Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclei and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to dGMP of DNA to form N2-[(4-carbamoylphenyl)azo]-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH2 moiety of the adduct at the nicotinamide-binding site of the enzyme.     

Benzamide-DNA interactions: deductions from binding, enzyme kinetics and from X-ray structural analysis of a 9-ethyladenine-benzamide adduct

The interaction of benzamide with the isolated components of calf thymus poly(ADP-ribose) polymerase and with liver nuclei has been investigated. A benzamide-agarose affinity gel matrix was prepared by coupling o-aminobenzoic acid with Affi-Gel 10, followed by amidation. The benzamide-agarose matrix bound the DNA that is coenzymic with poly(ADP-ribose) polymerase; the matrix, however, did not bind the purified poly(ADP-ribose) polymerase protein. A highly radioactive derivative of benzamide, the 125I-labelled adduct of o-aminobenzamide and the Bolton-Hunter reagent, was prepared and its binding to liver nuclear DNA, calf thymus DNA and specific coenzymic DNA of poly(ADP-ribose) polymerase was compared. The binding of labelled benzamide to coenzymic DNA was several-fold higher than its binding to unfractionated calf thymus DNA. A DNA-related enzyme inhibitory site of benzamide was demonstrated in a reconstructed poly(ADP-ribose) polymerase system, made up from purified enzyme protein and varying concentrations of a synthetic octadeoxynucleotide that serves as coenzyme. As a model for benzamide binding to DNA, a crystalline complex of 9-ethyladenine and benzamide was prepared and its X-ray crystallographic structure was determined; this indicated a specific hydrogen bond between an amide hydrogen atom and N-3 of adenine. The benzamide also formed a hydrogen bond to another benzamide molecule. The aromatic ring of benzamide does not intercalate between ethyladenine molecules, but lies nearly perpendicular to the planes of stacking ethyladenine molecules in a manner reminiscent of the binding of ethidium bromide to polynucleotides. Thus we have identified DNA as a site of binding of benzamide; this binding is critically dependent on the nature of the DNA and is high for coenzymic DNA that is isolated with the purified enzyme as a tightly associated species. A possible model for such binding has been suggested from the structural analysis of a benzamide-ethyladenine complex.     

Inhibition of HIV-1 IIIb replication in AA-2 and MT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.     

Dependence of trans-ADP-ribosylation and nuclear glycolysis on the Arg 34-ATP complex of Zn2+ finger I of poly-ADP-ribose polymerase-1

The H-bonded complex of ATP with Arg 34 of Zn2+ finger I of poly-ADP-ribose polymerase-1 (PARP-1) determines trans-oligo-ADP-ribosylation from NAD+ to proteins other than PARP-1. This mechanism was tested in lysolecithin fractions of non-malignant and cancer cells separately and after their recombination. Cellular PARP-1 activity was recovered when the centrifugal sediment was recombined with the supernatant fraction containing cellular ADP-ribose oligomer acceptor proteins. Combination of the matrix fraction (Mx) of cancer cells (lacking OXPHOS) with its supernatant had the same PARP-1 activity as the Mx alone. The supernatant of non-malignant cells was replaced by glycolytic enzymes as ADP-ribose acceptor. The hexokinase activity of the supernatant increased when OXPHOS of intact cells was uncoupled by carbonyl cyanide 4-(trifluoro methoxy) phenylhydrazone. trans-ADP-ribosylation was demonstrated by polyacrylamide gel electrophoresis.     

Beer from the early dynasties (3500–3400 cal B.C.) of Upper Egypt,detected by archaeochemical methods

Physical and chemical analyses of beer residues recovered from a vat site at Hierakonpolis (Upper Egypt) were carried out. Radiocarbon dates of the residues suggest a dating of 3500–3400 cal B.C. and are believed to represent the oldest known beer in the world. Macroscopic and microscopic examinations of the residues revealed the presence of intact remains of grains and spikelets of wheat and barley, as well as fragments of dates and grape pips. Chemical analyses included percentages of sample ingredients, pH and total soluble ions, quantitative determinations of sugars, carboxylic acids and free amino acids. A total of 25 compounds were identified, which are components of fermentation processes that are believed to have formed in connection with the preparation of what is called Nekhen-Hoffman beer.     

Revision ofFagonia species (Zygophyllaceae) with tri- to unifoliolate and simple leaves

Summary A taxonomical revision of3 Fagonia groups, viz. the complexes ofF. arabica, F. bruguieri andF. indica, including the interrelationships between their species, is presented. These complexes are characterized by their compound tri- or reduced unifoliolate leaves. Among the truly simple-leaved species, viz.F. socotrana, F. harpago andF. ovalifolia, the confusion betweenF. socotrana and some other taxa described is considered. The different patterns of the leaf trace of simple and compound tri- to unifoliolate leaves may prove to be of systematic value.     

Structure elucidation of a 6-methylbenzo[a]pyrene-DNA adduct formed by horseradish peroxidase in vitro and mouse skin in vivo

Activation of polycyclic aromatic hydrocarbons (PAH) by horseradish peroxidase (HRP) with H₂O₂ has been studied as a model system for one-electron oxidation. This peroxidase has been used to catalyze binding of $\text{6-}\text{[}{}^{14}\text{C]methylbenzo}\text{[a]}\text{pyrene}$ (BP-6-CH₃) to DNA, which was purified, hydrolyzed to deoxyribonucleosides and analyzed by high pressure liquid chromatography (HPLC). The predominant hydrocarbon-DNA adduct observed was identified as BP-6-CH₃ bound at the 6-methyl group to the 2-amino group of dG, confirming that activation by HRP occurs by one-electron oxidation. When DNA from mouse skin treated in vivo with [¹⁴C]BP-6-CH₃ was purified, hydrolyzed and analyzed by HPLC, a profile was observed which was qualitatively similar to that from the peroxidase system. In particular, the identified adduct with the hydrocarbon bound at the 6-methyl group to the 2-amino group of dG was obtained. These results demonstrate that one-electron oxidation is the mechanism of activation by HRP for aromatic hydrocarbons and indicate that the same mechanism may occur in mouse skin, a target tissue for hydrocarbon carcinogenesis.     

Systemic responses to prolonged hemorrhagic hypotension

Studies are needed to provide a rigorous examination of the relevance of monitored variables during prolonged hemorrhagic hypotension (HH). This study was designed to investigate the parameters that describe biochemical and O2 transport patterns in animals subjected to HH. Systemic parameters that could differentiate survivors from nonsurvivors were identified. An aortic flow probe was implanted in rats (n = 21) for continuous measurement of cardiac output. Experiments were performed 6-9 days after surgery. Rats were bled to a mean arterial pressure of 40 mmHg and kept at that level using Ringer-lactate solution. Arterial and venous blood pressures, gases, acid-base status, glucose, lactate, electrolytes, hemoglobin, O2 saturation, heart and respiratory rates, total peripheral resistance, and O2 delivery and consumption were measured before hemorrhage, soon after 40 mmHg was reached, and 0.5, 1, 2, 3, and 4 h later. Fifty-three percent of rats survived > or =3 h (survivors); others were considered nonsurvivors. Nonsurvivors showed a significantly greater degree of metabolic acidosis than survivors. Arterial PO2, respiratory rate, O2 saturation, O2 content, glucose, and pH were significantly higher in survivors. The rate of Ringer-lactate infusion, arterial K+, and PCO2 were lower in survivors. Arterial K+ and respiratory rate were the only parameters significantly different between survivors and nonsurvivors at all time points during HH. Arterial levels of K+ showed the clearest distinction between survivors and nonsurvivors and may explain the sudden death experienced by animals during HH. The data suggest that early respiratory and metabolic compensations are essential for survival of prolonged HH.     

Evidence for Fossilized Subsurface Microbial Communities at the TAG Hydrothermal Mound

Scanning electron microscope examinations have revealed fossilized cell-like structures randomly distributed in near-surface oxidized deposits of red and gray Fe-rich chert and Fe-Si oxyhydroxides of the Trans-Atlantic Geotraverse (TAG) hydrothermal mound, Mid-Atlantic Ridge at 26°08'N. Chemically, these structures are carbon-based with the morphology of half-spheroids that are 2 to 3 w m in diameter and are mostly arranged in the form of clusters and long thread-like cellular masses that resemble single-celled microorganisms. The wide range of intracrystalline silica concentration, which seems to replace the original chemistry, suggests that the microorganisms were subjected to various degrees of silica mineralization, which was probably controlled by the thermal development of this hydrothermal site.