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.

Reaction of apurinic/apyrimidinic sites with [14C]methoxyamine. A method for the quantitative assay of AP sites in DNA.

A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of [14C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37 degrees C, pH 7.2) untreated DNA is almost unreactive and the [14C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.

Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites.

Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.

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.

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.

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.

Characterization of damage in gamma-irradiated and OsO4-treated DNA using methoxyamine.

Unlabelled and radiolabelled methoxyamine have been used to characterize DNA damage caused by gamma-rays or by the chemical reagent osmium tetroxide (OsO4). Both treatments introduce in DNA a number of methoxyamine-binding sites proportional to the dose. Whereas the number of these sites remains constant after the OsO4 treatment it increases during postirradiation incubation; the postirradiation appearance of methoxyamine-binding sites is enhanced by the presence of methoxyamine. OsO4 treatment and gamma-irradiation also induce the formation of alkali-labile sites in DNA. Whereas the number of these sites remains constant after OsO4 treatment, it increases during postirradiation incubation and an alkaline medium accelerates their formation. A fraction of the alkali-labile sites found in gamma-irradiated DNA is methoxyamine-labile; by contrast, the OsO4-treated DNA is stable in the presence of methoxyamine.

[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.

Base-excision repair in carrot cells. Partial purification and characterization of uracil-DNA glycosylase and apurinic/apyrimidinic endodeoxyribonuclease.

Uracil-DNA glycosylase and apurinic/apyrimidinic (AP) endodeoxyribonuclease have been purified from cultured carrot cells. The two enzymes, separated by affinity chromatography on Sepharose-poly(rU), were found to have properties similar to those of the homologous bacterial and mammalian enzymes. The action of AP endodeoxyribonuclease on (dA)230 . (dT, dU)230 partially depyrimidinated by uracil-DNA glycosylase suggests that these two enzymes might act successively to initiate the repair of uracil-containing DNA.

A new approach to the study of the base-excision repair pathway using methoxyamine.

This paper describes the use of methoxyamine to study the enzymatic reactions catalyzed by uracil-DNA glycosylase and by AP (apurinic/apyrimidinic) endodeoxyribonuclease isolated from mammalian cells. [14C]Methoxyamine permits one to follow the formation of AP sites in a uracil-containing polydeoxyribonucleotide incubated with calf thymus uracil-DNA glycosylase. The number of methoxyamine-reacted AP sites is equal to that of uracil released. Methoxyamine has no effect on the uracil-DNA glycosylase activity and may be added together with the enzyme in order to block the AP sites and prevent the degradation of the polynucleotide by the AP endonucleases that may be present in a crude preparation. Addition of methoxyamine to AP sites prevents not only the enzymatic hydrolysis of the adjacent phosphodiester bond but also the degradation of the polynucleotide by NaOH. This protective effect disappears after methoxyamine is removed by acetaldehyde.

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.

Chemical carcinogenesis. II. Imidazole-ring-opened derivatives from 7-ethyl- and 1,7-diethylguanosine.

The UV-spectral and chromatographic analyses of the derivatives of the two synthetic standards 7-ethylguanosine and 1,7-diethylguanosine are here reported. The derivatives obtained from the dialkyl compound exhibit a striking similarity to those found in the "pyrimidine-nucleotide-like" fraction of rat liver tRNA ethylated in vivo by ethionine. The finding of imidazole-ring-opened products in tRNA ethylation by ethionine could be significant from the point of view of chemical carcinogenesis: in fact, imidazole-ring-opening of 1,7-dialkylguanosines directly at level of RNA with consequent formation of substituted pyrimidines is a transversion, i.e. a mutagenic event which would cause a change in the expression of genetic information since a purine has been transformed into a pyrimidine.

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.

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage.

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.

Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. II. Phosphorylation by phage T4 polynucleotide kinase.

Phage T4 polynucleotide kinase (EC 2.7.1.78) proved incapable of catalyzing the phosphorylation of thymidylyl-(3'----5')-thymidine containing either a cis-syn-cyclobutane pyrimidine dimer (d-T less than p greater than T) or a 6-4'-[pyrimidin-2'-one]pyrimidine photoproduct (d-T[p]-T), and similarly the UV-modified compounds of (dT)3 bearing either photoproduct at their 5'-end (d-T less than p greater than TpT and d-T[p]TpT). In contrast, the 3'-structural isomers of these trinucleotides (d-TpT less than p greater than T and d-TpT[p]T) were phosphorylated at the same rate as the parent compound. These phosphorylatable lesion-containing oligonucleotides are quantitatively released from UV-irradiated poly(dA):poly(dT) by enzymatic hydrolysis with snake venom phosphodiesterase and alkaline phosphatase (Liuzzi, M., Weinfeld, M., and Paterson, M. C. (1989) J. Biol. Chem. 264, 6355-6363). By combining this digestion regimen with phosphorylation by polynucleotide kinase and [gamma-32P]ATP, pyrimidine dimers were quantitated at the fmol level following exposure of poly(dA):poly(dT) and herring sperm DNA to biologically relevant UV fluences. The rate of dimer induction in the synthetic polymer, approximately 10 dimers/10(6) nucleotides/Jm-2, was in close agreement with that obtained by conventional methods. Dimers were induced at one-fourth of this rate in the natural DNA. Further treatment of the phosphorylated oligonucleotides derived from irradiated herring sperm DNA with nuclease P1 released the labeled 5'-nucleotide, thus permitting analysis of the nearest-neighbor bases 5' to the lesions. We observed a ratio for pyrimidine-to-purine bases of almost 6:1, implicating tripyrimidine stretches as hotspots for UV-induced DNA damage.

Selective inhibition by methoxyamine of the apurinic/apyrimidinic endonuclease activity associated with pyrimidine dimer-DNA glycosylases from Micrococcus luteus and bacteriophage T4.

The UV endonucleases [endodeoxyribonuclease (pyrimidine dimer), EC 3.1.25.1] from Micrococcus luteus and bacteriophage T4 possess two catalytic activities specific for the site of cyclobutane pyrimidine dimers in UV-irradiated DNA: a DNA glycosylase that cleaves the 5'-glycosyl bond of the dimerized pyrimidines and an apurinic/apyrimidinic (AP) endonuclease that thereupon incises the phosphodiester bond 3' to the resulting apyrimidinic site. We have explored the potential use of methoxyamine, a chemical that reacts at neutral pH with AP sites in DNA, as a selective inhibitor of the AP endonuclease activities residing in the M. luteus and T4 enzymes. The presence of 50 mM methoxyamine during incubation of UV- (4 kJ/m2, 254 nm) treated, [3H]thymine-labeled poly(dA).poly(dT) with either enzyme preparation was found to protect completely the irradiated copolymer from endonucleolytic attack at dimer sites, as assayed by yield of acid-soluble radioactivity. In contrast, the dimer-DNA glycosylase activity of each enzyme remained fully functional, as monitored retrospectively by release of free thymine after either photochemical- (5 kJ/m2, 254 nm) or photoenzymic- (Escherichia coli photolyase plus visible light) induced reversal of pyrimidine dimers in the UV-damaged substrate. Our data demonstrate that the inhibition of the strand-incision reaction arises because of chemical modification of the AP sites and is not due to inactivation of the enzyme by methoxyamine. Our results, combined with earlier findings for 5'-acting AP endonucleases, strongly suggest that methoxyamine is a highly specific inhibitor of virtually all AP endonucleases, irrespective of their modes of action, and may therefore prove useful in a wide variety of DNA repair studies.

Methylmethanesulfonate-induced DNA damage and its repair in cultured human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum (group A) cells.

Using conventional alkaline sucrose sedimentation analysis, we have compared the initial yield and subsequent enzymatic repair of DNA damage induced in cultured human [normal (GM38 and GM43) and xeroderma pigmentosum (XP12BE)] fibroblasts by the monofunctional alkylating agent methylmethanesulfonate (MMS). Exposure of both cell types to MMS (0-20 mM) resulted in a linear dose-response relationship for the formation of DNA alkali-labile sites (i.e. structurally altered sites that appeared as single-strand interruptions at alkaline pH). The majority (approximately 90%) of the sites detected in the normal cells immediately after chemical treatment (less than or equal to 8 mM) disappeared rapidly, with a half-life of less than or equal to 3 h; the remainder, however, persisted in genomic DNA for at least 72 h. Approximately 40% of the alkali-labile sites induced by 5 mM MMS could be stabilized by methoxyamine, a chemical which is known to react with apurinic/apyrimidinic (AP) sites in DNA so as to prevent alkali-catalyzed beta-elimination; thus this fraction of the alkali-labile sites, which is estimated to constitute approximately 4% of the total genomic injury inflicted by the chemical, may be ascribable to AP sites. XP12BE cells responded normally to MMS exposure as judged by: (i) the rate of initial induction of alkali-labile sites, including those (AP sites) subject to methoxyamine stabilization; (ii) the incidence of alkali-labile sites in cellular DNA at various times (0-72 h) after administration of the alkylating agent; and (iii) the capacity to execute the long-patch mode of excision repair as measured by accumulation of 1-beta-D-arabinofuranosylcytosine-induced strand breaks during post-treatment cell incubation. In addition, we have found that a significant portion of the genetic material in human fibroblasts undergoes degradation upon sustaining MMS damage, as indicated by the appearance of small DNA fragments (sedimenting near the top of alkaline sucrose gradients) in chemically treated cultures incubated for 24 h. Interestingly, the extent of this type of DNA injury proved to be markedly greater in XP12BE than in GM38 cells, and in exponentially growing than in G2-arrested normal cultures.

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.