Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.     

Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.     

The 7[prime]-Methyl Group of Abscisic Acid Is Critical for Biological Activity in Wheat Embryo Germination

Wheat (Triticum aestivum L.) embryo germination is inhibited by natural (S)-(+)-abscisic acid (ABA). In this report we have determined critical structural features of the ABA molecule, particularly the methyl and ketone groups of the ABA ring, required for inhibitory activity. To examine the ring residues a series of new optically active ABA analogs have been synthesized in which the 4[prime]-keto, 7[prime]-, 8[prime]-, or 8[prime]- and 9[prime]-carbons have been replaced with hydrogen atoms. Each of the analogs was tested over a range of concentrations as a germination inhibitor. Enantiomers of the analogs altered at the 4[prime]-keto or 8[prime]- and 9[prime]-methyl groups were active, but less so than ABA. Both enantiomers of 7[prime]-demethylABA were inactive as germination inhibitors. The results show that the 7[prime]-methyl group is absolutely required for activity, but that the other residues are less critical for hormone recognition.     

Comparison of the solution conformations of human [Zn7]-metallothionein-2 and [Cd7]-metallothionein-2 using nuclear magnetic resonance spectroscopy.

The solution structure of native human [Zn7]-metallothionein-2 has been compared with the previously determined structure of human [Cd7]-metallothionein-2. The comparison was based on complete sequence-specific 1H nuclear magnetic resonance assignments for human [Zn7]-metallothionein-2 obtained using the sequential assignment method. The secondary structure was found to be very similar in the [Zn7]- and [Cd7]- forms of the protein. Only seven amide protons in [Zn7]- metallothionein-2 were found to have exchange rates lower than approximately 0.2 min-1 at pH 7.0 and 10 degrees C, which corresponds closely to the results of amide proton exchange studies with the [Cd7]- form of the protein. Finally, the 1H-1H distance constraints determined from nuclear Overhauser enhancement spectroscopy for human [Zn7]-metallothionein-2 were checked for compatibility with the [Cd7]-metallothionein-2 structure. Overall, although no direct method is available for identifying the metal-polypeptide co-ordinative bonds in the Zn(2+)-containing protein, these measurements provided several independent lines of evidence showing that the [Zn7]- and [Cd7]- forms of human metallothionein-2 have the same molecular architecture.     

The formation of dihydrodiols by the chemical or enzymic oxidation of 7-hydroxymethyl-12-methylbenz[alpha]anthracene and the possible role of hydroxymethyl dihydrodiols in the metabolic activation of 7,12-dimethylbenz[alpha]anthracene.

The formation of dihydrodiols from 7-hydroxymethyl-12-methylbenz[alpha]anthracene by rat-liver microsomal fractions, by mouse skin in short-term organ culture and by chemical oxidation in an ascorbic acid/ferrous sulphate/EDTA system has been studied using a combination of thin-layer chromatography and high pressure liquie chromatography. The 3,4-, 8,9- and 10,11-dihydrodiols were formed in all three systems. The 5,6-dihydrodiol was formed in rat-liver microsomal fractions and in chemical oxidation but was not detected as a metabolite of [7-3H]hydroxymethyl-12-methylbenz[alpha]anthracene when this compound was incubated with mouse skin in short-term organ culture. The possible role of hydroxymethyl dihydrodiols in the in vivo metabolic activation of 7,12-dimethylbenz[alpha]anthracene in mouse skin has been studied using Sephadex LH-20 column chromatography. The results show that the hydrocarbon-nucleic acid products formed following the treatment of mouse skin in vivo with [7,12-3H]dimethylbenz[alpha]anthracene are not the same as those that are formed following the treatment of mouse skin under the same conditions with either 7-hydroxymethyl-12-methylbenz[alpha]anthracene or 7-methyl-12-hydroxymethylbenz[alpha]anthracene.     

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF).     

The specificity of different classes of ethylating agents toward various sites in RNA.

The alkyl products of neutral in vitro ethylation of TMV-RNA by [14C]diethyl sulfate, [14C]ethyl methanesulfonate, and [14C]ethylnitrosourea have been determined and found to differ significantly depending on the ethylating agent. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of TMV-RNA in the following order: 7-ethylguanine greater than 1-ethyladenine, 3-ethylcytidine greater than 7-ethyladenine, 3-ethyladenine, O6-ethylguanosine, 3-ethylguanine. Ethyl methanesulfonate was more specific for the 7 position of guanine, and other derivatives were found in lesser amounts than with diethyl sulfate. Neither reagent caused the formation of detectable amounts (smaller than 0.26 percent) of 1-ethylguanine, 1,7-diethylguanine, N2-ethylguanine, N6-ethyladenine, N4-ethylcytidine, or 3-ethyluridine. Identified ethyl bases account for over 85% of the total radioactivity of [14C]ethyl methanesulfonate and [14C]diethyl sulfate treated TMV-RNA. Phosphate alkylation accounts for about 13 and 1%, respectively, In contrast, [14C]ethylnitrosourea-treated TMV-RNA, while reacting to a similar extent (15-70 ethyl groups/6400 nucleotides), is found to cause considerably more phosphate alkylation. Upon either U4A RNase or acid hydrolysis up to 60% of the radioactivity is found as volatile ethyl groupw in the form of [14C]ethanol, and a further 15% appears to be primarily ethyl phosphate and nucleosides with ethylated phosphate. Of the remaining radioactivity, half is found as O6-ethylguanosine, the major identified ethyl nucleoside. Other ethyl bases found in ethylnitrosourea-treated TMV-RNA are 7-ethylguanine greater than 1-ethyladenine, 3-ethyladenine, 7-ethyladenine, 3-ethylcytidine, and 3-ethylguanine. It appears that ethylnitrosourea preferentially alkylates oxygens, and that formation of phosphotriesters is by far the predominant chemical event. Since the number of ethyl groups introduced into TMV-RNA by ethylnitrosourea is similar to the number of lethal events, one may conclude that phosphate alkylation leads to loss of infectivity. None of the three ethylating agents studied are strongly mutagenic on TMV-RNA or TMV. The role of phosphate alkylation in regard to in vivo mutagenesis and oncogenesis remains to be established. At present it appears possible that the extent of this reaction may correlate better with the oncogenic effectiveness of different ethylating agents, than the extent of any base reaction. Unfractionated HeLa cell RNA is ethylated primarily in acid labile manner even by diethyl sulfate and ethyl methanesulfonate, a fact that is attributed to its high content of low molecular weight trna rich in terminal phosphates which alkylate readily.     

Characterization of high density lipoprotein subspecies: structural studies by single vertical spin ultracentrifugation and immunoaffinity chromatography

Affinity columns containing anti-apolipoprotein A-I or A-II were used to fractionate plasma into subpopulations of lipoprotein particles containing: a) apoA-I [Lp(A-I)], b) apoA-I and A-II [Lp(A-I with A-II)], and c) apoA-I but no A-II [Lp(A-I without A-II)]. Single vertical spin and electron microscopy analyses of these HDL subpopulations demonstrated that acid elution from the affinity columns caused no detectable change in size and density of the three subpopulation particles. Analysis by nondenaturing gradient gel electrophoresis of the three subpopulations found in four normal subjects identified nine HDL subspecies, designated [1] through [9] in order of increasing size; [3-7] were the major subspecies. Lp(A-I with A-II) is composed primarily of subspecies [3],[5], and [6], and may contain some subspecies [2] and [7], while Lp(A-I without A-II) represents mainly [4] and [7] and the minor subspecies [1],[2],[8], and [9]. HDL subspecies [4],[5], and [6] are found in the standard sequential flotation density cuts for both HDL3 and HDL2, which illustrates the limitations of the latter terminology. Using single vertical spin ultracentrifugation, HDL fractions were located and isolated for physical and chemical analyses, including immunoassay for apoA-I, A-II, and C-II. The distribution of the Lp(A-I without A-II) particles corresponded closely to the apoC-II distribution. An apoA-I-rich, cholesteryl ester- and phospholipid-poor subspecies was identified in the dense HDL fractions. HDL subspecies [7] was found to contain at least three separate subspecies designated [7a], [7b], and [7c]. Based on these and previously published results (Brouillette, C. G., et al. 1984. Biochemistry. 23: 359-367), we propose that the HDL subspecies adjacent in size generally differ by the association/lack of association of a hinge-like domain of amphipathic helixes in a single molecule of apoA-I.     

Epimerization of benzo[a]pyrene-tetrols after acid hydrolysis, implications for determination of benzo[a]pyrene adducts in protein and DNA

Determination of benzo[a]pyrene-DNA or protein adducts with high performance liquid chromatography (HPLC) after acid hydrolysis at high temperature (90 degrees C) enables four isomers of benzo[a]pyrene tetrahydrotetrol to be identified and quantitated. We have investigated the effect of acid treatment of benzo[a]pyrene-tetrahydrotetrol isomers using HPLC and nuclear magnetic resonance spectroscopy (NMR) analysis. By HPLC, we found reversible epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and of (+/-)-benzo[a]pyrene-r-7,t-8,c-9, t-10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,c-9, 10-tetrahydrotetrol, but no interconversion between the two isomer groups. After acid hydrolysis, we found an equilibrium of 87% (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol and 9% (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and 68% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol and 20% (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. Minor amounts of two unknown compounds with similar chromatographic characteristics were also found. We have established a NMR method for determination of underivatized (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,9, 10-tetrahydrotetrol confirming the epimerization of (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol to (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10- tetrahydrotetrol. (+/-)-Benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol was treated with aqueous hydrochloric acid in tetrahydro- furan-d8 to give (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol at 57 degrees C while observing the 1H NMR resonances at 500 MHz. Gradient-selected correlation spectroscopy (COSY), heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) experiments were performed to confirm the assignments of the aliphatic hydrogens in the product (+/-)-benzo[a]pyrene-r-7,t-8,9, c-10-terahydrotetrol. Thus, when analyzing benzo[a]pyrene-DNA or protein adducts by cleaving the adducts with acid hydrolysis, the only ratio of biological significance is between (+/-)-benzo[a]pyrene-r-7,t-8,9,c-10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,9,10-tetrahydrotetrol and (+/-)-benzo[a]pyrene-r-7,t-8,c-9,10-tetrahydrotetrol plus (+/-)-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol, due to interconversion (epimerization) at C-10.     

Synthesis of dolichol derivatives in trypanosomatids. Characterization of enzymatic patterns

We have previously described that in certain parasitic protozoa, namely the trypanosomatids, the dolichol-P-P-linked oligosaccharides synthesized in vivo and transferred to protein are devoid of glucose residues and contain 6, 7, or 9 mannose units depending on the species. We have now conducted a cell-free characterization of the enzymatic patterns responsible for these phenotypes. Microsomes from Trypanosoma cruzi, Crithidia fasciculata, Leishmania enriettii, and Blastocrithidia culicis were found to synthesize dolichol-P-[14C]Man but not dolichol-P-[14C]Glc when incubated with rat liver dolichol-P and GDP-[14C]Man or UDP-[14C]Glc, thus providing for an explanation to the absence of glucosylated dolichol-P-P derivatives. Formation of dolichol-P-P-oligosaccharides was assayed in incubation mixtures containing rat liver dolichol-P, GDP-[14C]Man, microsomes, and unlabeled Man5-8GlcNAc2-P-P-dolichol from bovine liver. Membranes from species synthesizing dolichol-P-P-linked Man6GlcNAc2 or Man7GlcNAc2 in vivo were found to synthesize the same compounds but not the higher homologues in the cell-free assay. Species forming Man9GlcNAc2-P-P-dolichol in vivo were found to synthesize lipid-linked Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 in vitro. It is concluded that there are at least three and probably four different dolichol-P-Man-dependent enzymatic activities involved in the synthesis of dolichol-P-P-linked Man9GlcNAc2 and that microorganisms not forming that compound are devoid of all mannosyltransferases responsible for the addition of the missing residues and not only of the enzyme involved in the synthesis of the homologue higher than the oligosaccharide occurring in vivo by a single mannose unit.     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Solution 1H nuclear magnetic resonance determination of the distal pocket structure of cyanomet complexes of genetically engineered sperm whale myoglobin His64 (E7)-->Val, Thr67 (E10)-->Arg. The role of distal hydrogen bonding by Arg67 (E10) in modulating ligand tilt.

Sequence-specific 2D methodology has been used to assign the 1H NMR signals for all active site residues in the paramagnetic cyano-met complexes of sperm whale synthetic double mutant His64[E7]-->Val/Thr67[E10]-->Arg (VR-met-MbCN) and triple mutant His64[E7]-->Val/Thr67[E10]-->Arg/Arg45[CD3]-->Asn (VRN-metMbCN). The resulting dipolar shifts for noncoordinated proximal side residues were used to quantitatively determine the orientation of the paramagnetic susceptibility tensor in the molecular framework for the two mutants, which were found indistinguishable but distinct from those of both wild-type and the His64[E7]-->Val single point mutant (V-metMbCN). The observed dipolar shifts for the E helix backbone protons and Phe43[CD1], together with steady-state nuclear Overhauser effect between the E helix and the heme, were analyzed to show that both the E helix and Phe43[CD1] move slightly closer to the iron to minimize the vacancy resulting from the His64[E7]-->Val substitution, as found in V-metMbCN (Rajarathnam, K., J. Qin, G.N. LaMar, M. L. Chiu, and S. G. Sligar. 1993. Biochemistry. 32:5670-5680). The dipolar shifts of the mutated Val64[E7] and Arg67[E10] allow the determination of their orientations relative to the heme, and the latter residue is shown to insert into the pocket and provide a hydrogen bond to the coordinated ligand, as found in the naturally occurring ValE7/ArgE10 genetic variant, Aplysia limacina Mb. The oxy-complex of both A. limacina Mb and VR-Mb, VRN-Mb have been proposed to be stabilized by this hydrogen bonding interaction (Travaglini Allocatelli, C. et al. 1993. Biochemistry. 32:6041-6049). The magnitude of the tilt of the major magnetic axes from the heme normal in VR-metMbCN and VRN-metMbCN, which is related to the tilt of the ligand, is the same as in wild-type or V-metMbCN, but the direction of tilt is altered from that in V-metMbCN. It is concluded that the change in the direction of the ligand tilt in both the double and triple mutants, as compared to WT metMbCN and V-metMbCN single mutant, is due to the attractive hydrogen-bonding between ArgE10 and the bound cyanide.     

Spike protein VP8* of human rotavirus recognizes histo-blood group antigens in a type-specific manner

Rotaviruses (RVs), an important cause of severe diarrhea in children, have been found to recognize sialic acid as receptors for host cell attachment. While a few animal RVs (of P[1], P[2], P[3], and P[7]) are sialidase sensitive, human RVs and the majority of animal RVs are sialidase insensitive. In this study, we demonstrated that the surface spike protein VP8* of the major P genotypes of human RVs interacts with the secretor histo-blood group antigens (HBGAs). Strains of the P[4] and P[8] genotypes shared reactivity with the common antigens of Lewis b (Le(b)) and H type 1, while strains of the P[6] genotype bound the H type 1 antigen only. The bindings between recombinant VP8* and human saliva, milk, or synthetic HBGA oligosaccharides were demonstrated, which was confirmed by blockade of the bindings by monoclonal antibodies (MAbs) specific to Le(b) and/or H type 1. In addition, specific binding activities were observed when triple-layered particles of a P[8] (Wa) RV were tested. Our results suggest that the spike protein VP8* of RVs is involved in the recognition of human HBGAs that may function as ligands or receptors for RV attachment to host cells.     

Evidence that the regulation of diphtheria toxin production is directed at the level of transcription.

It has been known for several decades that iron inhibits the production of diphtheria toxin by Corynebacterium diphtheriae by preventing expression at maximal levels. We examined the inhibition kinetics of toxin production after the addition of either iron or rifampin to iron-limited cultures of C7 (betatox+). Iron-mediated inhibition of toxin production was found to be linear within the range of 16 nM to 16 micron. The inhibition kinetics following the addition of iron or rifampin was almost identical. [3H]RNA extracted from iron-limited toxigenic C. diphtheriae was found to hybridize to a greater extent to corynephage beta DNA than either [3H]RNA extracted from toxigenic C. diphtheriae before the onset of toxin production or [3H]RNA extracted from nonlysogenic, nontoxigenic C. diphtheriae.     

Biosynthesis of [7-3H]16 alpha-hydroxy-dehydroepiandrosterone having high specific activity.

Biosynthesis of [7-3H]16alpha-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-3H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 degrees C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-3H]16alpha-hydroxy-dehydroepiandrosterone (2.5 x 10(7) dpm) was obtained by microbial hydroxylation of substrate (1.9 X 10(9) dpm). In some cases [7-3H])5-androstene-3beta, 16alpha, 17beta-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

The specificity of different classes of ethylating agents toward various sites of HeLa cell DNA in vitro and in vivo.

The sites and extent of ethyl products of neutral ethylation of HeLa cell DNA by [14-C]diethyl sulfate, [14-C]ethyl methanesulfonate, and [14-C]ethylnitrosourea have been determined in vitro and in vivo, and found to differ significantly depending on the ethylating agents. Diethyl sulfate and ethyl methanesulfonate ethylate the bases of HeLa cell DNA in the following order: 7-ethylguanine greater than 3-ethyladenine greater than 1-ethyladenine, 7-ethyladenine greater than 3-ethylguanine, 3-ethylcytosine, O-6-ethylguanine. Ethyl bases accounted for 84-87% of the total ethyl groups associated with HeLa cell DNA. Ethylnitrosourea, in contrast, has particular affinity for the O-6 position of guanine. It ethylates the bases of HeLa cell DNA in the following order: O-6-ethylguanine, 7-ethylguanine greater than 3-ethyladenine greater than 3-ethylguanine, 3-ethylthymine greater than 1-ethyladenine, 7-ethyladenine, 3-ethylcytosine. Ethylation of the bases only accounts for 30% of the total ethylation in the case of ethylnitrosourea. The remaining 70% of the [14-C]ethyl groups, introduced in vivo and in vitro, are in the form of phosphotriesters which after perchloric acid hydrolysis are found as [14-CA1ethanol and [14-C]ethyl phosphate. In contrast, phosphotriesters amounted to only 8-20% of total ethylation in in vivo or in vitro diethyl sulfate and ethyl methanesulfonate treated HeLa cell DNA, and 25% of the total methylation in in vitro methylnitrosourea treated HeLa cell DNA. Alkylation at the N-7 and N-3 positions of purines in DNA destabilizes the glycosidic linkages. Part of 7-ethylguanine and 3-ethyladenine are found to be spontaneously released during the ethylation reaction. Incorporation of the 14-C of the alkylating agents into normal DNA bases of HeLa cells can be eliminated by performing the alkylations, in the presence of cytosine arabinoside, for 1 hr.     

The in vitro and in vivo reaction at the N7-position of guanine of the ultimate carcinogen derived from benzolalpyrene.

The previously reported reaction at N2- and N7- of guanine following addition of 7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to an aqueous solution of DNA has been studied in more detail. The extent of reaction and the relative yields of N2- and N7-products was measured over the range of pH 4--7. The depurination following reaction at the N7-position of guanine was found to have a half-life of 3 h. Reaction of the isomeric 7 alpha,8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (syn-BPDE) with DNA gave the expected N2- and no N7-guanine product. When either benzo[a]pyrene or anti-BPDE was added to mouse embryo or Chinese hamster V79 cells respectively, a major N2-guanine product and a very minor adenine product were isolated from the DNA, but no N7-guanine product was detected.     

Relations Between Cuticular Hydrocarbon (HC) Polymorphism, Resistance Against Desiccation and Breeding Temperature; A Model for HC Evolution in D. Melanogaster and D. Simulans

D. simulans and D. melanogaster present two types of polymorphism in their cuticular hydrocarbon (HC) composition. Especially both sexes of D. simulans, and D. melanogaster males display 7-tricosene (7T) as the major compound type [7T]s and [7T]m, or 7-pentacosene (7P) [7P]s and [7P]m. D. melanogaster females display 7,11-heptacosadiene (7,11HD) as the major compound: [7,11HD]m, or 5,9-heptacosadiene (5,9HD): [5,9HD]m. The [7P]s, [7P]m and [5,9HD]m are mainly present in central Africa. A significant correlation was found between latitude and the proportion of compounds with 23 and 25 carbon atoms, especially 7T and 7P in both sexes of D. melanogaster. [7P]m type of D. melanogaster, characterized with an excess of C25 compounds, presents a higher resistance against desiccation than [7T]m type, where C23 compounds are more abundant. These differences can be correlated with calculated HC fusion temperatures. Moreover, increasing the breeding temperature from 18 to 29 degrees C induces in D. melanogaster males an increase in 25C compounds and a decrease in 23C compounds, but the opposite effect in D. simulans. A mathematical model of biosynthesis, based on kinetics of elongation and decarboxylation enzymes, suggests that a simple variation of the efficiency of an elongation enzyme may account for the differences observed between the [7T]m and [7P]m types of D. melanogaster and [7T]s and [7P]s types D. simulans. Finally on the basis of the geographical distribution of the HC types of both Drosophila species, an evolutionary dispersal pathway is proposed and discussed in relation to the environment and reproductive behavior.     

Specificities of the Acyl-Acyl Carrier Protein (ACP) Thioesterase and Glycerol-3-Phosphate Acyltransferase for Octadecenoyl-ACP Isomers (Identification of a Petroselinoyl-ACP Thioesterase in Umbelliferae)

This study was designed to address the question: How specific for double bond position and conformation are plant enzymes that act on oleoyl-acyl carrier protein (ACP)? Octadecenoyl-ACPs with cis double bonds at positions [delta]6, [delta]7, [delta]8, [delta]9, [delta]10, [delta]11, or [delta]12 and elaidyl (18:1[delta]9trans)-ACP were synthesized and used to characterize the substrate specificity of the acyl-ACP thioesterase and acyl-ACP:sn-glycerol-3-phosphate acyltransferase. The two enzymes were found to be specific for the [delta]9 position of the double bond. The thioesterase was highly specific for the [delta]9 cis conformation, but the transferase was almost equally active with the cis and the trans isomer of 18:1[delta]9-ACP. In plants such as the Umbelliferae species coriander (Coriandrum sativum L.) that accumulate petroselinic acid (18:1[delta]6cis) in their seed triacylglycerols, a high petroselinoyl-ACP thioesterase activity was found in addition to the oleoyl-ACP thioesterase. The two activities could be separated by anion-exchange chromatography, indicating that the petroselinoyl-ACP thioesterase is represented by a distinct polypeptide.