A new species of Hippomonavella (Bryozoa: Cheilostomata) from the Holocene and Recent of Argentina and Uruguay (Southwest Atlantic).

Hippomonavella charrua n. sp. is introduced based on material from the continental shelf off Uruguay. Bilaminar fragments of this species were also found in mid-Holocene deposits of Destacamento Río Salado Member, Canal de las Escobas Formation (Buenos Aires Province, Argentina), ca. 6,000 yr BP. Hippomonavella charrua n. sp. resembles H. brasiliensis Ramalho, Muricy Taylor, 2008, but differs from this species in its more triangular and protruding avicularia occurring in just a small proportion of zooids. Hippomonavella charrua n. sp. is the third species of the genus with both fossil and Recent representatives. The tatiform ancestrula and the early astogeny are described for the first time in a species of Hippomonavella.

[Nocturnal hemodialysis: an alternative treatment for a better quality of life]. / Emodialisi notturna: un trattamento alternativo per una migliore qualità della vita.

The interest of investigators in intensified dialysis regimens has been growing in recent years, especially since the HEMO Study Group showed that a higher dose of thrice-weekly hemodialysis fails to reduce mortality and morbidity but improves clinical outcomes. Alternative hemodialysis strategies including short daily hemodialysis (SDHD), long hemodialysis (LHD) and nocturnal daily hemodialysis (NDHD) have been developed in the hope to improve patients' outcomes. A growing number of investigators are studying patients on alternative dialysis regimens and most publications in this field have reported significant improvements in clinical outcomes including left ventricular hypertrophy, blood pressure control, anemia, calcium-phosphate metabolism, and fluid and electrolyte balance; all of these parameters can be considered as indirect signs of improvement in quality of life. However, the strength of these results is often limited by shortcomings in study design. Indeed, in most of these studies an adequate control group is missing, the patient groups are not properly matched, and the number of patients enrolled is small. Similarly, most studies have evaluated the effects of NDHD and/or nocturnal LHD on health-related quality of life (HRQoL) by questionnaire administration. Even though better results might be achieved with nocturnal hemodialysis, no conclusive data exist to prove statistically significant differences in HRQoL between conventional and intensive hemodialysis. In conclusion, all of these novel dialysis strategies offer reliable opportunities for uremic patients, but further trials are needed to determine whether alternative hemodialysis can reduce morbidity and mortality in this high-risk population of patients.

Usefulness of endoscopic small intestinal biopsies in children with coeliac disease.

Small intestinal biopsy is the most important diagnostic method in the routine evaluation of children with chronic diarrhoea and malabsorption. At present morphological alterations are considered essential in the diagnosis of coeliac disease (CD) and the presence of a normal small bowel biopsy specimen, observed in patients eating a diet containing gluten, rules out the diagnosis of CD. The small intestinal biopsy can be carried out either by blind suction capsule or by endoscopic forceps. In everyday clinical practice endoscopic duodenal biopsies, if taken and handled suitably, are accepted as equivalent to capsule biopsies from the proximal jejunum. In the study we reported some patients in whom has been possible to demonstrate the presence of total villous atrophy in one biopsy, while other duodenal samples taken in different duodenal portions were normal or showed mild lymphocytes and plasmacells infiltrations of the lamina propria. In patients with this type of biopsy pathology, wherein flat mucosa has been found even close to normal mucosa, the possible explanation is mucosal patchiness. The occurrence of patchly distributed intestinal atrophy in children suffering of CD raises the question of the validity of using the peroral capsule, widely believed to be the best standard for the diagnosis of CD. In our opinion, small intestinal biopsies obtained via endoscopy are more reliable than the peroral capsule biopsies in order to identify patchy mucosal atrophy and could be very useful for a correct diagnosis in CD patients.

Expression of myelin basic protein (MBP) epitopes in human non-neural cells revealed by two anti-MBP IgM monoclonal antibodies.

Two monoclonal antibodies (1H6.2 and 45.30) were raised against MBP purified from human brain under experimental conditions that allowed MBP to retain binding to surrounding myelin lipids (human lipid-bound MBP (hLB-MBP)). 1H6.2 and 45.30 MoAbs were selected on the basis of their different binding properties to: hLB-MBP, human lipid-free-MBP (hLF-MBP) and bovine lipid-free-MBP (bLF-MBP). Although the isotype of both MoAbs was IgM, their specificity, as tested in ELISA assays against chemical haptens and unrelated protein antigens, was restricted to MBP. 1H6.2 and 45.30 MoAbs stained MBP from human brain white matter tissue extracts, as well as bLF-MBP, in Western blot assays. Both MoAbs stained oligodendrocytes and myelin in immunohistochemical analysis of white matter from human brain. Tissue sections from human peripheral nerves were labelled by 1H6.2 only, however, demonstrating that the MoAbs recognize two different epitopes. Epitopes recognized by 1H6.2 and 45.30 MoAbs were also expressed by a wide array of human non-neural cells of either normal or pathological origin, as evidenced by cytofluorimetric assays. In particular, MBP epitopes (MEs) were expressed by lymphoid cells as well as by cells which play a pivotal role in immune homeostasis and in the immune response, such as thymic epithelial cells and professional antigen-presenting cells. Both MoAbs were efficiently internalized by cells from a human B cell line, suggesting trafficking of MEs along the endocytic pathways. These findings support hypotheses regarding the role of MEs expressed by non-neural cells in establishing self-tolerance and/or in triggering the immune response against MBP antigen.

Radiation inactivation of ribonucleotide reductase, an enzyme with a stable free radical.

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128-153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of approximately 210 kDa and the other of approximately 20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Purification and characterization of a novel human acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase.

A novel N-glycosylated, mannose-rich protein has been purified approx. 4000-fold from human liver in a seven-step procedure including ion-exchange chromatography and fractionation on concanavalin A-Sepharose, Sephadex G-75 and oligo(dT)-cellulose matrices. The molecular mass of the protein is 46 kDa when measured by gel filtration (i.e. under non-denaturing conditions) and 60 kDa by SDS/PAGE (i.e. under denaturing conditions). The protein possesses two DNA backbone-incising activities, namely, the random introduction of single-strand breaks in native DNA and the rupture of the phosphodiester linkage internal to cyclobutyl pyrimidine dimers, the major class of DNA lesions induced by solar UV rays. Both activities are optimal at pH 5.0 in vitro, although the non-specific nuclease displays appreciable activity at neutral pH, depending on the buffer composition. The protein has been named acidic nuclease/intra-cyclobutyl-pyrimidine-dimer-DNA phosphodiesterase (AN/IDP). As a nuclease, the protein 'prefers' a linear DNA structure over a covalently closed circular molecule and is more proficient at digesting single-stranded than double-stranded DNA. The polynucleotide cleavage products of the nuclease contain 5'-OH and 3'-PO(4) termini, which are refractory to direct rejoining by DNA ligases. Depending on the substrate, the nuclease activity exhibits a temperature optimum of 50 degrees C or greater, and is neither stimulated by Mg(2+) or Ca(2+) nor inhibited by Zn(2+). AN/IDP is present in human liver and in cultured human cells of both fibroblastic and lymphocytic origins. Intracellularly, the protein can be readily detected in both the cytosolic and nuclear fractions, although much more (approx. 3-fold) is found in the latter fraction. We propose that this bifunctional enzyme may be involved in both apoptotic DNA digestion and metabolism of cyclobutyl pyrimidine dimers in UV-irradiated human cells.

[Non-Hodgkin's lymphoma in multiple places and uterine involvement]. / Linfoma non-Hodgkin a localizzazioni multiple con interessamento uterino.

Multiple locations of non-Hodgkin lymphoma, in cases of recurrence of disease, may affect all the lymph node stations. The case reported, sited in the uterus, constitutes a very rare event and whenever it occurs the preoperative diagnosis may present serious difficulties. Surgery, which must be prompt and radical, is mandatory for histopathological staging of the disease and for the implementation of an appropriate chemotherapy protocol. In the case reported here the diagnostic work-up enabled us to achieve correct preoperative staging.

Dose and duration-dependence of ganciclovir treatment against murine cytomegalovirus infection in severe combined immunodeficient mice.

The present study investigates the full dose-response curve and treatment duration dependence of ganciclovir (GCV) against murine cytomegalovirus (MCMV) infection in severe combined immunodeficiency (SCID) mice. Animals inoculated intraperitoneally with 6.3 x 10(3) pfu of MCMV per mouse developed typical wasting syndrome rapidly and died around day 12 post-inoculation. Once-daily treatment with subcutaneous GCV for 5 days dose dependently delayed MCMV-induced wasting syndrome and mortality at a dose range of 1-80 mg/kg per day, whereas a dose of 160 mg/kg per day induced reversible side-effects. The effect of GCV treatment on mean death day (MDD) was significantly correlated to reductions of viral titers in the lung (r = 0.969, P < 0.05). Treatment duration dependence was examined at the dose of GCV at 80 mg/kg per day for 1, 5, 8 and 12 days. The protective duration, over vehicle-treated mice, was constantly 3-4 days plus the duration of GCV treatment, as evidenced by the delay of viral replication, wasting syndrome and death. At a sub-optimally effective dose of 10 mg/kg per day of GCV, maximum protection was achieved with a 8-day treatment regimen. Prolongation of this treatment to 12 days failed to further delay mean death day and wasting syndrome that started on day 10, indicative of insufficient suppression of viral replication. Treatment with a single dose of GCV failed to show a complete dose-response curve since only minimal protective effects were observed at the dose of 80 mg/kg while side-effects were associated with the dose of 160 mg/kg. The treatment duration dependence and requirement for sufficient dosage of GCV against CMV infection observed in the current model are consistent with clinical observations. It also suggests that 5 8 days treatment duration may be a good balance considering the opportunity for identifying active compounds and speeding up the turnaround time in drug evaluations.

The antiviral activity of the ribonucleotide reductase inhibitor BILD 1351 SE in combination with acyclovir against HSV type-1 in cell culture.

BILD 1351 SE is a selective peptidomimetic subunit association inhibitor of the herpes simplex virus (HSV) ribonucleotide reductase (RR) with potent antiviral activity both in cell culture assays and animal models of HSV disease. The ability of BILD 1351 SE to inhibit the replication of HSV-1 when used in combination with acyclovir (ACV) for the treatment of HSV infections was investigated in baby hamster kidney cells using a 96-well enzyme-linked immunosorbent assay. The effective concentrations to achieve 50% inhibition (EC50) of virus replication by BILD 1351 SE in serum-starved and non serum-starved cells were 2 +/- 0.9 and 4.1 + 1.6 microM, respectively. The EC50 of ACV under both assay conditions was equal to 2.7 +/- 0.9 microM when tested alone. However, upon addition of BILD 1351 SE, the antiviral activity of ACV was potentiated in a synergistic manner as determined by the isobole method. At a concentration of BILD 1351 SE that produced 30% inhibition of HSV-1 replication, the EC50 of ACV decreased by about 15-fold in confluent cells and 17-fold in serum-starved cells. Similar conclusions were reached when evaluating drug interactions by the median dose-effect. Assuming mutually non-exclusive conditions at a drug ratio of ACV/BILD 1351 SE of 1/2, synergy was demonstrated in confluent cells with a drug enhancement index at EC50 of 14 and a combination index of 0.25. None of the drug combinations tested showed increased cytotoxicity in comparison with each drug alone. These results are consistent with the expected mode of action of a selective HSV RR inhibitor and support the strategy of combining these inhibitors with ACV for improved therapy of HSV infections.

Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo.

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10(6) and 10(7) PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1 dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum.

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.

Effects of ethanol administration on cerebral non-protein sulfhydryl content in rats exposed to styrene vapour.

Glutathione (GSH) and other non-protein sulfhydryls (NPS) are known to protect cells from oxidative stress and from potentially toxic electrophiles formed by biotransformation of xenobiotics. This study examined the effect of a simultaneous administration of styrene and ethanol on NPS content and lipid peroxidation in rat liver and brain. Hepatic cytochrome P450 and cytochrome b5 content, aniline hydroxylase and aminopyrine N-demethylase activities as well as the two major urinary metabolites of styrene, mandelic and phenylglyoxylic acids were also measured. Groups of rats given ethanol for 3 weeks in a liquid diet were exposed, starting from the second week, to 326 ppm of styrene (6 h daily, 5 days a week, for 2 weeks). In control pair-fed animals, styrene produced about 30% depletion of brain NPS and 50% depletion of hepatic NPS. Subchronic ethanol treatment did not affect hepatic NPS levels, but caused 23% depletion of brain NPS. Concomitant administration of ethanol and styrene caused a NPS depletion in brain tissue in the order of 60%. These results suggest that in the rat, simultaneous exposure to ethanol and styrene may lead to considerable depletion of brain NPS. This effect is seen when both compounds are given on a subchronic basis, a situation which better resembles possible human exposure.

The critical C-terminus of the small subunit of herpes simplex virus ribonucleotide reductase is mobile and conformationally similar to C-terminal peptides.

The C-terminus of the small subunit of class I ribonucleotide reductases is essential for subunit association and enzymatic activity. 1H NMR analysis of the small subunit (2 x 38 kDa as a homodimer) of herpes simplex virus ribonucleotide reductase shows that this critical binding site is mobile and exposed in relation to the rest of the protein. Assignments of six C-terminal amino acids are made by comparing the TOCSY and NOESY spectra of the small subunit with the spectra of an identical protein truncated by seven amino acids at the C-terminus and the spectra of an analogous 15 amino acid peptide. The mobility of the C-terminus may be important for subunit recognition and could be general for other ribonucleotide reductases. The spectral comparisons also suggest that the six C-terminal amino acids of the small subunit and peptide are conformationally similar. This observation may be important for the design of inhibitors of ribonucleotide reductase subunit association.

A potent peptidomimetic inhibitor of HSV ribonucleotide reductase with antiviral activity in vivo.

Herpes simplex viruses (HSV) types 1 and 2 encode their own ribonucleotide reductases (RNRs) (EC 1.17.4.1) to convert ribonucleoside diphosphates into the corresponding deoxyribonucleotides. Like other iron-dependent RNRs, the viral enzyme is formed by the reversible association of two distinct homodimeric subunits. The carboxy terminus of the RNR small subunit (R2) is critical for subunit association and synthetic peptides containing these amino-acid sequences selectively inhibit the viral enzyme by preventing subunit association. Increasing evidence indicates that the HSV RNR is important for virulence and reactivation from latency. Previously, we reported on the design of HSV RNR inhibitors with enhanced inhibitory potency in vitro. We now report on BILD 1263, which to our knowledge is the first HSV RNR subunit-association inhibitor with antiviral activity in vivo. This compound suppresses the replication of HSV-1, HSV-2 and acyclovir-resistant HSV strains in cell culture, and also strongly potentiates the antiviral activity of acyclovir. Most importantly, its anti-herpetic activity is shown in a murine ocular model of HSV-1-induced keratitis, providing an example of potent nonsubstrate-based antiviral agents that prevent protein-protein interactions. The unique antiviral properties of BILD 1263 may lead to the design of new strategies to treat herpesvirus infections in humans.

Metabolic processing of cyclobutyl pyrimidine dimers and (6-4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.

A new nuclease digestion assay was developed to elucidate the human excision-repair system operating on cyclobutyl pyrimidine dimers and (6-4) photoproducts. We analyzed lesions that accumulated in excised oligonucleotide fragments during incubation of UV-treated cultured fibroblasts. (6-4) photoproducts were removed intact, whereas excised cyclobutyl dimers often contained ruptured interpyrimidine phosphodiester bonds, raising the possibility that the intradimer backbone-cleavage reaction may help promote the bypass of unexcised dimers by the DNA replication or RNA transcription machinery. Cell strains representing eight different inherited forms of the cancer-prone skin disease xeroderma pigmentosum (XP) were generally found to exhibit characteristic abilities to excise the two classes of photolesions, ranging from total deficiency in groups A and G to normal proficiency in the variant. The capacity of any given XP group to act on one class of photoproducts in no way predicted its ability to act on the other. Finally, in those XP strains displaying significant levels of dimer removal, the ratio of intact-versus-modified dimers was normal, implying that rupture of the intradimer backbone linkage occurs independently of subsequent excision-repair reactions. Our data indicate that cyclobutyl dimers and (6-4) photoproducts are processed by distinct nucleotide-excision-repair pathways in human cells.

A solid-phase assay for the binding of peptidic subunit association inhibitors to the herpes simplex virus ribonucleotide reductase large subunit.

The herpes simplex virus (HSV) ribonucleotide reductase is comprised of two nonidentical homodimeric subunits whose association is essential for enzymatic activity. In order to evaluate the affinity of a series of peptidic inhibitors with the enzyme's large subunit (R1), we have developed a sensitive solid-phase binding assay. The assay entails the use of a nonneutralizing antibody directed against the R1 subunit of the enzyme to immobilize either the native holoenzyme from HSV-1-infected cells or a recombinantly expressed HSV-2 R1 subunit. In either case, the radioiodinated peptidic inhibitor 125I-desamino-Tyr-(N-methyl)-Val-Ile-Asp-(gamma-N,N-diethyl)-Asp-Leu demonstrated specific, saturable binding to the HSV R1 that could be competed by the nonapeptide Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu corresponding to the C-terminal sequence of the HSV ribonucleotide reductase small subunit (R2) or by recombinant HSV R2, but not by C-terminally truncated HSV R2 or murine R2. Our results provide direct evidence that inhibitors based on the carboxy-terminal amino acid sequence of HSV R2 compete with intact HSV R2 for a common binding site on HSV R1. The utility and sensitivity of this binding assay were further demonstrated by the ability to detect and discriminate ribonucleotide reductase inhibitors in the low nanomolar range.

Enzymatic analysis of oligonucleotides containing cyclobutane pyrimidine photodimers with a cleaved intradimer phosphodiester linkage.

Our recent studies indicate that enzymatic hydrolysis of the intradimer phosphodiester linkage constitutes an early reaction in processing UV light-induced cis-syn-cyclobutane pyrimidine dimers in cultured human fibroblasts. Before characterizing the resultant modified dimer sites in cellular DNA, it is necessary to establish experimental conditions that can distinguish backbone-nicked from intact dimers. We thus constructed a model substrate, i.e. p(dT) 10 <> p(dT)10 containing a dimer with a ruptured sugar-phosphate bond, and determined the products of its reaction with snake venom phosphodiesterase and alkaline phosphatase, an enzymatic digestion mixture known to release dimers from UV-treated poly(dA).poly(dT) within trinucleotides with the photoproduct intact at the 3'-end (d-TpT

T). The model substrate was prepared by (i) end labeling p(dT)9 using terminal deoxynucleotidyltransferase and [3H]thymine-labeled TTP; and (ii) annealing the chromatographically purified p(dT)10 oligomers to poly(dA) followed by UV (290 nm)-induced ligation. Photoligated 20-mers with one radioactive and modified internal dimer were isolated and enzymatically digested. High performance liquid chromatographic analysis of the reaction products revealed a novel trithymidylate with its backbone severed at the 3'-terminus (d-TpT<>dT), demonstrating that this procedure could discriminate between intact and modified dimers. The procedure was then exploited to show that (i) Escherichia coli DNA photolyase can monomerize, albeit inefficiently, backbone-ruptured dimers; and (ii) phage T4 polynucleotide kinase can catalyze the phosphorylation of d-TpT<>dT, thus facilitating the development of a sensitive postlabeling assay suitable for modified dimer detection under biologically relevant conditions.

Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA.

We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group). It was observed that the enzyme acted only on the latter isomer. Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase [Weinfeld, M., Liuzzi, M., & Paterson, M. C. (1989) Nucleic Acids Res. 17, 3735-3745], we were able to devise a postlabeling assay for these biologically important DNA lesions. The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites. Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour. This value compares favorably with previously published data of others, acquired by alternative approaches. The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour.

Selective hydrolysis by exo- and endonucleases of phosphodiester bonds adjacent to an apurinic site.

Partial depurination of d-ApA produced two UV260nm-absorbing isomers, d-SpA and d-ApS (where S represents the depurinated deoxyribose sugar), that provided simple model compounds with which to examine, by HPLC, the response of nucleases to phosphodiester bonds flanked 3' or 5' by an apurinic site. The structural identity of each compound was established by (i) reaction with methoxyamine to confirm the presence of an abasic deoxyribose group, and (ii) degradation of d-SpA under mild alkaline conditions to distinguish it from d-ApS. At an enzyme concentration which led to complete hydrolysis of d-ApA, snake venom phosphodiesterase readily cleaved d-SpA to 5'-dAMP but had no discernible effect on d-ApS. Calf spleen phosphodiesterase also failed to act on one isomer, in this instance d-SpA, but additionally reacted at a much slower rate (approximately 100 fold) with d-ApS than with d-ApA. Three single-strand specific endonucleases, nuclease P1, nuclease S1 and mung bean nuclease, all responded in an identical manner, hydrolysing d-ApS but not d-SpA. The possibility that the aldehyde group at the AP sites might be responsible for some of these observations was rejected after repeating the enzyme digestions with the methoxyamine-capped molecules and observing no differences from the reactions with d-SpA and d-ApS.