Infidelity of DNA synthesis as related to mutagenesis and carcinogenesis

An assay system has been developed for measuring the fidelity of DNA synthesis in vitro by using synthetic polynucleotide templates and purified DNA polymerases. Nearest-neighbor analysis of the synthesized product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The accuracy of DNA synthesis can be decreased by (1) prior alkylation of the template, (2) increasing the relative concentration of incorrect nucleotides, and (3) addition of specific metal salts to the reaction mixture. As an initial evaluation of the utility of this system, the effects of 31 metal salts on the fidelity of DNA synthesis have been determined. The results indicate that potential metal mutagens and/or carcinogens may be detected by measuring alterations in the fidelity of DNA synthesis.     

Anthrylvinyl-labeled phospholipids as membrane probes: the phosphatidylcholine-phosphatidylethanolamine system

The phase behavior of mixtures of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) identical or differing in their fatty acid composition has been investigated by using the steady-state fluorescence anisotropy of anthrylvinyl-labeled PC and PE (APC and APE) as well as of the non-lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to detect temperature-dependent changes in multilayer liposomes. APC, but not APE, was able to detect the pretransition of dimyristoyl-PC. The phospholipid probes APC and APE showed the main phase transition of their unlabeled disaturated analogues at temperatures almost identical with those revealed by differential scanning calorimetry, whereas the onset of the PE phase transition recorded by DPH was several degrees higher. In PC-PE mixtures with high content of PE the phase transitions shown by APC and APE were broader than those recorded by DPH. Comparison of phase diagrams constructed on the basis of fluorescence anisotropy and calorimetric data led to the conclusion that in biphasic PE and PC-PE systems DPH tends to partition into solid regions, whereas the anthrylvinyl-labeled phospholipids distribute more evenly between coexisting phases or prefer fluid domains. The use of anthrylvinyl phospholipid probes made it possible to demonstrate that PEs and PCs identical in their fatty acids are not miscible completely, not only below but also well above Tm of the higher melting component. Generally, APC and APE fluorescence anisotropy measurements correctly reflect headgroup-dependent phase segregations in mixtures of PC with PE, but may lead to ambiguous conclusions if demixing is caused by differences in the hydrocarbon chains.     

Isolation of specific DNA probes from Cryptococcus neoformans

OBJECTIVE: To construct plasmid library and screen specific DNA probes for Cryptococcus neoformans. METHODS: Serotype A Cryptococcus neoformans was used as the study strain, plasmid pUC18 as vector, and Escherichia coli JM103 as host cell. The plasmid library of cryptococcus neoformans was constructed (pCN). Other pathogenes causing affection diseases which should be distinguished from cryptococcusis clinically, and other fungi similar to Cryptococcus neoformans with physiological and biochemical characteristics were used as a distinguishing system, specific colonies were screened by hibridization in double steps. RESULTS: The inserts of the library were 280 to 1800 base pairs and 580 base pairs in average length. Repeated sequence was 32.43% and single copy sequence was 67.57% in genome of cryptococcus neoformans respectively. Three specific colonies were isolated from the library. Colony pCNII A6 was serotype A specific, pCNII B5 species specific and pCNIII G1-specific for var. neoformans. CONCLUSION: A rapid diagnosis of Cryptococcus neoformans infection at early stage can be made by using species-specific probe, and serotype and variaty of neoformans and gattii be distinguished in epidemic study.     

DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens

Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA. These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition. Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved. Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose. Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine [BrdUrd]) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression. Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels. Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control). Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis. In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment. Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity.     

Fluorescent probes for adenosine receptors: synthesis and biology of N6-dansylaminoalkyl-substituted NECA derivatives

New fluorescent ligands for adenosine receptors are described; these compounds were obtained by the insertion, in the N6 position of NECA (a potent adenosine agonist), of dansylaminoalkyl moieties with alkyl spacers of increasing carbon chain length (from 3 to 12). Among them, the compound with a C6 alkyl spacer proved to be the most interesting one, showing a marked selectivity for the A1 receptor subtype; furthermore, in fluorescence microscopy assays it proved to be able to visualize and localize this receptor subtype at the level of the molecular layer of the rate cerebellar cortex.     

Membrane skeleton restraint of surface shape change during fusion of erythrocyte membranes: evidence from use of osmotic and dielectrophoretic microforces as probes

The role of the spectrin-based membrane skeleton in cell fusion was studied by following the condition-dependent diameter versus time expansion signature of the fusion zone in electrofused pairs of erythrocyte ghost membranes. Previous work showed that the presence of the dielectrophoresis-inducing alternating electric field, which is used to bring membranes into contact through pearl chain formation, had a detectable promoting effect on fusion zone expansion. Two new dielectrophoresis protocols were used in the present work to utilize this externally generated and controllable microforce field to probe the forces intrinsic to the system that drives the expansion of the fusion zone. First, fusion zones expanded to a greater diameter in a strong AC field compared to a weak AC field, and they expanded to a greater diameter if erythrocyte ghosts received a prior heat treatment (42 degrees C, 20 min). Furthermore, flat diaphragm fusion zones broke down into open lumen fusion zones sooner (i.e., had shorter lifetimes) when they were expanding more quickly. Second, changing the AC field strength at specific times during the fusion zone expansion led to an immediate visco-elastic response. However, shifting the AC field strength to zero after 5 s of fusion zone expansion resulted in a subsequent decrease in the average fusion zone diameter. This suggests not only that the spectrin-based membrane skeleton actually tends to prevent the rounding up process but that it may be capable of generating an antirounding force, which has broad implications for the role of the membrane skeleton in cell fusion. These results are consistent with the hypothesis that flat diaphragm fusion zones induced in heat-treated membranes were very easily stretched and that membrane-based forces that control or drive the expansion process must originate from membrane area that is outside rather than inside the fusion zone. Lastly, when an outward-directed osmotic pressure-based microforce was present at the time that erythrocyte ghosts were fused, the fusion zone diameter underwent a greater expansion in the 0-1 s interval after fusion. This suggests that an osmotic pressure-based microforce can be used to experimentally calibrate the dielectrophoretic force.     

Detection of foodborne pathogens using DNA probes and a dipstick format

The detection of foodborne microorganisms has traditionally been done using microbiologically based methods. Such "gold standard" methods are generally reliable but have the disadvantages of being labor intensive, subjective, and time consuming. Over the last several years, the development of DNA probe-based methods has simplified the methods used to detect organisms such as Salmonella, Listeria, and E. coli by targeting the unique DNA or RNA sequences of these organisms using DNA probes and nonradioactive detection.     

A new method of nonradioactive labelling of oligonucleotides and their use as allele-specific probes for detecting mutations causing beta-thalassemia

We have developed simple and efficient methods for synthesis of biotin and horseradish peroxidase (HRP)-labeled oligonucleotides. Biotinylated oligonucleotides were obtained in quantitative yields, and oligonucleotide conjugates with HRP in 60-80% yields. Allele-specific oligonucleotide probes for the diagnostics of IVS 1-110 mutation in the beta-globin gene causing beta-thalassemia were thus obtained. Temperature conditions for the non-radioactive ASO hybridization with the amplified segment of the human beta-globin gene and wash conditions were selected. HRP-labelled probes were used in hybridization without preliminary separation after synthesis. To decrease nonspecific enzyme binding we have elaborated special conditions for membrane blocking. Detection of the biotinylated probe was carried out with the help of a streptavidin--HRP conjugate. O-Dianisidine was used as a chromogenic substrate. We have demonstrated the usefulness of this method in the analysis of amplified samples of DNA obtained from blood of patients homozygous in the mutant gene, and heterozygous carriers.     

Substituted glycals as probes of glycosidase mechanisms

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).     

Non-enzymatic hydrolyses of aryl esters of thymidine 3'-monophosphate as probes for the rate-determining step in DNA hydrolysis

In order to shed light on the rate-limiting step in DNA hydrolysis, non-enzymatic hydrolyses of aryl esters of thymidine 3'-monophosphate have been kinetically examined. In alkaline solutions, these dinucleotide analogues were hydrolyzed far faster than was thymidylyl(3'-5')thymidine (TpT), indicating that the departure of the leaving group from the phosphorus atom is rate-limiting. The CeIV- and CeIV/PrIII combination-mediated hydrolyses of these analogues were also faster than the corresponding hydrolysis of the dinucleotide.     

Population study in West Austria using DNA single locus probes

Allele frequency distribution of the single-locus system YNH 24 (n = 302), G3 (n = 251), MS 43A (n = 333), and MS 31 (n = 333) was determined in Western Austria. After digestion with Hinf I, electrophoresis and Southern blotting, the genomic DNA was hybridised with the probes YNH 24, G3, MS 43A, and MS 31. Blood samples were taken from 333 unrelated caucasians living in the area of Innsbruck, Tyrol, Austria. The fragment distribution was calculated for each of the 4 single-locus systems. A single fragment pattern, indicating homozygosity, was shown in 9.93% with YNH 24, 12.45% with G3, 9.61% with MS 43A, and 7.21% with MS 31; the corresponding heterozygosity rates were 90.07%, 87.55%, 90.39%, and 92.79%, respectively. Our data are compared with those from England [18], Germany [3], Greenland [7], and Denmark [14]. Discrimination indices (Table 2) were calculated and statistical parameters (Table 3) presented.     

Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology

Carbon nanotubes combine a range of properties that make them well suited for use as probe tips in applications such as atomic force microscopy (AFM). Their high aspect ratio, for example, opens up the possibility of probing the deep crevices that occur in microelectronic circuits, and the small effective radius of nanotube tips significantly improves the lateral resolution beyond what can be achieved using commercial silicon tips. Another characteristic feature of nanotubes is their ability to buckle elastically, which makes them very robust while limiting the maximum force that is applied to delicate organic and biological samples. Earlier investigations into the performance of nanotubes as scanning probe microscopy tips have focused on topographical imaging, but a potentially more significant issue is the question of whether nanotubes can be modified to create probes that can sense and manipulate matter at the molecular level. Here we demonstrate that nanotube tips with the capability of chemical and biological discrimination can be created with acidic functionality and by coupling basic or hydrophobic functionalities or biomolecular probes to the carboxyl groups that are present at the open tip ends. We have used these modified nanotubes as AFM tips to titrate the acid and base groups, to image patterned samples based on molecular interactions, and to measure the binding force between single protein-ligand pairs. As carboxyl groups are readily derivatized by a variety of reactions, the preparation of a wide range of functionalized nanotube tips should be possible, thus creating molecular probes with potential applications in many areas of chemistry and biology.     

Synthesis and use of iodinated nonsteroidal antiinflammatory drug analogs as crystallographic probes of the prostaglandin H2 synthase cyclooxygenase active site

The cyclooxygenase activity of the membrane protein prostaglandin H2 synthase isoform 1 (PGHS-1) is the target of the nonsteroidal antiinflammatory drugs (NSAIDs). The X-ray crystal structures of PGHS-1 in complex with the NSAIDs flurbiprofen and bromoaspirin have been determined previously [Picot, D., et al. (1994) Nature 367, 243-249; Loll, P. J., et al. (1995) Nat. Struct. Biol. 2, 637-643]. We report here the preparation and characterization of novel potent iodinated analogs of the NSAIDs indomethacin and suprofen, as well as the refined X-ray crystal structures of their complexes with PGHS-1. The PGHS-iodosuprofen complex structure has been refined at 3.5 A to an R-value of 0.189 and shows the suprofen analog to share a common mode of binding with flurbiprofen. The PGHS-iodoindomethacin complex structure has been refined at 4.5 A to an R-value of 0.254. The low resolution of the iodoindomethacin complex structure precludes detailed modeling of drug-enzyme interactions, but the electron-dense iodine atom of the inhibitor has been unambiguously located, allowing for the placement and approximate orientation of the inhibitor in the enzyme's active site. We have modeled two equally likely binding modes for iodoindomethacin, corresponding to the two principal conformers of the inhibitor. Like flurbiprofen, iodosuprofen and iodoindomethacin bind at the end of the long channel which leads into the enzyme active site. Binding at this site presumably blocks access of substrate to Tyr-385, a residue essential for catalysis. No evidence is seen for significant protein conformational differences between the iodoindomethacin and iodosuprofen of flurbiprofen complex structures.     

Design and synthesis of [111In]DTPA-folate for use as a tumor-targeted radiopharmaceutical

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.     

Update on the use of metabolic probes to quantify liver function: caffeine versus lidocaine

The search for continues for a safe, accurate and reliable method to quantify liver function similar in principle to renal creatinine clearance or pulmonary function spirometry tests. When evaluating patients in the more advanced stages of chronic liver disease, one's clinical judgement regarding the degree of liver dysfunction usually suffices, but in patients with early or only intermediate disease, and estimate based on routine blood tests and/or clinical severity scores is often unreliable. A more quantitative approach under investigation at present has been to monitor specific pharmacokinetic parameters of 'probe' drugs metabolized primarily by hepatic cytochrome P-450. These parameters include the plasma or salivary clearance rate of the parent compound and/or the formation rate of its metabolites. Following a review of basic hepatic pharmacology relevant to this topic, we shall explore the advantages and disadvantages of two 'metabolic probes' that have shown the most promise to date, caffeine and lidocaine.     

The use of 16S rRNA-targeted oligonucleotide probes to study competition between ruminal fibrolytic bacteria: development of probes for Ruminococcus species and evidence for bacteriocin production

A total of six oligonucleotide probes, complementary to the 16S rRNA, were evaluated for quantitative and determinative studies of Ruminococcus albus and Ruminococcus flavefaciens. On the basis of specificity studies, probes for R. albus (probe RAL196) and R. flavefaciens (probe RFL196) were selected to quantitate these species in mixed culture. In combination with a Fibrobacter succinogenes S85 subspecies probe (SUB1) and a domain Bacteria (formerly kingdom Eubacteria) probe (EUB338), they were used to quantitate these species competing in mixed cultures for cellobiose as the carbon source. In dicultures containing R. albus 8 and F. succinogenes S85, competition was not observed. However, R. flavefaciens FD-1 eventually outcompeted F. succinogenes S85 when cellobiose was the substrate. When R. albus 8 and R. flavefaciens FD-1 were grown together on cellobiose medium, R. albus 8 outcompeted R. flavefaciens FD-1, resulting in undetectable R. flavefaciens 16S rRNA only 1 to 3 h after inoculation, suggesting production of an antagonistic compound by R. albus 8 during rapid growth on soluble substrates. Further, when R. albus 8, R. flavefaciens FD-1, and F. succinogenes S85 were grown together in a triculture, R. flavefaciens FD-1 16S rRNA was detectable for only 2 h after inoculation, while R. albus 8 and F. succinogenes S85 showed a similar competition pattern to that of the dicultures. The results show that the Ruminococcus probes were effective in the measurement of relative populations of selected R. albus and R. flavefaciens strains during in vitro competition studies with F. succinogenes.(ABSTRACT TRUNCATED AT 250 WORDS)