Methylthioadenosine nucleoside phosphorylase deficiency in methylthio-dependent cancer cells.

The cleavage of the methylthio group from methylthioadenosine is shown to involve two enzymes, a nucleosidase which catalyses the phosphorolytic cleavage of methylthioadenosine to yield adenine and 5-methylthioribose-1-phosphate and an enzyme which uses the latter compound as substrate and catalyses the release of the methylthio group as an ether-extractable product. Three malignant murine hematopoietic cell lines which require methylthio group supplementation for proliferation in vitro are shown to lack methylthioadenosine nucleosidase activity while retaining activity of the second enzyme. Four cell lines which are methylthio-independent in vitro contain activity of both enzymes. The data suggest that the requirement for exogenous methylthio groups in certain cells is caused by the block in their biosynthetic pathway imposed by methylthioadenosine nucleoside phosphorylase deficiency. Secondarily, the data suggest that all cells require methylthio or related groups for division.     

31P n.m.r. studies of complexes of NADPH and NADP+ with Escherichia coli dihydrofolate reductase

We have measured the ³¹P n.m.r. spectra of NADP⁺ and NADPH in their binary complexes with Escherichia coli dihydrofolate reductase and in ternary complexes with the enzyme and folate or methotrexate. The ³¹P chemical shift of the 2′ phosphate group is the same in all complexes; its value indicates that it is binding in the dianionic state and its pH independence suggests that it is interacting strongly with cationic residue(s) on the enzyme. Similar behaviour has been noted previously for the complexes with the Lactobacillus casei enzyme although the ³¹P shift is somewhat different in this complex, possibly due to an interaction between the 2′ phosphate group and His 64 which is not conserved in the E. coli enzyme. For the coenzyme complexes with both enzymes ³¹POC₂¹H_2′ spin-spin interactions were detected (7.5–7.8 Hz) on the 2′ phosphate resonances, indicating a POC₂H_2′ dihedral angle of 30 or 330 : this is in good agreement with the value of 330° measured in crystallographic studies¹ (Matthews et al., 1978) on the L. casei enzyme. NADPH-MTX complex. The pyrophosphate resonances are shifted to different extents in the various complexes and there is evidence that there is more OPO bond angle distortion in the E. coli enzyme complexes than in those with the L. casei enzyme. The effects of ³¹POC₅¹H_5′ spin coupling were detected on one pyrophosphate resonance and indicate that the POC₅H_5′ torsion angle has changed by at least ～30° on binding to the E. coli enzyme: this is considerably less than the distortion (～50°) observed previously in the L. casei enzyme complex.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

The synthesis of technetium(V) complexes containing sterically bulky thiolate ligands. The molecular structure of tetrabutylammonium oxotetrakis(2,4,6-trimethylbenzenethiolato)technetate(V)

The reactions between [TcOCl₄]⁻ and the sterically bulky thiols ArSH (Ar = 2,4,6-Me₃C₆H₂, 2,4,6- Prⁱ₃C₆H₂ and 2,6-Ph₂C₆H₃) in methanol afford complexes of formula [TcO(SAr)₄]⁻ which may be isolated as salts with bulky organic cations. The molecular structure of [Buⁿ₄N][TcO(2,4,6-Me₃C₆H₂S)₄] was determined by X-ray diffraction methods. The Tc(V) centre was found to adopt the expected square pyramidal geometry in which an oxo group occupies the apical site and the four thiolate sulphurs the basal sites. The TcO distance is 1.659(11) Å and the average TcS distance 2.38(2) Å. The average cis STcS, trans STcS and OTcS angles are respectively 82.7(6)°, 138.4(3)° and 110.8(4)°.     

The crystal and molecular structures of dioxo mo(VI) complexes of tripodal,tetradentate N,S-donor ligands

The crystal and molecular structures of the complexes MoO₂((SCH₂CH₂)₂NCH₂CH₂SCH₃), I and MoO₂((SCH₂CH₂)₂NCH₂CH₂N(CH₃)₂), II, have been determined from X-ray intensity data collected by counter methods. Compound I crystallizes in two forms, Ia and Ib. In form Ia the space group is P2₁/n with cell parameters a = 7.235(2), b = 7.717(2), c = 24.527(6) Å, β = 119.86(2)°, V = 1188(1) ų, Z = 4. In form Ib the space group is P2₁/c with cell parameters a = 14.945(5), b = 11.925(5), c = 14.878(4) Å, β = 114.51(2)°, V = 2413(3) ų, Z = 8. The molecules of I in Ia and Ib are very similar having an octahedral structure with cis oxo groups, trans thiolates (cis to both oxo groups) and N and thioether sulfur atoms trans to oxo groups. Average ditances are MoO = 1.70, MoS (thiolate) = 2.40, MoN = 2.40 and MoS (thioether) = 2.79 Å. Molecule II crystallizes in space group P2₁2₁2₁ with a = 7.188(1), b = 22.708(8), c = 7.746(2) Å, V = 1246(1) ų and Z = 4. The coordination about Mo is octahedral with cis oxo groups, trans thiolates and N atoms trans to oxo. Distances in the first coordination sphere are MoO = 1.705(2), 1.699(2), MoS = 2.420(1), 2.409(1) and MoN = 2.372(2), 2.510(2) Å. The conformational features of the complexes are discussed. Complex I displays MoO and MoS distances which are very similar to those found by EXAFS in sulfite oxidase. This similarity is discussed.     

Unusual structural features of a siloxane

Crystals of the R, S diastereoisomer of [Cp(CO)₂-FeSiCH₃F]₂O are monoclinic, space group P2₁/c (No. 14), with a = 846.0(3) [836.4(1)], b = 768.0(3) [757.1(1)], c = 1548.5(4) [1522.3(2)] pro, β = 97.34(3)° [97.47(3)°] at 300 K [120 K] with Z = 2. Even at 120 K the SiOSi fragment is found to be strictly linear due to crystallographically imposed symmetry. To explain the unusual electron distribution derived from the X-ray data collected, several types of possible disorders are discussed, none of which leads to a satisfying explanation. Retaining the C_i symmetry (linear SiOSi fragment in the final model) the important bond lengths are FeSi 226.7(1) [226.5(1)] pm, SiF 160.9(2) [161.8(2)] pm, SiO 160.3(1) [161.1(1)] pm, SiC 185.0(3) [185.6(3)] pm. The electronic features of this compound were probed via molecular orbital calculations of the extended Hückel type. It was found that the lone pairs on the siloxane oxygen were tipped away from cylindrical symmetry. The tipping was directed toward the fluorine substituents on the silicon atoms and away from the CpFe(CO)₂ units. A pertubational approach was utilized to rationalize this effect.     

Raman study of crystalline peptides containing beta turns

The Raman spectra of crystalline H-ProLeuGlyNH₂ which has a type II β turn, crystalline S-benzylCysProLeuGlyNH₂ which has a type I β-turn, and crystalline gramicidin S which has two β turns and β-sheet structure in its conformation, were investigated. The amide I and amide III bands of the peptides with β turns were generally different from those which are diagnostic for α-helix and β-sheet conformations. The patterns of the amide I and amide III bands, when examined together, indicate that Raman spectra can provide diagnostic evidence for β-turn structure in peptides.     

Letter: Conformation of the cystine linkages in bovine alpha-lactalbumin as revealed by its Raman effect

In the Raman spectrum of 10% bovine α-lactalbumin in neutral aqueous solution, two Raman lines assignable to the SS stretching vibrations have been observed at 507 and 540 cm^?1, with an intensity ratio of 3:1. From this fact, it has been concluded that, of the four CCSSCC linkages in the molecule of this protein, three are in the gauche-gauehe-gauche conformation, whereas one is in the trans-gauche-trans conformation.     

Synthesis,structural characterization,and properties of the organothorium alkylthiolate complex [(CH3)5C5]2Th(SCH2CH2CH3)2

This contribution reports the synthesis and characterization of the organothorium alkylthiolate complex [(CH₃)₅C₅]₂Th(SCH₂CH₂CH₃)₂. This compound crystallizes in the monoclinic space group C2/c (#15) with four molecules in a cell of dimensions a=19.066(2), b=11.603(1), c=16.379(2) Å, and β=130.08(1)°. Least-squares refinement led to a value for the conventional R index (on F_o) of 0.040 for 132 variables and 2030 observations having F_o²⩾3σ(F_o²). The molecular structure consists of an unexceptional ‘bent sandwich’ [(CH₃)₅C₅]₂Th fragment coordinated to two n-propylthiolate ligands. The ThS bond distance is 2.718(3) Å; the SC(α) distance, 1.78(2) Å; the ThSC(α) angle, 108.3(5)°; and the SThS′ angle, 102.5(2)°. Contrasts are drawn with the structures of analogous actinide alkoxides     

Site-specific and temporally controlled initiation of DNA replication in a human cell-free system

We have recently established a cell-free system from human cells that initiates semi-conservative DNA replication in nuclei isolated from cells which are synchronised in late G₁ phase of the cell division cycle. We now investigate origin specificity of initiation using this system. New DNA replication foci are established upon incubation of late G₁ phase nuclei in a cytosolic extract from proliferating human cells. The intranuclear sites of replication foci initiated in vitro coincide with the sites of earliest replicating DNA sequences, where DNA replication had been initiated in these nuclei in vivo upon entry into S phase of the previous cell cycle. In contrast, intranuclear sites that replicate later in S phase in vivo do not initiate in vitro. DNA replication initiates in this cell-free system site-specifically at the lamin B2 DNA replication origin, which is also activated in vivo upon release of mimosine-arrested late G₁ phase cells into early S phase. In contrast, in the later replicating ribosomal DNA locus (rDNA) we neither detected replicating rDNA in the human in vitro initiation system nor upon entry of intact mimosine-arrested cells into S phase in vivo. As a control, replicating rDNA was detected in vivo after progression into mid S phase. These data indicate that early origin activity is faithfully recapitulated in the in vitro system and that late origins are not activated under these conditions, suggesting that early and late origins may be subject to different mechanisms of control.     

Preparation,properties, crystal and molecular structure of uranyl(VI) complexes with a new cyclic schiff base compartmental ligand

Some uranyl(VI) complexes with new acyclic and cyclic Schiff base compartmental ligands have been prepared and characterized. The ligands have been obtained by reaction of 4-chloro-2,6-diformylphenol and polyamines of the type NH₂(CH₂)₂X (CH₂)₂NH₂ (X= NH, S). The structure of the uranyl(VI) complex with the ligand 1,7,15,21-tetra- aza-4,18-dithia-11,25-dichloro 8,22-bis-metadiphenyl cyclophane-gb-7,14,21,28 has been determined by X-ray crystallography. The compound crystallizes in the orthorhombic space group Pbca with eight formula units in a cell of dimensions a = 26.654(3), b = 22.871(3), c = 8.875(5) Å. The structure was solved by standard methods and refined by full- matrix least squares to the conventional R index of 4.6% for 2678 independent observed reflexions. Five donor atoms (including sulphur) of the ligand are equatorially bonded to the uranyl group to form discrete monomeric molecules with the seven-coordinated metal in the usual distorted pentagonal bipyramidal coordination geometry. Selected bond distances are: UO (equatorial), 2.22(1) and 2.25(1) Å; UN, 2.60(1) and 2.59(1) Å; US, 3.018(4) Å.     

Kinetic studies on the chymotrypsin Aα-catalyzed hydrolysis of a series of N-acetyl-l-phenylalanyl peptides

A series of N-acetyl-l-phenylalanyl peptides of general formula Ac-Phe-(Gly)_n-NH₂ (n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A_α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH₂, AcPheAlaGlyNH₂, and AcPheGlyAlaNH₂. Values of k_cat and K_m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.     

Butyrate-induced glycolipid biosynthesis in HeLa cells: properties of the induced sialyltransferase.

CMP-sialic acid:lactosylceramide sialyltransferase is induced in HeLa cells by butyrate which also causes the cells to undergo morphological changes including the extension of neurite-like processes. The activity of this enzyme is more than 20-fold higher in butyrate-treated cells than in cells grown without this short chain fatty acid. In vitro synthesis of hematoside from endogenous acceptors is also elevated in cells grown in the presence of butyrate. The levels of induced enzyme activity are influenced by the pH of the culture medium, being higher in more acidic cultures, but are not affected markedly by varying the cell density over a wide range. Detergent is required for in vitro sialyltransferase activity, and this activity is stimulated almost fivefold by cardiolipid. The optimum pH for in vitro activity is 6.0 and the apparent K_m value for lactosylceramide is 3.5 × 10^?5m. Although there are several sialyltransferase activities in HeLa cells, the induced enzyme is specific for lactosylceramide.     

Purification and properties of multiple forms of mouse trypsinogen

Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low K_m values (3.2?6.5 μM) for the substrates, BzArgOEt and TosArgOMe, and the dame pH optimum profile between pH 8.0–10.0. However, the ratios of catalytic rate constants, k_cat (s^?1), with BzArgOEt as substrates compared to that with TosArgOMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with TosArgOMe than with BzArgOEt, and the values of Try-II were much higher with TosArgOMe than with BzArgOEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in k_cat (s^?1) for the two substrates, compared to the trypsinogen of CFO mice.     

In vitro cultivation of Schistosoma japonicum-parasites and cells

Schistosomiasis is a serious parasitic zoonosis caused by blood-dwelling flukes of the genus Schistosoma. Understanding functions of genes and proteins of this parasite is important for uncovering this pathogen's complex biology, which will provide valuable information to design new strategies for schistosomiasis control. Effective applications of molecular tools reported to investigate schistosome gene function, such as inhibitor studies and transgenesis, rely on the developments of in vitro cultivation system of this parasite and cells. Besides the in vitro culture studies dealing with Schistosoma mansoni, there are also numerous excellent studies about the in vitro cultivation of Schistosoma japonicum, which were performed by Chinese researchers and published in Chinese journals. Nearly every stage of the life-cycle of S. japonicum, including miracidia, mother sporocysts, cercariae, schistosomula, and egg-laying adult worms, was employed for developing in vitro cultivation methods, being accompanied by the introduction of several media and supplements that helped to improve culture conditions. It was not only possible to generate mother sporocysts from miracidia in vitro, but also to obtain adult worms from cercariae through in vitro cultivation. The main obstacles to complete the life cycle of S. japonicum in the lab are the transition from mother sporocysts to cercariae, and the production of fertilized and completely developed eggs by adult worms generated in vitro. With regard to cells from S. japonicum, besides established isolation protocols and morphological observations, media optimizations were conducted by using different chemical reagents, biological supplements and physical treatment. Among these, mutagens like N-methyl-N-nitro-N-nitrosoguanidine and the addition of extracellular matrix were found to be able to induce mitogenic activities. Although enzyme activities or the level of silver-stained nucleolar region associated protein in cultured cells indicated still suboptimal conditions, the achievements made point to the possibility of reaching the aim of establishing cell lines for S. japonicum. Both the improvements of the in vitro culture of larval and adult worms of S. japonicum as well as the access of cells of this parasite provide excellent advances for research on this important parasite in the future.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

Metalloproteins in the evolution of photosynthesis

Certain metalloproteins are common to all photosynthetic electron transfer chains. These include soluble proteins such as ferredoxins and cytochromes of the c₂ type, and membrane-bound components such as cytochrome b, c₁ and the Rieske iron-sulphur protein. The sequence of electron transfer Quinone → (cyt b, FeS, cyt c₁) → cyt c₂ indicates a common precursor to these systems and to the mitochondrial respiratory chain. In cyanobacteria the cytochrome c₁, can be interchanged with the copper protein plastocyanin, and furthermore in chloroplasts of higher plants the latter is used exclusively. The ferredoxins in anaerobic photosynthetic bacteria are mostly of the [4Fe4S] type, probably derived from those of the fermentative bacteria. These could readily be formed in the earliest cells from iron, sulphide and a very simple peptide. In the oxygen-evolving cyanobacteria and the aerobic halobacteria the [2Fe2S] ferredoxins predominate. The electron transfer chains of the cyanobacteria have been incorporated almost unchanged into the chloroplasts of plants. The electron transfer chains of purple photosynthetic bacteria were probably the precursors of the mitochondrial respiratory chain, as shown by similarities of cytochromes c₂ and succinate dehydrogenase. However a different origin of the eukaryotic cytoplasm is indicated by the presence of the copper/zinc superoxide dismutase.     

Lysosomal carboxypeptidase B from rabbit lung purification and characterization

Lysosomal carboxypeptidase B has been purified from rabbit lung acetone powder by acid precipitation and ammonium sulfate fractionation followed by further purification on Sephadex G-100, DEAE-Sephadex, Organomercurial-Sepharose, preparative isoelectric focusing, Sephadex G-75, and carboxymethyl-Sephadex. This procedure resulted in a homogeneous preparation as determined by polyacrylamide gel electrophoresis at pH 4.5, 8.3 and with sodium dodecyl sulfate. This enzyme has a molecular weight of 52,000, is composed of two subunits of approximately equivalent molecular weight, and is a glycoprotein with a carbohydrate content estimated to be 10% by weight. The amino acid composition is also reported. The enzyme is active on two synthetic substrates, α-N-benyoyl-l-arginineamide and hippuryl-l-arginine. With these two substrates, respectively, lysosomal carboxypeptidase B has pH optima of 5.7 and 5.0, temperature optima of 40 and 50 °C, and K_m values of 10 and 16 mm. With each substrate, the enzyme requires the presence of a reducing agent for maximal activity and is inhibited to the same extent with several inhibitors. The most potent inhibitors were leupeptin and antipain at low concentrations (1 μm). Iodoacetate and Ac-(Ala)₃-Ala-chloro-methyl ketone also inhibited at higher concentrations (10 μm). However, compounds such as leucyl-chloromethyl ketone, bestatin, pepstatin, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and α-1-antitrypsin did not inhibit. When tested with peptides as substrates, this proteinase exhibited strong carboxypeptidase activity on the tetrapeptide, ThrProArgLys, and on angiotensin I, AspArgValTyrIle HisProPheHisLeu, liberating Lys, and Leu, respectively. Substance P (containing 11 amino acids plus a C-terminal amide group) was virtually inactive as a substrate for this enzyme. However, with oxidized insulin B chain as substrate, lysosomal carboxypeptidase B exhibited significant carboxypeptidase and endopeptidase activities.     

Photoreactivating enzyme from Euglena and the control of its intracellular level

Photoreactivating (PR) enzyme activity has already been demonstrated by us in cell-free extracts of Euglena gracilis var. bacillaris Pringsheim using the Hemophilus transformation assay. This activity can also be detected in extracts using a direct non-biological assay for the photorepair of thymine dimers in DNA. PR enzyme is found in extracts of both wild-type cells and cells of an aplastidic mutant, W₃BUL, lacking detectable chloroplast DNA, indicating that the PR enzyme is neither coded nor translated exclusively in the chloroplast, but is probably coded in the nucleus and translated in the cytoplasm. Growing cultures of wild-type cells manifest a large increase in PR enzyme activity in vitro upon entering stationary phase. This correlates with the increased photoreactivability of chloroplast inheritance in vivo in stationary phase cells, previously found for Euglena, and suggests that a substantial part of the newly synthesized PR enzyme is available to repair plastid DNA. When dark-grown nondividing wild-type cells are exposed to light, there is a large increase in the specific activity of PR enzyme measured in vitro. This increase is prevented by cycloheximide but not by chloramphenicol or streptomycin, indicating that the enzyme is synthesized on 87s cytoplasmic ribosomes rather than 68s chloroplast ribosomes. Wavelengths of light effective for PR of chloroplast DNA in vivo are also effective for the light induction of PR enzyme. A brief illumination (45 min) of dark-grown nondividing wild-type cells triggers the synthesis of PR enzyme which continues in the absence of light. Growing cultures of W₃BUL also exhibit a preferential synthesis of PR enzyme in the staionary phase of growth, but the specific activity in vitro is consistently ten times higher than that of wild-type. Dark-grown non-dividing cultures of W₃BUL also show a cycloheximide-sensitive light induction of PR enzyme synthesis which, however, is dependent on the continued presence of light. The light induction of PR enzyme synthesis can be regarded as the induction of an enzyme by one of its substrates.     

Hydrogen Peroxide Contributes to the Epithelial Cell Death Induced by the Oral Mitis Group of Streptococci

Members of the mitis group of streptococci are normal inhabitants of the commensal flora of the oral cavity and upper respiratory tract of humans. Some mitis group species, such as Streptococcus oralis and Streptococcus sanguinis, are primary colonizers of the human oral cavity. Recently, we found that hydrogen peroxide (H₂O₂) produced by S. oralis is cytotoxic to human macrophages, suggesting that streptococcus-derived H₂O₂ may act as a cytotoxin. Since epithelial cells provide a physical barrier against pathogenic microbes, we investigated their susceptibility to infection by H₂O₂-producing streptococci in this study. Infection by S. oralis and S. sanguinis was found to stimulate cell death of Detroit 562, Calu-3 and HeLa epithelial cell lines at a multiplicity of infection greater than 100. Catalase, an enzyme that catalyzes the decomposition of H₂O₂, inhibited S. oralis cytotoxicity, and H₂O₂ alone was capable of eliciting epithelial cell death. Moreover, S. oralis mutants lacking the spxB gene encoding pyruvate oxidase, which are deficient in H₂O₂ production, exhibited reduced cytotoxicity toward Detroit 562 epithelial cells. In addition, enzyme-linked immunosorbent assays revealed that both S. oralis and H₂O₂ induced interleukin-6 production in Detroit 562 epithelial cells. These results suggest that streptococcal H₂O₂ is cytotoxic to epithelial cells, and promotes bacterial evasion of the host defense systems in the oral cavity and upper respiratory tracts.