Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development

REV3 is the catalytic subunit of DNA polymerase ζ (pol ζ), which is responsible for the damage-induced mutagenesis that arises during error-prone translesion synthesis in eukaryotes. The related REV3L genes in human and mouse encode proteins of approximately 350 kDa, twice as large as yeast REV3, but full-length REV3L has not been identified in any vertebrate cell. We report that Xenopus laevis REV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340 kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65 pM is much lower than those of other replication and repair proteins including the accessory pol ζ subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a pattern of regulation during development. These observations indicate the existence of REV3L as a scarce protein, of approximately the full predicted size, whose level may impose severe constraints on the assembly of pol ζ in X. laevis.     

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2′-deoxyguanosine 5′-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Roles of translesion synthesis DNA polymerases in the potent mutagenicity of tobacco-specific nitrosamine-derived O2-alkylthymidines in human cells

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent human carcinogen. Metabolic activation of NNK generates a number of DNA adducts including O²-methylthymidine (O²-Me-dT) and O²-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O²-POB-dT). To investigate the biological effects of these O²-alkylthymidines in humans, we have replicated plasmids containing a site-specifically incorporated O²-Me-dT or O²-POB-dT in human embryonic kidney 293T (HEK293T) cells. The bulkier O²-POB-dT exhibited high genotoxicity and only 26% translesion synthesis (TLS) occurred, while O²-Me-dT was less genotoxic and allowed 55% TLS. However, O²-Me-dT was 20% more mutagenic (mutation frequency (MF) 64%) compared to O²-POB-dT (MF 53%) in HEK293T cells. The major type of mutations in each case was targeted T → A transversions (56% and 47%, respectively, for O²-Me-dT and O²-POB-dT). Both lesions induced a much lower frequency of T → G, the dominant mutation in bacteria. siRNA knockdown of the TLS polymerases (pols) indicated that pol η, pol ζ, and Rev1 are involved in the lesion bypass of O²-Me-dT and O²-POB-dT as the TLS efficiency decreased with knockdown of each pol. In contrast, MF of O²-Me-dT was decreased in pol ζ and Rev1 knockdown cells by 24% and 25%, respectively, while for O²-POB-dT, it was decreased by 44% in pol ζ knockdown cells, indicating that these TLS pols are critical for mutagenesis. Additional decrease in both TLS efficiency and MF was observed in cells deficient in pol ζ plus other Y-family pols. This study provided important mechanistic details on how these lesions are bypassed in human cells in both error-free and error-prone manner.     

Role of damage-specific DNA polymerases in M13 phage mutagenesis induced by a major lipid peroxidation product trans-4-hydroxy-2-nonenal

One of the major lipid peroxidation products trans-4-hydroxy-2-nonenal (HNE), forms cyclic propano- or ethenoadducts bearing six- or seven-carbon atom side chains to G > C ? A > T. To specify the role of SOS DNA polymerases in HNE-induced mutations, we tested survival and mutation spectra in the lacZα gene of M13mp18 phage, whose DNA was treated in vitro with HNE, and which was grown in uvrA^? Escherichia coli strains, carrying one, two or all three SOS DNA polymerases. When Pol IV was the only DNA SOS polymerase in the bacterial host, survival of HNE-treated M13 DNA was similar to, but mutation frequency was lower than in the strain containing all SOS DNA polymerases. When only Pol II or Pol V were present in host bacteria, phage survival decreased dramatically. Simultaneously, mutation frequency was substantially increased, but exclusively in the strain carrying only Pol V, suggesting that induction of mutations by HNE is mainly dependent on Pol V. To determine the role of Pol II and Pol IV in HNE induced mutagenesis, Pol II or Pol IV were expressed together with Pol V. This resulted in decrease of mutation frequency, suggesting that both enzymes can compete with Pol V, and bypass HNE-DNA adducts in an error-free manner. However, HNE-DNA adducts were easily bypassed by Pol IV and only infrequently by Pol II.Mutation spectrum established for strains expressing only Pol V, showed that in uvrA^? bacteria the frequency of base substitutions and recombination increased in relation to NER proficient strains, particularly mutations at adenine sites. Among base substitutions A:T → C:G, A:T → G:C, G:C → A:T and G:C → T:A prevailed.The results suggest that Pol V can infrequently bypass HNE-DNA adducts inducing mutations at G, C and A sites, while bypass by Pol IV and Pol II is error-free, but for Pol II infrequent.     

2-Hydroxy-2'-deoxyadenosine 5'-triphosphate enhances A.T --> C.G mutations caused by 8-hydroxy-2'-deoxyguanosine 5'-triphosphate by suppressing its degradation upon replication in a HeLa extract

The coexistence effects of multiple kinds of oxidized deoxyribonucleotides were examined using an SV40 origin-dependent in vitro replication system with a HeLa extract. Oxidized dGTP and dATP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP), were used in this study. The mutation frequency synergistically increased when the two oxidized deoxyribonucleotides were together in the reaction. 2-OH-dATP enhanced the mutagenicity of 8-OH-dGTP, since the induced mutations were A.T --> C.G transversions. The contribution of the highly error-prone DNA polymerase eta was unlikely, since similar results were observed with an XP-V cell extract. The possible involvement of 2-hydroxyadenine in the complementary (template) strand was excluded on the basis of experiments using plasmids containing 2-hydroxyadenine as templates in the reactions with 8-OH-dGTP. 2-OH-dATP suppressed hydrolysis of 8-OH-dGTP, suggesting that the inhibition of the MTH1 protein played the major role in the enhancement. These results highlight the importance of specific hydrolysis of 8-OH-dGTP for the suppression of its induced mutation.     

Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90 kDa consistent with the 90,470 Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70–75 °C. The enzyme possessed 3′ → 5′ exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.     

Single-electron photoreduction of the PM intermediate of cytochrome c oxidase

The P_M → F transition of the catalytic cycle of cytochrome c oxidase from bovine heart was investigated using single-electron photoreduction and monitoring the subsequent events using spectroscopic and electometric techniques. The P_M state of the oxidase was generated by exposing the oxidized enzyme to CO plus O₂. Photoreduction results in rapid electron transfer from heme a to oxoferryl heme a₃ with a time constant of about 0.3 ms, as indicated by transients at 605 nm and 580 nm. This rate is ∼ 5-fold more rapid than the rate of electron transfer from heme a to heme a₃ in the F → O transition, but is significantly slower than formation of the F state from the P_R intermediate in the reaction of the fully reduced enzyme with O₂ to form state F (70–90 μs). The ∼ 0.3 ms P_M → F transition is coincident with a rapid photonic phase of transmembrane voltage generation, but a significant part of the voltage associated with the P_M → F transition is generated much later, with a time constant of 1.3 ms. In addition, the P_M → F transition of the R. sphaeroides oxidase was also measured and also was shown to have two phases of electrogenic proton transfer, with τ values of 0.18 and 0.85 ms.     

Different contributions of side-chains in β-d-(1 → 3,6)-galactans on intestinal Peyer’s patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge

Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer’s patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with β-d-(1 → 3)-galactan moieties branched with β-d-(1 → 6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the β-d-(1 → 3)-galactan or β-d-(1 → 6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with β-d-(1 → 3,6)-galactosyl side-chains having terminal β-d-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.     

A novel variant of DNA polymerase ζ, Rev3ΔC,highlights differential regulation of Pol32 as a subunit of polymerase δ versus ζ in Saccharomyces cerevisiae

Unrepaired DNA lesions often stall replicative DNA polymerases and are bypassed by translesion synthesis (TLS) to prevent replication fork collapse. Mechanisms of TLS are lesion- and species-specific, with a prominent role of specialized DNA polymerases with relaxed active sites. After nucleotide(s) are incorporated across from the altered base(s), the aberrant primer termini are typically extended by DNA polymerase ζ (pol ζ). As a result, pol ζ is responsible for most DNA damage-induced mutations. The mechanisms of sequential DNA polymerase switches in vivo remain unclear. The major replicative DNA polymerase δ (pol δ) shares two accessory subunits, called Pol31/Pol32 in yeast, with pol ζ. Inclusion of Pol31/Pol32 in the pol δ/pol ζ holoenzymes requires a [4Fe–4S] cluster in C-termini of the catalytic subunits. Disruption of this cluster in Pol ζ or deletion of POL32 attenuates induced mutagenesis. Here we describe a novel mutation affecting the catalytic subunit of pol ζ, rev3ΔC, which provides insight into the regulation of pol switches. Strains with Rev3ΔC, lacking the entire C-terminal domain and therefore the platform for Pol31/Pol32 binding, are partially proficient in Pol32-dependent UV-induced mutagenesis. This suggests an additional role of Pol32 in TLS, beyond being a pol ζ subunit, related to pol δ. In search for members of this regulatory pathway, we examined the effects of Maintenance of Genome Stability 1 (Mgs1) protein on mutagenesis in the absence of Rev3–Pol31/Pol32 interaction. Mgs1 may compete with Pol32 for binding to PCNA. Mgs1 overproduction suppresses induced mutagenesis, but had no effect on UV-mutagenesis in the rev3ΔC strain, suggesting that Mgs1 exerts its inhibitory effect by acting specifically on Pol32 bound to pol ζ. The evidence for differential regulation of Pol32 in pol δ and pol ζ emphasizes the complexity of polymerase switches.     

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis,but enhances DNA polymerase ζ – dependent spontaneous mutagenesis

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.     

Characterization and PCR optimization of the thermostable family B DNA polymerase from Thermococcus guaymasensis

The gene encoding Thermococcus guaymasensis DNA polymerase (Tgu DNA polymerase) was cloned and sequenced. The 2328 bp Tgu DNA polymerase gene encoded a 775 amino acid residue protein. Alignment of the entire amino acid sequence revealed a high degree of sequence homology between Tgu DNA polymerase and other archaeal family B DNA polymerases. The Tgu DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by two steps of chromatography. The optimum pH and temperature were 7.5 and 80 °C, respectively. The optimal buffer for PCR with Tgu DNA polymerase consisted of 50 mM Tris–HCl (pH 8.2), 4 mM MgCl₂, 50 mM KCl, and 0.02% Triton X-100. Tgu DNA polymerase revealed 4-fold higher fidelity (3.17 × 10^?6) than Taq DNA polymerase (12.13 × 10^?6) and a faster amplification rate than Taq and Pfu DNA polymerases. Tgu DNA polymerase had an extension rate of 30 bases/s and a processivity of 150 nucleotides (nt). Thus, Tgu DNA polymerase has some faster elongation rate and a higher processivity than Pfu DNA polymerase. Use of different ratios of Taq and Tgu DNA polymerases determined that a ratio of 4:1 efficiently facilitated long PCR (approximately 15 kb) and a 3-fold lower error rate (4.44 × 10^?6) than Taq DNA polymerase.     

Chemical constituents from Tribulus terrestris and screening of their antioxidant activity

Two oligosaccharides (1, 2) and a stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of Tribulus terrestris along with five known compounds (4–8). The structures of the compounds were established as O-β-d-fructofuranosyl-(2 → 6)-α-d-glucopyranosyl-(1 → 6)-β-d-fructofuranosyl-(2 → 6)-β-d-fructofuranosyl-(2 → 1)-α-d-glucopyranosyl-(6 → 2)-β-d-fructofuranoside (1), O-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 2)-β-d-fructofuranoside (2), 4,5-di-p-cis-coumaroylquinic acid (3) by different spectroscopic methods including 1D NMR (¹H, ¹³C and DEPT) and 2D NMR (COSY, TOCSY, HMQC and HMBC) experiments as well as ESI-MS analysis. This is the first report for the complete NMR spectral data of the known 4,5-di-p-trans-coumaroylquinic acid (4).The antioxidant activity represented as DPPH free radical scavenging activity was investigated revealing that the di-p-coumaroylquinic acid derivatives possess potent antioxidant activity so considered the major constituents contributing to the antioxidant effect of the plant.     

Crystal structure of human MTH1 and the 8-oxo-dGMP product complex

MTH1 hydrolyzes oxidized nucleotide triphosphates, thereby preventing them from being incorporated into DNA. We here present the structures of human MTH1 (1.9 Å) and its complex with the product 8-oxo-dGMP (1.8 Å). Unexpectedly MTH1 binds the nucleotide in the anti conformation with no direct interaction between the 8-oxo group and the protein. We suggest that the specificity depends on the stabilization of an enol tautomer of the 8-oxo form of dGTP. The binding of the product induces no major structural changes. The structures reveal the mode of nucleotide binding in MTH1 and provide the structural basis for inhibitor design.     

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3′ → 5′ exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3′ → 5′ exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.     

The effect of dissolved oxygen concentrations on (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium sp. IMI 383068 in batch culture

The influence of dissolved oxygen tension (DOT) on the production of extracellular (1 → 3)- and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 was investigated in batch culture. The controlled DOT levels at which cultures were grown affected the measured (1 → 3)-β-glucanase specific activities, but these effects were less marked on the measured (1 → 6)-β-glucanase specific activities. There appeared to be no direct link between the branching frequency of the fungus as reflected by its hyphal growth unit, and changing DOT levels. Whether pustulan or scleroglucan were used as the sole carbon source also affected production of these enzymes and their response to varying DOT. The measured (1 → 3)-β-glucanase specific activities were generally higher with scleroglucan, mainly because of the production of an extra active (1 → 3)-β-glucanase whereas pustulan grown cells produced a corresponding inactive protein with an identical electrophoretic mobility by SDS-PAGE and N-terminal amino acid sequence. With pustulan grown cells, DOT levels had comparatively little influence on the optimal measured specific activities of the (1 → 3)-β-glucanases, while with scleroglucan, they increased as DOT increased.     

Methanolysis of triterpenoid saponin from Ardisia gigantifolia stapf. and structure–activity relationship study against cancer cells

Thirteen 13,28-epoxy triterpenoid saponins were isolated from Ardisia gigantifolia stapf. and one potential anti-tumor saponin was methanolysised by H₂SO₄ to afford four new compounds. The seventeen compounds were evaluated for their anti-proliferative activity on A549, HCT-8 and Bel-7402 cells. The structure–activity relationship analysis indicated that the incorporation of O group at C-16, l-rhamnose at R⁵ and acetyl group at OH-6 of the d-glucose lead to a significant increase of the cytotoxic activity on A549 and HCT-8 but significant reduction of the cytotoxic activity on Bel-7402 cells. The synthesized saponins losing 13,28-epoxy and CHO at C-30, losed their cytotoxicities on A549 and HCT-8 cells, suggesting that the two moieties play an essential role for activity. 3β-O-α-l-rhamnopyranosyl-(1 → 3)-[β-d-xylopyranosyl-(1 → 2)]-β-d-glucopyranosyl-(1 → 4)-[β-d-glucopyranosyl-(1 → 2)]-α-l-arabinopyranoside-16α-hydroxy-13,28-epoxy-oleanane (2) showed better inhibitory activity to Bel-7402 (IC₅₀ 0.86 μM) than that of 5-FU (IC₅₀ 8.30 μM), which indicate that five saccharide and methyl moiety at C-30 are important for anti-proliferative activity. The activities of saponins 15 > 14, 17 > 16, suggested that the configuration of 28,30-epoxy is preferable to be 30(R) rather than 30(S) on Bel-7402 cells. Further molecular mechanism studies of saponins 1 and 2 were carried out on the cell cycle distribution of Bel-7402 cells.