The molecular mechanism of a novel derivative of BTO-956 induced apoptosis in human myelomonocytic lymphoma cells.

Acute myeloid leukemia (AML) is a malignant cancer of the hematopoietic system. Although the effectiveness of arsenic compounds has been recognized and applied clinically, some patients are still found resistant to this chemotherapy. In this study, we investigated that a synthetic thyroid hormone analog (TA), 2-iodo-4-nitro-1-(o-tolyloxy) benzene, had a strong apoptosis effect on U937 cells. U937 cells were treated with TA, and examinted the generation of reactive oxygen species (ROS), dysfunction of mitochondria, expression of pro-apoptosis and anti-apoptosis, and cleavage of caspase-3 and Poly (ADP-ribose) polymerase (PARP). Further, it is also evaluated that insight molecular mechanism and signaling pathways involved in the study. It is found that TA significantly induced apoptosis in U937 cells through production of ROS, dysfunction of mitochondria, and activation of caspase cascade. It was also observed that MAPK signaling pathway including ERK, JNK, and P38 signals are involved in the induction of apoptosis. Moreover, marked activation of autophagy and ER stress markers such as LC3, P62, Beclin1 and GRP78, CHOP were observed, respectively. Pretreatment with ER stress inhibitor tauroursodeoxycholic acid (TUDCA) and autophagy inhibitor 3-Methyladenine (3-MA) have successfully attenuated and aggravated TA-induced apoptosis, respectively. We further confirmed the active involvement of ER stress and autophagy signals. In conclusion, TA induced apoptosis through ER stress and activation of autophagy, and the latter is not conducive to TA-induced cell death. Our results may provide a new insight into the strategic development of novel therapy for the treatment of AML.

Novel tetrahydroacridine derivatives with iodobenzoic moieties induce G0/G1 cell cycle arrest and apoptosis in A549 non-small lung cancer and HT-29 colorectal cancer cells.

A series of nine tetrahydroacridine derivatives with iodobenzoic moiety were synthesized and evaluated for their cytotoxic activity against cancer cell lines-A549 (human lung adenocarcinoma), HT-29 (human colorectal adenocarcinoma) and somatic cell line-EA.hy926 (human umbilical vein cell line). All compounds displayed high cytotoxicity activity against A549 (IC50 59.12-14.87 µM) and HT-29 (IC50 17.32-5.90 µM) cell lines, higher than control agents-etoposide and 5-fluorouracil. Structure-activity relationship showed that the position of iodine in the substituent in the para position and longer linker most strongly enhanced the cytotoxic effect. Among derivatives, 1i turned out to be the most cytotoxic and displayed IC50 values of 14.87 µM against A549 and 5.90 µM against HT-29 cell lines. In hyaluronidase inhibition assay, all compounds presented anti-inflammatory activity, however, slightly lower than reference compound. ADMET prediction showed that almost all compounds had good pharmacokinetic profiles. 1b, 1c and 1f compounds turned out to act against chemoresistance in cisplatin-resistant 253J B-V cells. Compounds intercalated into DNA and inhibited cell cycle in G0/G1 phase-the strongest inhibition was observed for 1i in A549 and 1c in HT-29. Among compounds, the highest apoptotic effect in both cell lines was observed after treatment with 1i. Compounds caused DNA damage and H2AX phosphorylation, which was detected in A549 and HT-29 cells. All research confirmed anticancer properties of novel tetrahydroacridine derivatives and explained a few pathways of their mechanism of cytotoxic action.

Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization.

Effect of two antiandrogens as protectors of prostate and brain in a Huntington's animal model.

The purpose of this work is to know the effect of flutamide and a novel synthetic steroid 3ß-p-Iodobenzoyloxypregnan-4,16- diene-6,20-dione (IBP) on the levels of dopamine, 5-HIAA (5-hydroxyindole acetic acid), and some oxidative stress markers in animal model with Huntington disease. Thirty male Wistar rats divided in groups of 6 animals each were subjected to the following treatment: group A, 3-nitro propionic acid (3-NPA, as inducer of Huntington); group B, flutamide; group C, 3-NPA + flutamide; group D, IBP; and group E, 3-NPA + IBP. Treatment scheme for all groups were at 4 mg/kg/day administered intraperitoneally. The measurement of haemoglobin was carried out from blood while the concentrations of ATPase, 5α-reductase, reduced glutathione (GSH), calcium, H2O2, 5-HIAA, and dopamine were determined from brain and prostate tissues using validated methods. The results depicted a significant decrease of dopamine and GSH in cerebellum/Medulla oblongata of animals treated with IBP. The prostate gland of the same group of treatment also showed a significant decrease in the concentrations of TBARS, H2O2, and total ATPase. In hemispheres of groups D and E, dopamine, H2O2, and total ATPase decreased significantly while in prostate, hemispheres, and cerebellum/Medulla oblongata of groups B and C; calcium, 5α-reductase, ATPase, H2O2, and TBARS were found to witness a significant decrease. Results showed an antiandrogenic activity of flutamide, while the novel steroid IBP showed neuroprotective properties by changes on oxidative stress biomarkers as critical pathways leading to prostate and brain degeneration. Probably steroid homeostasis disequilibrium could have led to alterations in dopamine metabolism GSH in Huntington's disease animal models.

Photophysical studies on the supramolecular photochirogenesis for the photocyclodimerization of 2-anthracenecarboxylate within human serum albumin.

The mechanism for the chirogenesis in the photocyclodimerization of 2-anthracenecarboxylate (AC) bound to human serum albumin (HSA) was investigated using time-resolved fluorescence measurements in the presence of HSA inhibitors and/or an AC singlet excited state quencher. The photophysical studies were correlated with product studies to explain the high enantiomeric excess (ee) observed for the chiral photoproducts. AC binds to HSA in five different binding sites with decreasing affinities. AC bound to the sites with the highest affinity (sites 1 and 2) is unreactive, and the AC can be displaced from these sites by the use of known inhibitors. Time-resolved fluorescence studies isolated a singlet excited state AC bound to a site which exhibited moderate protection from interactions with species in the aqueous phase. This site was assigned to binding site 3, where the chiral photoproducts are formed with a high ee based on the correlation of the photophysical studies with product studies in the presence of a quencher. These results show that the use of inhibitors for multiple binding site proteins is useful to uncover the properties of binding sites for which guest binding has only moderate affinity and where the photophysical characterization of these binding sites is not possible in the absence of inhibitors.

The relationship between chemical structure and antimicrobial activity of selected nicotinates, p-iodobenzoates, picolinates and isonicotinates.

Alkaline metal, calcium and magnesium p-iodobenzoates and alkaline metal nicotinates, as well as sodium and potassium picolinic and isonicotinates were investigated by means of their antimicrobial and chemical properties. The quality estimation of the influence of metal cation coordinated to the carboxylic anion of the series of studied compounds on their antimicrobial activity as well as on the vibrational structure of whole complex in water solution was done. The changes in antimicrobial properties and in charge distribution of the complex along the position of nitrogen atom in the aromatic ring in sodium and potassium complexes were investigated. The analysis of influence of iodine substituent in para position on the change of electronic charge distribution of carboxylate anion and aromatic ring was done. The relationship between electronic properties estimated by vibrational spectroscopy and antimicrobial activity of studied complexes was investigated.

Synthesis of the catechols of natural and synthetic estrogens by using 2-iodoxybenzoic acid (IBX) as the oxidizing agent.

A method for the synthesis of 2-hydroxyestrone/estradiol, 4-hydroxyestrone/estradiol, 3'-hydroxydiethylstilbestrol, 3'-hydroxyhexestrol, and 3'-hydroxydienestrol is reported, in which 2-iodoxybenzoic acid (IBX) and the corresponding phenolic estrogen are reacted. Treatment of the natural estrogens, estrone/estradiol, with stoichiometric amounts of IBX in dimethylformamide initially yielded a mixture of estrone/estradiol-2,3- and -3,4-quinones, which were reduced in situ to the corresponding catechols by treatment with a 1 M aqueous solution of ascorbic acid. Chromatographic separation of the reaction products afforded 2- and 4-hydroxyestrone/estradiol in good overall yields (79%). In the case of the synthetic estrogens containing two identical phenolic rings, protection of one ring is a prerequisite for the synthesis of the monocatechol. Thus, diethylstilbestrol and dienestrol were protected at one phenol ring as their methyl ethers. The resulting monophenols were treated with stoichiometric amounts of IBX for 1 h, followed by treatment with 1 M aqueous ascorbic acid to obtain the corresponding catechols in more than 70% yield. Furthermore, the catechol of diethylstilbestrol, protected at one ring, was reduced by catalytic hydrogenation at the C3-C4 double bond to obtain 3'-hydroxyhexestrol in 90% yield. Removal of the protected methoxy groups of the synthetic estrogen catechols was carried out by treatment with a 1 M solution of boron tribromide in dichloromethane. This method is highly efficient for the preparative scale synthesis of catechols of both natural and synthetic estrogens.

Mechanisms of antitumor action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate: drug-induced protein dephosphorylations and inhibition of the permissive action of ceramide on growth factor induced cell proliferation.

The tumoricidal mechanism of methyl-3,5-diiodo-4-(4'-methoxypropoxy)benzoate (DIME), or DIPE, has been analyzed beyond its first recognized cellular site, which is the inhibition of tubulin polymerization. DIME (or DIPE) pretreatment of Eras cells for 3 days abolished ceramide basic fibroblast growth factor (bFGF)-induced glycolysis, coinciding with a block produced by the phosphoprotein dephosphorylation of cdc 25 by protein phosphatase 2A (PP2A). Protein dephosphorylation is directly activated by DIME (or DIPE), and enzyme activities that are dependent on P-proteins are significantly down-regulated (e.g. Topo I and II, MAP-kinase, and cdc-cyclin kinase). Purified PP2A is one target of activation by DIME (or DIPE), and an alkaline phosphatase isoenzyme is also induced by the drug. It is proposed that the pleiotropic effects of DIME (or DIPE) on cancer cells involve the activation of protein dephosphorylations, as well as inhibition of tubulin polymerization.

The novel tubulin-binding drug BTO-956 inhibits R3230AC mammary carcinoma growth and angiogenesis in Fischer 344 rats.

BTO-956 [methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate], a novel tubulin-binding drug and thyroid hormone analogue, was originally found to inhibit human carcinoma cell proliferation in vitro and to have potent growth delay activity in human breast and ovarian carcinoma xenografts in nude mice. Here we report that BTO-956 given to Fischer 344 rats also inhibits corneal angiogenesis and the growth and neovascularization of the R3230Ac rat mammary carcinoma tumor implanted in skin-fold window chambers. Hydron pellets containing recombinant human basic fibroblast growth factor (50 ng) and Sucralfate (20 microg) were implanted into surgically created corneal micropockets (day 0). BTO-956 was administrated by oral gavage (500 mg/kg, twice a day for 6 days) on days 1-6 (controls received vehicle alone). On day 7, rats received retrograde infusions of India ink via the thoracic aorta to visualize the corneal vasculature. Digitized images of slide-mounted corneas from control and treated animals were taken with a microscope. For the tumor growth and angiogenesis study, small pieces of R3230Ac tumor from a donor rat were implanted into surgically prepared window chambers (day 0). BTO-956 was given during days 5-11, and images of the tumors and their vasculature were recorded on day 12. No body weight loss was observed in either study. BTO-956 significantly inhibited corneal angiogenesis (by 50-80%), as assessed by measurements of limbal circumference displaying neovascularization, vessel length, vascularized area, and vascular area density. In the window chamber assay, tumors from treated animals were >50% smaller than tumors in control animals. In addition, vascular length densities in peripheral tumor zones were 30% less in treated compared with control animals. Together, these findings demonstrate that BTO-956 can inhibit angiogenesis induced by a growth factor in the rat cornea and in the peripheral area of implanted tumors, where tumor angiogenesis is most active.

m-Iodobenzoic acid complexes with selected metals: molecular structure and antimicrobial activity.

Complexes of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, manganese, and zinc with m-iodobenzoic acid were studied. The FT-IR and FT-Raman spectra of the mentioned compounds in the solid state and water solutions were recorded and analyzed. Principal component analysis (PCA) was performed on the wavenumbers of selected bands (eight bands) occurring in the vibrational spectra. The numbers obtained as a result of this procedure characterize the electronic properties of the molecule of each complex. The antimicrobial activity of the studied compounds against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Saccharomyces cerevisiae and Hansenula anomala) was estimated. The relationship between the chemical properties (as characterized by PCA of the IR spectra) and antimicrobial properties of the compounds was examined, and a good correlation between the two factors was found.

Cancer cell selectivity of 5-iodo-6-aminobenzopyrone (INH2BP) and methyl-3,5-diiodo-4(4'-methoxyphenoxy) benzoate (DIME).

The cellular pharmacologic actions, as measured by cell killing, of INH2BP, DIME and INO2BA (+ BSO) were determined in three types of cancer cells and compared to their action on quiescent confluent human foreskin fibroblast (HSF) and pre-confluent growing fibroblasts. The confluent HSF cells were completely refractory to the action of INH2BP and DIME, but were killed by INO2BA (+ BSO). Proliferating HSF and all three tumor cell types were killed by all three drugs. The apparent in vivo tumor specificity of INH2BP and DIME is explained by preferential cell cycle dependent selective drug uptake into tumor cells and by drug metabolism that reverses drug action in less vigorously cycling normal cells. The covalent binding of iodonitrosobenzamide (formed from INO2BA) and its toxicity are regulated by the concentration of GSH, and exhibit no cell cycle selectivity.

Molecular pharmacology of methyl-3,5-diiodo-4 (4'methoxyphenoxy) benzoate (DIME) and its non-hydrolyzible ethanone analog (DIPE) (Review).

A molecular structural relationship of thyroid hormones to methyl-3,5-diiodo-4-(4'-methoxy-phenoxy) benzoate (DIME) and 1-[3,5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone) (DIPE) and to apoptosis-mediated metamorphogenic mechanisms is postulated. DIME disrupts microtubule assembly already in anaphase, preparing cells for G2/M block, chromosome aggregation and caspase-3 mediated apoptosis. Cooperative action of DIME and vincristine, defining mutually exclusive cellular sites, identifies microtubules as primary drug targets followed by downstream cellular consequences, leading to cell death. Absence of in vivo toxicity of DIME appears to be related to impermeability to DIME of normal cells, but not of tumor cells in vivo. Normal tissue cells hydrolyze DIME but most tumor cells, except lung cancer cells, do not. DIPE, being resistant to enzymatic hydrolysis, is equally effective in all tumor cells.

Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite.

Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was inactivated by peroxynitrite under biologically relevant conditions. The decrease of enzymatic activity followed an exponential function, and the concentration of peroxynitrite needed to inactivate 50% of 7 microM GAPDH (IC50) was 17 microM. Hydroxyl radical scavengers did not protect GAPDH from inactivation, but molecules that react directly with peroxynitrite such as cysteine, glutathione, or methionine and the substrate, glyceraldehyde 3-phosphate, afforded significant protection. Assuming simple competition kinetics between scavengers and the enzyme, we estimated a second-order rate constant of (2.5 +/- 0.5) x 10(5) M-1 s-1 at 25 degreesC and pH 7.4 for the GAPDH tetramer. The loss of enzyme activity was accompanied by protein thiol oxidation (two thiols oxidized per subunit) with only one critical thiol responsible of enzyme inactivation. Indeed, the pH profile of inactivation was consistent with the reaction of GAPDH sulfhydryls (GAPDH-SH) with peroxynitrite. Peroxynitrite-inactivated GAPDH was resistant to arsenite reduction and only 15% recovered by 20 mM dithiothreitol, suggesting that GAPDH-SH has been mainly oxidized to sulfinic or sulfonic acid, with a minor proportion yielding a disulfide. On the other hand, under anaerobic conditions the peroxynitrite precursor, nitric oxide (*NO), only slowly inactivated GAPDH with a rate constant of 11 M-1 s-1. The remarkable reactivity of the critical thiol group in GAPDH (Cys-149) toward peroxynitrite, which is one order of magnitude higher than that of previously studied sulfhydryls, indicate that it may constitute a preferential intracellular target for peroxynitrite.

Unusual potentiation by vinca alkaloids of the cytostatic and cytocidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) and its nonhydrolyzable ethanone analog (DIPE) on MDA-MB-231 human mammary cancer cells.

Drug interaction between DIME or DIPE ¿1-[3, 5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone¿ with vincristine and vinblastine on the growth rate of MDA-MB-231 human mammary cancer cells was determined by the median effect kinetic method. Mutually exclusive cellular binding sites were identified kinetically and isobologram analyses showed potentiation. The combind effect of 0.75 MICROM DIME and 2 nM vincristine demonstrated a nearly type of mutual activation. It was shown that the nonhydrolyzable DIME derivative DIPE is equivalent to DIME, but because of its biological stability is a preferred drug candidate. Vinblastine-DIME cooperative action is similar to that of vincristine-DIME (or DIPE). Activation of caspase 3 by both DIME and vincristine is greatly potentiated when both drugs are added simultaneously in a given proportion. We propose that following a primary binding of DIME and vinca alkaloids to microtubules, an as yet unrecognized mutual activation of caspase 3 apoptotic path is initiated, explaining DNA fragmentation and cell death. A subpopulation of cancer cells, capable of slow growth at 1.5 microM DIME was identified. This cell type was also killed by the DIME-vincristine drug combination.

Nitric oxide and thiol redox regulation of Janus kinase activity.

The activation of Janus kinases (JAKs) is crucial for propagation of the proliferative response initiated by many cytokines. The proliferation of various cell lines, particularly those of hematopoietic origin, is also modulated by mediators of oxidative stress such as nitric oxide and thiol redox reagents. Herein we demonstrate that nitric oxide and other thiol oxidants can inhibit the autokinase activity of rat JAK2 in vitro, presumably through oxidation of crucial dithiols to disulfides within JAK2. The reduced form of JAK2 is the most active form, and the oxidized JAK2 form is inactive. Nitric oxide pretreatment of quiescent Ba/F3 cells also inhibits the interleukin 3-triggered in vivo activation of JAK2, a phenomenon that correlates with inhibited proliferation. Furthermore, we observed that the autokinase activity of JAK3 responds in a similar fashion to thiol redox reagents in vitro and to nitric oxide donors in vivo. We suggest that the thiol redox regulation of JAKs may partially explain the generally immunosuppressive effects of nitric oxide and of other thiol oxidants.

In vivo tissue compatibility of two radio-opaque polymeric biomaterials.

Polymeric biomaterials featuring intrinsic radio-opacity continue to attract considerable scientific attention. This work focusses on two polymers that contain covalently bound iodine, rendering the materials radio-opaque. The first material is hard, transparent and glass-like, and consists of methyl methacrylate, 2-(2'-iodobenzoyl)-ethyl methacrylate (1) and 2-hydroxyethyl methacrylate (HEMA), in the molar ratio 65:20:15, respectively. The second material is a cross-linked hydrophilic network, consisting of HEMA and 1, in the molar ratio 80:20, respectively. Both materials were characterized by means of different physico-chemical techniques, including magic-angle-spinning solid state NMR spectroscopy, infrared spectroscopy and differential scanning calorimetry. Moreover, both materials were implanted subcutaneously in rats for 24 days. Upon explanation and histological examination, it appeared that both materials were well tolerated. No tissue necrosis, abscess formation or inflammation were observed. The samples were found to be surrounded by a vascularized capsule consisting of connective tissue cells. The results reveal excellent tissue compatibility for both materials. This is an important observation, since tissue compatibility is absolutely necessary for the applications which are foreseen for this type of radio-opaque biomaterials.

Redox regulation of maize NADP-malic enzyme by thiol-disulfide interchange: effect of reduced thioredoxin on activity.

Incubation of C4 NADP-malic enzyme from maize leaves with the oxidant o-iodosobenzoate leads to the reversible and complete inactivation of the enzyme. The time-course of inactivation is biphasic with the rate depending on the o-iodosobenzoate concentration. The inactivation is partially prevented by L-malate, NADP and Mg2+ alone, while NADP plus Mg2+ afford total protection. The complete reversal of the inactivation by the reductive agents dithiothreitol and 2-mercaptoethanol suggests that the modification of the enzyme by o-iodosobenzoate occurs concomitant with the oxidation of one or more pairs of sulfhydryl groups to the disulfide state, producing a conformationally altered form of the protein or directly modifying the active site. Titration of free thiol groups before and after inactivation of maize malic enzyme by o-iodosobenzoate shows a decrease in the accessible groups from 7 to 5, suggesting inactivation is accompanied by oxidation of two vicinal thiols. The oxidized form of the enzyme is rapidly reactivated by incubation with chemical and photochemically reduced thioredoxin in vitro, while the 'dark' activity of the enzyme is enhanced to the level of the 'light' activity by dithiothreitol. This evidence suggests that a reversible reduction and oxidation of disulfide bonds may take place during the regulation of the enzyme, indicating that the redox state of the disulfide bonds of C4 NADP-malic enzyme from maize leaves is important for the expression of maximal catalytic activity.

o-Iodosobenzoic oxidation and cleavage of myosin subfragment 1.

1. o-Iodosobenzoic acid (IOB) caused the formation of a disulfide bridge between SH1 and SH2 groups of myosin SF1 rendering inactive its ATPase activity. 2. IOB at high concentrations provoked fragmentation of SF1 at its tryptophan residues. 3. The main fragmentation point was located at 15 K from the amino terminus of the myosin heavy chain. 4. Actin was not fragmented by IOB. It protected SF1 tryptophans from IOB attack. 5. These results suggest a possible use of IOB as a reagent to study protein tryptophan under nondenaturing conditions.

Reduction of nitroaromatic compounds by anaerobic bacteria isolated from the human gastrointestinal tract.

Human intestinal microbial flora were screened for their abilities to reduce nitroaromatic compounds by growing them on brain heart infusion agar plates containing 1-nitropyrene. Bacteria metabolizing 1-nitropyrene, detected by the appearance of clear zones around the colonies, were identified as Clostridium leptum, Clostridium paraputrificum, Clostridium clostridiiforme, another Clostridium sp., and a Eubacterium sp. These bacteria produced aromatic amines from nitroaromatic compounds, as shown by thin-layer chromatography, high-pressure liquid chromatography, and biochemical tests. Incubation of three of these bacteria with 1-nitropyrene, 1,3-dinitropyrene, and 1,6-dinitropyrene inactivated the direct-acting mutagenicity associated with these compounds. Menadione and o-iodosobenzoic acid inhibited nitroreductase activity in all of the isolates, indicating the involvement of sulfhydryl groups in the active site of the enzyme. The optimum pH for nitroreductase activity was 8.0. Only the Clostridium sp. required added flavin adenine dinucleotide for nitroreductase activity. The nitroreductases were constitutive and extracellular. An activity stain for the detection of nitroreductase on anaerobic native polyacrylamide gels was developed. This activity stain revealed only one isozyme in each bacterium but showed that the nitroreductases from different bacteria had distinct electrophoretic mobilities.

Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions.

When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.