequence↗

PubMed1994

Properties of three proteinases functioning at G1, S and G2 phases in HeLa cells and their inhibition by guanidino- and amidino-acid esters.

HeLa cells were synchronized by double thymidine-block and allowed to grow after removal of thymidine. Three proteinases, tryptase 17:17, proteinase In and late G2 proteinase, were prepared from the HeLa cells harvested at the time when each proteinase appeared in the cell cycle of the cells. All of them were suggested to be trypsin-like serine proteinases, because they hydrolyzed trypsin-specific fluorogenic substrates and their activities were inhibited by benzamidine, soybean trypsin inhibitor, leupeptin, tosyl-L-lysine chloromethan (TLCK) and diisopropylfluorophosphate (DEP). However, the actions of these proteinases on the substrates and inhibitors suggested that they were three different proteinases. They were strongly inhibited by 4-tert-butylphenyl and biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, amidinopiperidine-4-acetic and 4-propionic acids, which retard the second DNA synthetic (S) and mitotic (M) phases for 3h, 4-tert-butylphenyl ester of amidinopiperidin-4-carboxylic acid, which blocks initiation of S phase, the ester of amidinopiperidine-4-butyric acid, which suppresses the second S and M phases, and the esters of trans-4-amidinocyclohexanecarboxylic and 4-propionic acids which inhibit M phase.

Amino Acid Sequence↗

PubMed1994

Inhibition of trypsin, plasmin, thrombin and kallikrein by various esters of guanidino- and amidino-acids.

Inhibitory effects of various phenolic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, amidino-piperidine-4-alkanoic acids or trans-4-amidinocyclohexane-4-alkanoic acids on trypsin, thrombin, plasmin and pancreatic kallikrein were examined. Their inhibitory effects were strongly affected by the acid portion and phenolic group constituting the esters. The effects of the acidic portion and phenolic group on the inhibitory effect varied with each protease; they were most effective on thrombin and plasmin and least effective on kallikrein. The inhibitory effect of these esters on trypsin was affected mainly by acid portion.

Amidines↗

PubMed1994

Partial purification of a trypsin-like proteinase in platelets.

Among various fluorogenic substrates for trypsin-like proteinases, tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide was strongly hydrolyzed by a crude extract of rabbit platelets. The proteinase was partially purified (92-fold) from rabbit platelets by successive chromatographic separations on phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. Its molecular mass was found to be greater than 200 kDa by analytical gel filtration and its optimal pH was approximately 9. The proteinase activity was strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, tosyl-L-lysine chrolomethyl ketone, leupeptin, p-nitrophenyl-p-guanidinobenzoate, and also by the 2,4-dimethylphenyl ester of amidinopiperidine-4-propionic acid and the 4-tert-butylphenyl ester of trans-4-guanidinomethylcyclohexanecarboxylic acid which strongly inhibit platelet aggregation induced by various stimuli.

Amino Acid Sequence↗

PubMed1994

Antibacterial effects of esters of guanidino- and amidino-acids trypsin inhibitors.

Inhibitory effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid and the 4-tert- butylphenyl esters of amidinopiperidine-4-alkanoic, trans-4- amidinocyclohexanealkanoic, trans-4-guanidinoethylcyclohexanecarboxylic and trans-4-guanidinocyclohexanealkanoic acids, all trypsin inhibitors, on the growth of E. coli, B. subtilis, S. aureus and S. epidermidis were examined. 4-tert-Butylphenyl esters strongly inhibited the growth of E. coli and the order of the inhibitory effects correlated with that for the inhibitory effects on proteinase In, which appears immediately before initiation of DNA synthesis in E. coli and closely correlates with the onset of DNA synthesis. No correlation was observed between the inhibitory effects and Ki values for trypsin. The 4-tert-butylphenyl esters also strongly inhibited B. subtilis, S. aureus and S. epidermidis, and the order, of the inhibitory effects on these bacteria roughly coincided with that on E. coli. The order of the inhibitory effects of each ester, on these bacteria was S. epidermidis > S. aureus > B. subtilis > E. coli. Among the esters examined, the biphenyl ester of trans-4-guanidinoethylcyclohexanecarboxylic acid was the most inhibitory on these four bacteria and proteinase In. Hydrolysis of tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide, which is a substrate for proteinase In, in crude extracts of E. coli, B. subtilis and S. epidermidis was examined. The order of this activity in these bacteria was E. coli > B. subtilis > S. epidermidis.

Bacteria↗

PubMed1993

Effects of synthetic trypsin inhibitors on the growth of Escherichia coli.

The inhibitory effects of various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), potent trypsin inhibitors, on the growth of Escherichia coli were examined and the effects were compared with those of well known synthetic trypsin inhibitors. Various GMCHA esters strongly inhibited the growth of E. coli and their effects were markedly affected by the species and position of substitution on the phenyl nucleus of the GMCHA phenyl esters. No correlation was observed between the effects on the growth of E. coli and Ki's for trypsin. Inhibitory effects of benzamidine, phenylmethane sulfonylfluoride and diisopropyl fluorophosphate were less than 10% at 200 microM. 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPhtBu), a representative of various GMCHA esters, dose-dependently inhibited the growth of E. coli and the growth inhibition was preceded by a dose- and time-dependent inhibition of DNA synthesis. Tosyl-L-lysine chloromethyl ketone (TLCK) and pentamidine isethionate, potent trypsin inhibitors, also dose-dependently inhibited the growth of E. coli and the DNA synthesis. However, their effects were transient and disappeared after a short while. These results indicate that the effects of TLCK and pentamidine isethionate differ from those of GMCHA esters. GMCHA-OPhtBu and pentamidine isethionate also inhibited RNA and protein synthesis.

Bacterial Proteins↗

PubMed1993

Effects of various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, trypsin inhibitors, on the growth of Bacillus subtilis.

Various aromatic esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA), trypsin inhibitors, strongly inhibited the growth of Bacillus subtilis 558 and their effects were markedly affected by the species of substitution on the phenyl nucleus of the GMCHA phenyl esters. 4-tert-Butylphenyl ester of GMCHA (GMCHA-OPhtBu), a representative of various GMCHA esters, dose-dependently inhibited the growth of B. subtilis and DNA, RNA and protein syntheses in the cells. The growth inhibition was preceded by suppressive effect of GMCHA-OPhtBu on DNA synthesis. These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis. A trypsin-like proteinase was partially purified from B. subtilis 558 by DEAE-cellulose column chromatography, ammonium sulfate fractionation and successive chromatographies on Sephadex G-200, phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. The properties were compared with those of proteinase In, which momentarily appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E. coli K-12 IAM 1264. The properties of the proteinase from B. subtilis 558 were similar to proteinase In, however, the molecular mass (110000) was different from that of the latter (66000). Various GMCHA esters strongly inhibited the proteinase activity and the order of the effects was closely correlated with that on the cell growth. The proteinase was tentatively called subtilis proteinase In.

Amino Acid Sequence↗

PubMed1993

Effects of synthetic trypsin inhibitors on the cell cycle of synchronized HeLa cells.

HeLa cells were synchronized by a double-thymidine block. After removal of thymidine, the cells immediately caused the uptake of [3H]thymidine into DNA and reached a half-maximum. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and between 35 and 36 h after removal of thymidine. Thus, the initiation time of S phase could be fixed. A trypsin-like proteinase appearing at around 17 h 17 min after removal of thymidine and correlated with the onset of the second S phase, tryptase 17:17 [cf., M. Muramatu et al., Biochim. Biophys. Acta, 1087, 87 (1990)], was obtained. 4-tert-Butylphenyl and 4-biphenyl esters of trans-4-guanidinomethylcyclohexanecarboxylic acid (GMCHA) and amidinopiperidine-4-alkanoic acids, trypsin inhibitors, strongly inhibited the tryptase activity, and these esters exert different effects on the cell cycle of HeLa cells at concentrations showing complete inhibition or maximal inhibitory effect on the tryptase. Both esters of GMCHA elongated the onset of the second S phase for 3 h. Esters of amidinopiperidine-4-acetic and 4-propionic acids showed a similar effect at lower concentrations than GMCHA esters. 4-tert-Butylphenyl esters of amidinopiperidine-4-propionic acid and butyric acids strongly suppressed the second S and M phases by probably affecting the G1 late phase, since they have no effect on the first S and M phases. The addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester 0 min after removal of thymidine into the cells completely suppressed the first S and M phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗

PubMed1993

A trypsin inhibitor trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester suppresses the onset of DNA synthesis in Escherichia coli cells synchronized by phosphate starvation.

trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPh'Bu), a trypsin inhibitor, dose-dependently inhibited the growth of Escherichia coli K-12 IAM1264. Growth inhibition was preceded by dose- and time-dependent inhibition of DNA synthesis. These results strongly suggested participation of a trypsin-like proteinase in DNA synthesis. To clarify this suggestion, the effects of GMCHA-OPh'Bu on the doubling time and on the uptake of [methyl-3H]thymidine into DNA were examined with E. coli K-12 IAM1264 synchronized by a modified version of phosphate starvation. The synchrony lasted for two or three cycles with a doubling time of 55 min and a cell division period of 15 min. The cell cycle of E. coli was divided into three periods, cell division period (P), the period between cell division and initiation of chromosome replication (Q) and the period between initiation of chromosome replication and cell division (R). The R period was subdivided into two periods, R1 in which the rate of thymidine uptake into DNA was increasing, and R2 in which it was constant. The addition of GMCHA-OPh'Bu at the R1 period did not affect the already-initiated round of cell division, however, it retarded the next round. The addition at P, Q or R2 retarded the cell division in the same round, causing prolongation of the R1 period. A sharp and momentary appearance of trypsin-like proteinase activity peaked at the Q/R1 boundary in one cell cycle, and inhibition of the activity prolonged the R1 period.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester, a trypsin inhibitor, suppresses the onset of DNA synthesis in HeLa cells synchronized by a double-thymidine block.

Release of HeLa cells arrested at the G1/S boundary by double-thymidine block caused abrupt uptake of [methyl-3H]thymidine into DNA after 5 min, and two sharp high activity peaks, peak I and peak II, were observed 8 and 23 min after removal of the thymidine block and this was followed by a gradual uptake of [3H]thymidine. The duration of the cell cycle was 23 h, and definite changes in cell density were observed between 12 and 13 h and also between 35 and 36 h after removal of the thymidine. Addition of amidinopiperidine-4-carboxylic acid 4-tert-butylphenyl ester (APCA-OPh'Bu), a trypsin inhibitor, immediately after removal of the arrest strongly suppressed DNA synthesis and mitosis. In contrast, addition of APCA-OPh'Bu 10 min after removal of the arrest, and hence also after the appearance of peak I, had no effect on peak II nor on the uptake of thymidine occurring during the remainder of the first cell cycle, nor on mitosis. However, it strongly suppressed the second DNA synthesis and mitosis. These results suggest participation of a trypsin-like proteinase at the onset of DNA synthesis. Removal of thymidine from the arrested cells at a cell density of 2% (4 x 10(3) cells/cm2) induced an immediate and rapid rise in trypsin-like proteinase activity. However, the activity decreased with increasing cell density. No clear increase in the activity was seen at a cell density of 20% (4 x 10(4) cells/cm2). However, both trypsin-like proteinases obtained at cell densities of 2% and 20% were strongly inhibited by APCA-OPh'Bu and these inhibitory effects were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗

PubMed1993

Effect of trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester, a trypsin inhibitor, on the growth of various strains of Escherichia coli.

Similarly to Escherichia coli K-12 IAM 1264, trans-4- guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhtBu), a trypsin inhibitor, had a strong inhibitory effect on the growth of various strains of E. coli K-12, such as W 3350, AB 1157, JM 103, W 3110, C 600r-m- and C 600r+m+, preceded by suppressive effects of GMCHA-OPhtBu on DNA synthesis in these strains, although the inhibitory effects varied from strain to strain. These results suggested the possible involvement of a trypsin-like proteinase in DNA synthesis. A trypsin-like proteinase was partially purified from E. coli K-12 AB 1157 by ammonium fractionation and successive chromatographies on DEAE-cellulose, Sephadex G-100 and arginine-Sepharose 4B columns. The properties were compared with those of proteinase In, which instantly appears just before the onset of DNA synthesis and seems to participate in the initiation of DNA replication, and which was purified from E. coli K-12 IAM 1264. The proteinase from E. coli K-12 AB 1157 was identified with proteinase In. These results suggest that proteinase In must be widely distributed in various E. coli strains and plays a pivotal role in the onset of DNA replication.

Bacterial Proteins↗

PubMed1993

trans-4-amidinocyclohexanecarboxylic acid 4-tert-butylphenyl ester, a trypsin inhibitor, blocks entry of HeLa cells from G2 phase into mitosis.

Release of HeLa cells arrested at the G1/S boundary by double-thymidine block immediately caused uptake of [3H]thymidine into DNA. The duration of the cell cycle time was 23 h and definite changes in cell density were observed between 12 h and 13 h and also between 35 h and 36 h after removal of thymidine. Addition of trans-4-amidino-cyclohexanecarboxylic acid 4-tert-butylphenyl ester (ACHCA-OPhBut), immediately after removal of the arrest had no effect on DNA synthesis, although it dose-dependently suppressed the first mitosis and the next round of DNA synthesis. While the addition of ACHCA-OPhBut at any time from 0 to 10 h after removal of thymidine suppressed mitosis, its addition after 11 h did not. A trypsin-like proteinase sharply appeared around 10 h 30 min and vanished within a few minutes. The proteinase activity seemed to be density dependent and was strongly inhibited by ACHCA-OPhBut. The effects of trans-4-amidinocyclohexanepropionic acid 4-tert-butylphenyl ester (ACHPA-OPhBut), another trypsin inhibitor, on the proteinase activity and mitosis were more potent than those of ACHCA-OPhBut. These results suggest the involvement of the proteinase in the entry of HeLa cells from the G2 late phase into mitosis. The proteinase was named late G2 proteinase.

Cell Cycle↗

PubMed1992

Purification and characterization of proteinase In, a trypsin-like proteinase, in Escherichia coli.

We previously found a trypsin-like proteinase which momentarily appears immediately before DNA synthesis in the cell cycle of Escherichia coli synchronized by phosphate starvation and which is closely related to the initiation of DNA replication (Kato, M., Irisawa, T., Morimoto, Y. and Muramatu, M., unpublished results). The proteinase was named proteinase In. It was purified approximately 2880-fold with a recovery of 15%. The isolated enzyme appeared homogeneous by gel filtration and electrophoresis. Its molecular mass was estimated by analytical gel filtration and SDS/PAGE as approximately 66 kDa. The isoelectric point of proteinase In is 4.9 and its optimal pH is approximately 9. Although protein In hydrolyzes fluorogenic substrate for trypsin, its hydrolytic activity seems markedly affected by amino-acid sequence lying towards the N-terminal from the P1 (lysine, arginine) residue. The proteinase does not hydrolyze N2-benzoyl-D,L-arginine-4-nitronanilide and fluorogenic substrates for chymotrypsin and elastase. The proteinase activity is inhibited by leupeptin, antipain and 4-nitrophenyl 4-guanidinobenzoate, but the effects of tosyl-L-lysine chloromethane, diisopropylfluorophosphate, benzamidine and pentamidine isethionate on the proteinase activity are weak or not inhibitory. Its activity is strongly affected in the presence of NaCl and KCl, and at a concentration of 1.5 M, these increase the activity 14-fold and 13-fold, respectively, above that without salt. Proteinase In was strongly inhibited by various esters of trans-4-guanidinomethylcyclohexanecarboxylic acid, and their inhibitory effects were roughly correlated with those on growth of E. coli. Proteinase activity was found in the cytoplasmic fraction.

Amino Acid Sequence↗

PubMed1991

Inhibition of gastric H+, K(+)-ATPase and acid secretion by ellagic acid.

The effects of ellagic acid on gastric H+, K(+)-ATPase, acid secretion, and the occurrence of gastric ulcers were studied. Ellagic acid inhibited hog gastric H+, K(+)-ATPase activity with a 50% inhibition at 2.1 x 10(-6)M; kinetic studies showed that the inhibition of H+, K(+)-ATPase by ellagic acid is competitive with respect to ATP and is noncompetitive with respect to K+. The effect on gastric ulcers was investigated by using a stress ulcer model. Intraperitoneal administration of ellagic acid at above 5 mg/kg markedly reduced the occurrence of gastric lesion. Ellagic acid significantly reduced acid secretion at the same doses. These results suggest that ellagic acid has a marked inhibitory effect on acid secretion and the occurrence of stress-induced gastric lesions, and these effects may be attributed to the inhibition of H+, K(+)-ATPase activity.

Adenosine Triphosphatases↗

PubMed1990

Inhibition of phospholipid methylation by an anti-allergic agent, NCO-650, during histamine release.

Antigen, anti-IgE and concanavalin A (Con A) induced an increase in both the incorporation of the 3H-methyl moiety into phospholipids and histamine release. Maximal incorporation of the 3H-methyl moiety into the lipid fraction of the cells was observed within 15 sec and 1 min after being challenged with antigen (100 micrograms/mL) and anti-IgE (200 micrograms/mL) respectively. However, the methylated phospholipid decreased rapidly. The addition of Con A (10 micrograms/mL) also increased phospholipid methylation, which reached a maximum at 5 min after challenge. Trans-4-guanidinomethylcyclohexanecarboxylic acid p-tert-butylphenyl ester hydrochloride (NCO-650; 27 microM) strongly inhibited the incorporation of the 3H-methyl moiety into phospholipid by antigen, anti-IgE and Con A. The IC50 values of NCO-650 for phospholipid methylation in response to antigen, anti-IgE and Con A were 1.5, 4.7 and 1.1 microM respectively. Although the Ca2(+)-ionophore A23187 did not induce phospholipid methylation, it caused histamine release.

Animals↗

PubMed1990

A trypsin-like proteinase appearing at 17 h and 17 min in the cell cycle time of HeLa cells correlates with the onset of DNA synthesis.

A trypsin-like proteinase appearing sharply at 17 h 17 min (17:17) in the cell cycle time of the synchronized and growing HeLa cells correlates with the onset of DNA synthesis, and the inhibition of the proteinase activity by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut) results in a 3 h retardation of the onset of the DNA synthesis. The proteinase activity is cell density dependent and completely inhibited by 100 microM GMCHA-OPhBut. The proteinase was named HeLa tryptase 17:17. The fact that the DNA synthesis occurred after the 3 h retardation in the presence of GMCHA-OPhBut strongly suggests the involvement of the alternative trigger for DNA synthesis in HeLa cells.

Cell Cycle↗