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PubMedDate not supplied

Laser pulse-induced photochemical strand cleavage of site-specifically and covalently modified (+)-anti-benzo[a]pyrene diol epoxide-oligonucleotide adducts.

The specificity of the laser pulse-induced photocleavage method [Boles, T. C., and Hogan, M. E. (1986) Biochemistry 25, 3039] for detecting the sites of covalent binding of benzo[a]pyrene diol epoxide in DNA was investigated using site-specifically modified oligonucleotide duplexes d(CTCACAT[G*]TACACTCT).d(GAGAGTGTACATGTGA), where [G*] is the adducted guanine residue (+)-trans-anti-BPDE-N2-dG (anti-BPDE = 7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene). The appropriate oligonucleotide strands were 32P-end-labeled, and the duplexes were irradiated with a pulse train of 355 nm Nd:YAG laser pulses (approximately 300 mW/cm2). The products of the photocleavage reaction were analyzed by denaturing gel electrophoresis. The major observed products included the intact oligonucleotide strand that had lost the BPDE residue, and shorter oligonucleotide fragments arising from strand scission at the BPDE-modified guanine residue and at nearby flanking bases. Photocleavage at the BPDE-modified G is dominant (approximately 50 +/- 5% of the sum of all of the shorter oligonucleotide fragments), and cleavage extends to at least 4 bases on the 5'-side, and 7 bases on the 3'-side of the BPDE-modified G residue; the probability of cleavage diminishes with increasing distance from the modified G residue. On the unmodified complementary strand, nonspecific strand cleavage is also observed, but the probability of cleavage is > or = 20 times smaller than at the BPDE-modified G residue on the modified strand. The photocleavage method thus preferentially causes strand scission at the sites of BPDE modification, but the occurrence of strand cleavage with lower probabilities at neighboring sites is also significant.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMedDate not supplied

Base sequence-dependent bends in site-specific benzo[a]pyrene diol epoxide-modified oligonucleotide duplexes.

The site specifically modified oligonucleotides 5'-d(TCCTCCTG1G2CCTCTC) (I) and 5'-d(CTATG1G2G3TATC) (II) were synthesized with single modified guanine residues at positions G1, G2, or G3, derived from the covalent binding reaction of 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) with the exocyclic amino groups of the guanine residues. In denaturing 20% polyacrylamide gels, the electrophoretic mobilities of the (+)-anti-BPDE-modified oligonucleotides I and II are slower than the mobilities of the respective unmodified oligonucleotides and independent of the positions of the BPDE-modified guanines. However, in the double-stranded forms in native 8% polyacrylamide gels, the electrophoretic mobilities of the duplexes with lesions at G2 or G3 are remarkably slower (reductions in mobilities up to approximately 40%) than to duplexes with lesions at G1 and are attributed to physical bends or flexible hinge joints at the sites of the BPDE lesions. These sequence-dependent mobility effects occur whenever the BPDE-modified guanine residues with (+)-trans-stereochemistry are flanked by unmodified G's on the 5'-side. These retarded electrophoretic mobilities are attributed to bending induced by steric hindrance effects involving the bulky 5'-flanking guanines and the pyrenyl residues that are known to point into the 5'-direction relative to the modified G [Cosman, M., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1914-1918]. These anomalous electrophoretic mobility effects are not observed in the case of (-)-anti-BPDE-modified sequences I with trans-(-)-anti-BPDE-N2-dG adduct stereochemistry.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1994

Activity of cyclosporins as resistance modifiers in primary cultures of human haematological and solid tumours.

The semiautomated fluorimetric microculture cytotoxicity assay (FMCA) was used for evaluation of the ability of cyclosporin A (CsA) and its novel non-immunosuppressive derivative SDZ PSC 833 (PSC) to modify the response to doxorubicin or vincristine in vitro in different haematological and solid human tumour types. Primary cultures of 322 tumour samples were analysed. Both cyclosporins showed resistance-modifying activity in all haematological tumours tested, and in solid tumours activity was observed in ovarian carcinoma and childhood tumours. Little or no effect was found in the remaining tumour types, including breast, renal and adrenal cortical carcinomas and adult sarcomas. In most of the responsive cases the interaction between the modifier and the cytotoxic drug was synergistic. There was a tendency to higher activity in samples from previously treated patients, and an inverse relationship between degree of cytotoxic drug resistance and resistance-modifying activity was noted. No difference in potency between CsA and PSC could be discerned. The results indicate differential in vitro resistance-modifying activity of the cyclosporins depending on tumour type. The results also suggest that treatment with resistance modifiers should be considered also for primary therapy of drug-sensitive tumours. Drug resistance assays such as the FMCA may become useful in preclinical evaluation of resistance modifiers.

Antineoplastic Agents↗

PubMed1975

The preparation and some properties of mammalian cytochrome c modified with 2-hydroxy-5-nitrobenzyl bromide.

2-Hydroxy-5-nitrobenzyl bromide reacts with horse heart cytochrome c at acid pH to yield a chemically modified protein. Chromatography of the protein on CM-cellulose allows separation of a single chemically modified species. This species is shown by gel chromatography to be monomeric, and isoelectric focusing shows the pI to be lowered from 10.5 to 9.8 on introduction of the reagent molecule. The changes observed in the u.v. region of the spectrum are consistent with the introduction of a single residue of the reagent, and the normal fluorescence of tryptophan is lost. The chemically modified protein exhibits marked changes in its functional properties as compared with native cytochrome c. Unlike the native monomer, the modified cytochrome c has a pH-dependent spectrum which is typical of a high-spin species in the alpha/beta region at low pH, changing to a low-spin species with an apparent pK of 7.5. The modified protein is autoxidizable and the ferrous form binds CO at neutral pH with an affinity constant of 2.6 X 10(5)M-1. The ferrous form of the modified cytochrome c binds CN- at pH 10.0 with an affinity constant of 3.5 X 10(2)M-1. The modified cytochrome c was incapable of restoring the electron-transfer activity to mitochondria depleted of cytochrome c.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed1999

Comparative inhibitory potential of differently modified antisense oligodeoxynucleotides on hepatitis C virus translation.

BACKGROUND: A completely modified phosphorothioate antisense oligodeoxynucleotide (cS-ODN 4) directed against nucleotides 326-348 of the hepatitis C virus (HCV) 5' non-coding region (NCR) efficiently inhibits viral gene expression. As cS-ODN exerts undesired side-effects in vivo, we synthesized partially modified ODN 4 that contained only six modified nucleotides which are located at the ODN termini or are scattered along the molecule. The tested modifications were polar phosphorothioates (S) and non-polar methyl- (M) or benzylphosphonates (B). RESULTS: In an in vitro translation system, specific inhibition of HCV gene expression by M-ODN 4 or B-ODN 4 was observed if terminally modified ODN were used; the maximal inhibition was 92.3% +/- 1.9% and 87.1% +/- 3.7%, respectively, at 10 microgram mol L-1 concentration. S-ODN 4 specifically suppressed viral translation irrespective of the location of the modifications, resulting in a maximal inhibition of 86.3% +/- 3.3%. For all terminally modified ODNs the therapeutic index was high, with tB-ODN 4 the second best at 3.8. Inhibition correlated with efficient RNase H-associated cleavage of target RNA. In transient co-transfection experiments of HepG2 cells with a reporter gene construct and the ODN, terminally modified B-ODN 4 was the most effective and specific inhibitor. At a concentration of 5 microgram mol L-1 the suppression of HCV translation was 96.3% +/- 0.7%. CONCLUSION: These data demonstrate that terminally modified B-ODN 4 is a potent inhibitor of HCV gene expression in vitro and in HepG2 cell culture and may be valuable for future antiviral treatment.

Animals↗

PubMed1975

On the role of the H-2 histocompatibility complex in determining the specificity of cytotoxic effector cells sensitized against syngeneic trinitrophenyl-modified targets.

Spleen cells cultured with syngeneic trinitrophenyl (TNP)-modified stimulator cells display a cytotoxic effect against syngeneic TNP-modified targets, but not against modified targets from unrelated H-2 haplotypes. Targets that share the K and I region of the H-2 complex with the stimulator (or effector) cell are lysed to the same extent as the specific targets, while targets that share the I region only are not. When only the D region is shared, a weak cytotoxic effect is observed. Therefore, the stimulator (or effector) and target cell must share the K or D but not the I region of the H-2 complex in order for optimal cytotoxicity to occur. Spleen cells sensitized to irradiated TNP-modified H-2-allogeneic cells are cytotoxic to these specific cells. Coculture of F1 hybrid cells with irradiated TNP-modified parental cells result in a cytotoxic effect against only those specific parental cells and not TNP-modified cells from the other parent. The cytotoxic effect of the F1 effector cells in the cell-mediated lympholysis test is blocked by the addition of unlabeled TNP-modified targets that are H-2 syngeneic with the sensitizing parental strain, but not H-2 syngeneic with the other parental strain. These data demonstrate that the specificity of the effector cell in this syngeneic cytotoxicity system is directed against altered self H-2-controlled-gene products, rather than a requirement for sharing of histocompatibility genes between effector and target cell in order for lysis to occur. The role of H-2 antigens in determining the sensitivity of a target cell to T-cell-mediated lysis is discussed.

Animals↗

PubMed1977

Specificity of cytotoxic effector cells directed against trinitrobenzene sulfonate-modified syngeneic cells. Failure to recognize cell surface-bound trinitrophenyl dextran.

Mouse splenic lymphocytes and lymphoid tumor cells were modified with the trinitrophenyl (TNP) group either by treatment with trinitrobenzene sulfonate (TNBS) (which covalently modifies cell surface proteins) or with TNP stearoyl dextran (TSD) (which binds to the cell by noncovalent forces). These cell preparations were compared for their ability to: (a) sensitive syngeneic splenic lymphocytes leading to the generation of cytotoxic effector cells; (b) serve as lysable targets in a 4-h(51)Cr- release assay for effector cells generated in (a); and (c) act as blocking cells in the lysis of TNBS-medified targets lysed by TNP self effector cells generated in (a). In none of these three experimental systems did TSD-medified syngeneic spleen or H-2-matched tumor cells act either as a sensitizing immunogen or as a target antigen, despite the demonstration that quantitatively equivalent mounts of TNP were exposed on the cell surface in the TNBS- and TSD-modified cells. In contrast, TNBS-modified spleen cells sensitized syngeneic lymphocytes to generate effectors against TNBS-modified syageneic targets. Furthermore, TNBS- modified, H-2-matched cells served as specific lysable targets and as inhibiting cells for such effectors. These results indicate that the manner in which TNP is associated with the cell surface is important in the immunogenicity and antigenicity of hapten-modified syngeneic stimulating cells in generating H-2-associated cell-mediated lympholysis (CML) reactions. These findings raise the possibility that a covalent or at least a stable linkage with cell surface proteins (possibly H-2- controlled products) is important for immunological function. Furthermore, these observations do not favor the dual receptor model for H-2-restricted syngeneic CML if it is assumed in such a model that one receptor is specific for the TNP moiety and the second for unmodified self major histocompatibility products.

Animals↗

PubMed1978

T cells specific for hapten-modified self are precommitted for self major histocompatibility complex antigens before encounter with the hapten.

The technique of antigen-driven, 5-bromo-deoxyuridine and light suicide has been adapted to eliminate the precursors of cytotoxic effector cells both for alloantigen and for 2,4,6-trinitrophenyl(TNP)-modified stimulator and target cells. Using this technique, the following observations have been made. Precursors of killer cells specific for alloantigen can be suicided independently of precursors of killer cells specific for TNP-modified self cells. The loss of activity during this procedure is not due to either specific or nonspecific suppressor cells, as judged by mixing experiments. With responder cells from F1 animals, it has been possible to show that precursors specific for TNP-modified cells from one parent are suicided independently of precursors specific for TNP-modified cells of the other parent, but only if the parental strains differ in the K and D regions of the H-2 complex. Cells of F1 mice derived from K and D identical, I region different, parental strains were specifically suicided by TNP-modified stimulator cells from either parent. However, the cross-reactive killing of TNP-self targets induced by stimulation with allogeneic cells is not eliminated by first suiciding with TNP-parental cells, suggesting that the precursors of these two types of TNP-self killer cells are different. This is compatible with reported differences in their specificity, as confirmed in this report. Finally, deletion of alloreactive cells by this technique reveals little or no reactivity specific for TNP-modified allogeneic stimulator cells. In summary, these results strongly suggest that recognition of self MHC antigens is preprogrammed in peripheral T cells of normal animals, and is not acquired during the immunization process. They also suggest that cells specific for modified alloantigen are relatively rare in the strains of mice studied.

Animals↗

PubMed1992

Altered stereoselectivity of cocaine and bupivacaine isomers in normal and batrachotoxin-modified Na+ channels.

The inhibitory effects of local anesthetics (LAs) of cocaine and bupivacaine optical isomers on Na+ currents were studied in clonal GH3 cells under whole-cell patch clamp conditions. At holding potential of -100 mV, all four isomers inhibited peak Na+ currents when the cell was stimulated infrequently. The dose-response curves of this tonic block of peak Na+ currents by (-)/(+) cocaine and (-)/(+) bupivacaine were well fitted by the Langmuir isotherm, suggesting that one LA isomer blocked one Na+ channel. Each pair of isomers showed no greater than a twofold difference in stereoselectivity toward Na+ channels. Additional block of Na+ currents occurred when the cell was stimulated at 2 Hz. This use-dependent block was also observed in all four isomers, which again displayed little stereoselectivity. The voltage dependence of the use-dependent block produced by cocaine isomers did not overlap with the activation of Na+ channels but did overlap with the steady-state inactivation (h infinity), indicating that cocaine can bind directly to the inactivated state of Na+ channels before channel opening. In comparison, the peak batrachotoxin (BTX)-modified Na+ currents were little inhibited by cocaine and bupivacaine isomers. However, the maintained BTX-modified Na+ currents were highly sensitive toward the (-) form of cocaine and bupivacaine isomers during a prolonged depolarization. As a result, a profound time-dependent block of BTX-modified Na+ currents was evident in the presence of these LA isomers. The estimated values of the equilibrium dissociation constant (KD in micromolar) at +50 mV were 35.8, 661, 7.0, and 222 for (-)/(+) cocaine and (-)/(+) bupivacaine, respectively. Although chloramine-T (CT) also modified the fast inactivation of Na+ channels and gave rise to a maintained Na+ current during a prolonged depolarization, LA isomers showed no greater stereoselectivity in blocking this maintained current than in blocking the normal transient Na+ current. We conclude that (a) cocaine and bupivacaine isomers exhibit only weak stereoselectivity toward the LA receptor in normal and CT-treated Na+ channels, (b) BTX drastically modifies the configuration of the LA binding site so that the LA stereoselectivity of the open Na+ channels is altered by an order of magnitude, and (c) the (-) forms of cocaine and bupivacaine interact strongly with the open state of BTX-modified Na+ channels but only weakly, if at all, with the closed state. The last finding may explain why most LA drugs were reported to be less effective toward BTX-modified Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Batrachotoxins↗

PubMed1995

Rapid electrogenic sulfate-chloride exchange mediated by chemically modified band 3 in human erythrocytes.

One of the modes of action of the red blood cell anion transport protein is the electrically silent net exchange of 1 Cl- for 1 SO4= and 1 H+. Net SO4(=)-Cl- exchange is accelerated by low pH or by conversion of the side chain of glutamate 681 into an alcohol by treatment of intact cells with Woodward's reagent K (WRK) and BH4-. The studies described here were performed to characterize the electrical properties of net SO4(=)-Cl- exchange in cells modified with WRK/BH4-. The SO4= conductance measured in 100 mM SO4= medium is smaller in modified cells than in control cells. However, the efflux of [35S] SO4= into a 150-mM KCl medium is 80-fold larger in modified cells than in control cells and is inhibited 99% by 10 microM H2DIDS. No detectable H+ flux is associated with SO4(=)-Cl- exchange in modified cells. In the presence of gramicidin to increase the cation permeability, the stoichiometry of SO4(=)-Cl- exchange is not distinguishable from 1:1. In modified cells loaded with SO4=, the valinomycin-mediated efflux of 86Rb+ into an Na-gluconate medium is immediately stimulated by the addition of 5 mM extracellular Cl-. Therefore, SO4(=)-Cl- exchange in modified cells causes an outward movement of negative charge, as expected for an obligatory 1:1 SO4(=)-Cl- exchange. This is the first example of an obligatory, electrogenic exchange process in band 3 and demonstrates that the coupling between influx and efflux does not require that the overall exchange be electrically neutral. The effects of membrane potential on SO4(=)-SO4= exchange and SO4(=)-Cl- exchange in modified cells are consistent with a model in which nearly a full net positive charge moves inward through the transmembrane field during the inward Cl- translocation event, and a small net negative charge moves with SO4= during the SO4= translocation event. This result suggests that, in normal cells, the negative charge on Glu 681 traverses most of the transmembrane electric field, accompanied by Cl- and the equivalent of two protein-bound positive charges.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1989

Rapid and slow gating of veratridine-modified sodium channels in frog myelinated nerve.

The properties of voltage-dependent Na channels modified by veratridine (VTD) were studied in voltage-clamped nodes of Ranvier of the frog Rana pipiens. Two modes of gating of VTD-modified channels are described. The first, occurring on a time scale of milliseconds, is shown to be the transition of channels between a modified resting state and a modified open state. There are important qualitative and quantitative differences of this gating process in nerve compared with that in muscle (Leibowitz et al., 1986). A second gating process occurring on a time scale of seconds, was originally described as a modified activation process (Ulbricht, 1969). This process is further analyzed here, and a model is presented in which the slow process represents the gating of VTD-modified channels between open and inactivated states. An expanded model is a step in the direction of unifying the known rapid and slow physiologic processes of Na channels modified by VTD and related alkaloid neurotoxins.

Animals↗

PubMed1991

Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide.

We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.

Base Sequence↗

PubMed1982

In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide.

Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO). It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed. For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides. This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released. The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used. The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.

4-Hydroxyaminoquinoline-1-oxide↗

PubMed1988

Interlaboratory comparison of antisera and immunoassays for benzo[a]pyrene-diol-epoxide-I-modified DNA.

An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1988

Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect.

Twelve dominant enhancers of position effect variegation, representing four loci on the second and third chromosomes of Drosophila melanogaster, have been induced by P-element mutagenesis. Instead of simple transposon insertions, seven of these mutations are cytologically visible duplications and three are deficiencies. The duplications define two distinct regions, each coinciding with a locus that also behaves as a dominant haplo-dependent suppressor of variegation. Conversely, two of the deficiencies overlap with a region that contains a haplo-dependent enhancer of variegation while duplications of this same region act to suppress variegation. The third deficiency defines another haplo-dependent enhancer. These data indicate that loci capable of modifying variegation do so in an antipodal fashion through changes in the wild-type gene copy number and may be divided into two reciprocally acting classes. Class I modifiers enhance variegation when duplicated or suppress variegation when deficient. Class II modifiers enhance when deficient but suppress when duplicated. From our data, and those of others, we propose that in Drosophila there are about 20 to 30 dominant loci that modify variegation. Most appear to be of the class I type whereas only two class II modifiers have been identified so far. From these observations we put forth a model, based on the law of mass action, for understanding how such suppressor-enhancer loci function. We propose that each class I modifier codes for a structural protein component of heterochromatin and their effects on variegation are a consequence of their dosage dependent influence on the extent of the assembly of heterochromatin at the chromosomal site of the position effect. It is further proposed that class II modifiers may inhibit the class I products directly, bind to hypothetical termination sites that define heterochromatin boundaries or promote euchromatin formation. Consistent with our mass action model we find that combining two enhancers together produce additive and not epistatic effects. Also, since different enhancers have different relative strengths on different variegating mutants, we suggest that heterochromatic domains are constructed by a combinatorial association of proteins. The mass action model proposed here is of general significance for any assembly driven reaction and has implications for understanding a wide variety of biological phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1984

Conformations of poly(dG-dC).poly(dG-dC) modified by the O-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) has been modified by reaction with 4-acetoxyaminoquinoline 1-oxide (Ac-4 HAQO), the ultimate carcinogen of 4-nitroquinoline 1-oxide. The circular dichroism (CD) spectra of the modified and unmodified polymers have been compared under various experimental conditions. The CD spectra were recorded in 1 mM phosphate, 50% (v/v) ethanol, 3.8 M LiCl and 95% (v/v) ethanol, conditions in which poly(dG-dC).poly(dG-dC) adopts the B-, Z-, C- and A-form respectively. In 1 mM phosphate buffer, poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO seems not to contain regions in the Z-form. Z-form induction could be progressively obtained by the addition of ethanol as follows: in the buffer with about 30% ethanol the modified polymer started to adopt the Z structure, while 40% of ethanol in the buffer was necessary for the unmodified polymer. In the 50% ethanol-1 mM phosphate buffer mixture (v/v), poly(dG-dC).poly(dG-dC) was entirely in the Z-form while poly(dG-dC).poly(dG-dC) modified by Ac-4 HAQO remained partially in the B-form. Enzymatic digestions with the nuclease S1 which is specific of the single-stranded DNA were carried out in order to support the modified poly(dG-dC).poly(dG-dC) CD study conclusions. The role played by the two major adducts on the conformational characteristics of modified polymer is discussed.

Aminoquinolines↗

PubMed1988

Separation of SH-modified myosin subfragment-1 (A1) isozyme into two distinct equimolar fractions by an affinity chromatography.

Our previous kinetic studies indicated that SH-modified myosin subfragment-1 A1 isozyme (S1(A1] contains at least two different types of active sites (Emoto, Y., Kawamura, T., & Tawada, K. (1985) J. Biochem. 98, 735-745). In those studies we have modified highly reactive SH-groups in S1(A1) with thimerosal. In this work, we separated the modified S1(A1) into two equimolar fractions by affinity chromatography with agarose-ADP. For the separation, Mg2+ in the elution buffer was indispensable. Although the two fractions appeared to have the same number of modified SH-groups per mol of S1, they had different enzymic and fluorescent properties. SH-modification with an excess of thimerosal for a much longer duration did not change any of the results: not the chromatographic profile, the properties of the two fractions, nor the number of modified SH-groups. Hence the two different populations were not generated by incomplete modification. After reduction with dithiothreitol, however, the differences between the two fractions disappeared. When we separately re-modified the reduced fractions and re-chromatographed them, in each case we again obtained two fractions, which had the same properties as the two fractions obtained from the original modification with thimerosal. These results demonstrate that the active site heterogeneity in SH-modified S1(A1) had no intrinsic origin in the unmodified S1: it was introduced by the SH-modification, but by an unknown mechanism(s) other than incomplete modification.

Adenosine Triphosphatases↗

PubMed1999

Use of abciximab-modified thrombelastography in patients undergoing cardiac surgery.

UNLABELLED: Thrombelastography (TEG) is a reliable coagulation monitoring system that can guide blood product transfusion in cardiac surgery. The maximum amplitude (MA) of TEG measures clot strength, which is dependent on both fibrinogen level and platelet function. Inhibition of platelet function with abciximab-fab is suggested to permit quantitative assessment of the contribution of fibrinogen to clot strength. We hypothesized that abciximab-modified TEG permits prediction of plasma fibrinogen levels and that the difference of standard MA and abciximab-modified MA (deltaMA) is a correlate for platelet function. We correlated abciximab-modified MA with plasma fibrinogen levels and deltaMA with platelet count in patients undergoing coronary revascularization. Correlation between plasma fibrinogen levels and abciximab-modified MA was significant (adjusted r2: 0.8; P < 0.0001). Correlation of deltaMA with platelet count was not significant when calculated in millimeters (adjusted r2: 0.04; P = 0.73). However, when deltaMA was calculated in dynes per square centimeter (deltaGMA), it correlated significantly with platelet count (adjusted r2: 0.51; P < 0.0001). We conclude that abciximab-modified TEG may therefore help to discriminate between hypofibrinogenemia and platelet dysfunction as a cause of decreased MA. IMPLICATIONS: We examined the use of abciximab-modified thrombelastography in patients undergoing cardiac surgery. Modification of thrombelastography with abciximab-fab allows prediction of fibrinogen levels, despite coagulation altered by cardiac surgery. The difference of standard maximum amplitude and abciximab-modified maximum amplitude correlates with platelet function when expressed in dynes per square centimeter.

Abciximab↗