Age-related changes in adenosine-mediated relaxation of coronary and aortic smooth muscle.

We tested whether adenosine mediates nitric oxide (NO)-dependent and NO-independent dilation in coronary and aortic smooth muscle and whether age selectively impairs NO-dependent adenosine relaxation. Responses to adenosine and the relatively nonselective analog 5'-N-ethylcarboxamidoadenosine (NECA) were studied in coronary vessels and aortas from immature (1-2 mo), mature (3-4 mo), and moderately aged (12-18 mo) Wistar and Sprague-Dawley rats. Adenosine and NECA induced biphasic concentration-dependent coronary vasodilation, with data supporting high-sensitivity (pEC(50) = 5.2-5.8) and low-sensitivity (pEC(50) = 2.3-2.4) adenosine sites. Although sensitivity to adenosine and NECA was unaltered by age, response magnitude declined significantly. Treatment with 50 microM N(G)-nitro-L-arginine methyl ester (L-NAME) markedly inhibited the high-sensitivity site, although response magnitude still declined with age. Aortic sensitivity to adenosine declined with age (pEC(50) = 4.7 +/- 0.2, 3.5 +/- 0.2, and 2.9 +/- 0.1 in immature, mature, and moderately aged aortas, respectively), and the adenosine receptor transduction maximum also decreased (16.1 +/- 0.8, 12.9 +/- 0.7, and 9.6 +/- 0.7 mN/mm(2) in immature, mature, and moderately aged aortas, respectively). L-NAME decreased aortic sensitivity to adenosine in immature and mature tissues but was ineffective in the moderately aged aorta. Data collectively indicate that 1) adenosine mediates NO-dependent and NO-independent coronary and aortic relaxation, 2) maturation and aging reduce NO-independent and NO-dependent adenosine responses, and 3) the age-related decline in aortic response also involves a reduction in the adenosine receptor transduction maximum.

Acute but not chronic caffeine impairs functional responses to ischaemia-reperfusion in rat isolated perfused heart.

1. The effect of acute (50 micromol/L) and chronic (0.06% in drinking water for 14 days) caffeine on the response to ischaemia-reperfusion was studied in Wistar rat isolated perfused hearts. 2. Neither acute nor chronic caffeine modified normoxic heart rate or left ventricular pressures. However, acute caffeine decreased coronary flow by up to 20%, while chronic caffeine consumption increased coronary flow by approximately 15% and abolished the vasoconstrictor effect of acute caffeine (P<0.05). 3. After 15 min global ischaemia, chronic caffeine treatment did not alter the recovery of left ventricular diastolic pressure (LVDP), end-diastolic pressure (EDP) or heart rate during reperfusion, but did enhance coronary flow rate (P<0.05). Acute caffeine inhibited the recovery of LVDP and elevated postischaemic EDP in both caffeine-naive and chronic caffeine-treated groups. Acute caffeine also significantly inhibited coronary reflow in naive but not chronic caffeine-treated groups and produced a transient tachycardia during reperfusion in hearts from chronic caffeine-treated rats. 4. The incidence of arrhythmias was unaltered by chronic caffeine treatment, but was increased by acute caffeine in both naive and chronic caffeine hearts. 5. In conclusion, chronic caffeine intake alone has no detrimental effects on recovery from ischaemia; however, acute caffeine worsens postischaemic contractile function in hearts from naive and chronic caffeine-treated rats.

Age-related changes in A(1)-adenosine receptor-mediated bradycardia.

The impact of age on functional sensitivity to A(1)-adenosine receptor activation was studied in Langendorff-perfused hearts from young (1-2 mo) and old (12-18 mo) male Wistar rats. Adenosine mediated bradycardia in young and old hearts, with sensitivity enhanced approximately 10-fold in old [negative logarithm of EC(50) (pEC(50)) = 4.56 +/- 0.11] versus young hearts (pEC(50) = 3.70 +/- 0. 09). Alternatively, the nonmetabolized A(1) agonists N(6)-cyclohexyladenosine and (R)-N(6)-phenylisopropyladenosine were equipotent in young (pEC(50) = 7.43 +/- 0.12 and 6.61 +/- 0.19, respectively) and old hearts (pEC(50) = 7.07 +/- 0.10 and 6.80 +/- 0. 11, respectively), suggesting a role for uptake and/or catabolism in age-related changes in adenosine sensitivity. In support of this suggestion, [(3)H]-adenosine uptake was approximately twofold greater in young than in old hearts (from 3-100 microM adenosine). However, although inhibition of adenosine deaminase and adenosine transport with 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and 10 microM S-(4-nitrobenzyl)-6-thioinosine increased adenosine sensitivity three- to fourfold, it failed to abolish the sensitivity difference in old (pEC(50) = 4.95 +/- 0.08) versus young (pEC(50) = 4.29 +/- 0.13) hearts. Data indicate that 1) age increases functional A(1) receptor sensitivity to adenosine without altering the sensitivity of the A(1) receptor itself, and 2) age impairs adenosine transport and/or catabolism, but this does not explain differing functional sensitivity to adenosine. This increased functional sensitivity to adenosine may have physiological significance in the older heart.

3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures.

DNA probes with conjugated minor groove binder (MGB) groups form extremely stable duplexes with single-stranded DNA targets, allowing shorter probes to be used for hybridization based assays. In this paper, sequence specificity of 3'-MGB probes was explored. In comparison with unmodified DNA, MGB probes had higher melting temperature (T(m)) and increased specificity, especially when a mismatch was in the MGB region of the duplex. To exploit these properties, fluorogenic MGB probes were prepared and investigated in the 5'-nuclease PCR assay (real-time PCR assay, TaqMan assay). A 12mer MGB probe had the same T(m)(65 degrees C) as a no-MGB 27mer probe. The fluorogenic MGB probes were more specific for single base mismatches and fluorescence quenching was more efficient, giving increased sensitivity. A/T rich duplexes were stabilized more than G/C rich duplexes, thereby leveling probe T(m)and simplifying design. In summary, MGB probes were more sequence specific than standard DNA probes, especially for single base mismatches at elevated hybridization temperatures.

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.

Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation.

Age-related changes in adenosine in rat coronary resistance vessels.

1. The vasodilator effects of adenosine receptor agonists, isoprenaline and histamine were examined in perfused heart preparations from young (4-6 weeks) and mature (12-20 weeks) rats. 2. Adenosine induced a biphasic concentration-dependent decrease in KCl (35 mM) raised coronary perfusion pressure in hearts from young and mature rats, suggesting the presence of both high- and low-affinity sites for adenosine receptors in the two age groups tested. In heart preparations from mature rats, vasodilator responses to adenosine were significantly reduced compared with responses observed in young rats. 3. Responses to 5'-N-ethylcarboxamidoadenosine (NECA) and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680) were reduced in preparations from mature rats, whereas the vasodilator actions of N6-cyclopentyladenosine (CPA) and N6-2-(4-aminophenyl)ethyladenosine (APNEA) did not change with age. 4. The results presented in this study suggest that several adenosine receptor subtypes mediate vasodilator responses in the coronary circulation of the rat and that a reduction in response to adenosine with age may be due to changes in the high-affinity receptor site.

Electromechanical Fredericks effects in nematic gels.

The solid nematic equivalent of the Fredericks transition is found to depend on a critical field rather than a critical voltage as in the classical case. This arises because director anchoring is principally to the solid rubbery matrix of the nematic gel rather than to the sample surfaces. Moreover, above the threshold field, we find a competition between quartic (soft) and conventional harmonic elasticity which dictates the director response. By including a small degree of initial director misorientation, the calculated field variation of optical anisotropy agrees well with the conoscopy measurements of Chang et al. [Phys. Rev. E 56, 595 (1997)] of the electro-optical response of nematic gels.

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.

Solution structure of a highly stable DNA duplex conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

Synthesis and reactivity of aryl nitrogen mustard-oligodeoxyribonucleotide conjugates.

A versatile method is described for preparing aryl nitrogen mustard-oligodeoxyribonucleotide (mustard-ODN) conjugates under anhydrous conditions. The chemistry uses DMSO soluble triethylammonium or tributylammonium salts of the ODNs. A G/A motif triplex forming ODN was chosen for study since it had been shown earlier to bind with high affinity and specificity to a duplex DNA target. A 5'-hexylamine derivative of this ODN was reacted with three different 2,3,5,6-tetrafluorophenyl ester derivatives of aryl nitrogen mustards which were designed to have different alkylation rates. An HPLC assay was used to determine reaction rates of these mustard-ODNs under various conditions. The reactivity of the mustard groups depended on chloride concentration and the presence of nucleophiles. Conjugation of mustards to G/A-containing ODNs decreased their aqueous stability. Hydrolysis and alkylation rates of these agents were consistent with reaction via an aziridinium intermediate. Rates of sequence specific alkylation within a triplex were determined by denaturing gel electrophoresis and shown to depend on inherent reactivity of the mustard group. The improved synthesis and chemical characterization of mustard-ODNs should facilitate their use as sequence specific alkylating agents and as probes for nucleic acid structure.

Genetic testing legislation: a review of the past two years.

The American Council of Life Insurance is a national trade association that represents the interests of legal reserve life insurance companies in legislative, regulatory and judicial matters at the federal, state and municipal levels of government and at the National Association of Insurance Commissioners. Its member companies hold more than 90 percent of the life insurance in force in the United States.

Modulation of Cm/T, G/A, and G/T triplex stability by conjugate groups in the presence and absence of KCl.

Apparent equilibrium association constants were determined by gel mobility shift analysis for triple strand formation between a duplex target containing a 21 base long A-rich homopurine run and several end-modified C(m)/T (pyrimidine motif; C(m) = 5-methylcytosine), G/A (purine motif), and G/T (purine-pyrimidine motif) triplex-forming oligonucleotides (TFOs). Incubations were carried out for 24 h at 37 degrees C in 20 mM HEPES, pH 7.2, 10 mM MgCl2, and 1 mM spermine. The purine motif triplex was the most stable (Ka = 6.2 x 10(8) M-1) even though the TFO self-associated as a linear duplex. Conjugation of a terminal hexanol or cholesterol group to the G/A-containing TFO reduced triplex stability by 1.6- or 13-fold, whereas an aminohexyl group or intercalating agent (acridine or psoralen) increased triplex stability by 1.3- or 13-fold. These end groups produced similar effects in C(m)/T and G/T triplexes, although the magnitude of the effect sometimes differed. Addition of 140 mM KCl to mimic physiological conditions decreased stability of the G/A triplex by 1900-fold, making it less stable than the C(m)/T triplex. The inhibitory effect of KCl on G/A triplex formation could be partially compensated for by conjugating the TFO to an intercalating agent (30-350-fold stabilization) or by adding the triplex selective intercalator coralyne (1000-fold stabilization). Although the G/T triplex responded similarly to these agents, the stability of the C(m)/T triplex was unaffected by the presence of coralyne and was only enhanced 1.4-2.8-fold when the TFO was linked to an intercalating agent. In physiological buffer supplemented with 40 microM coralyne, the G/A triplex (Ka = 3.0 x 10(8) M-1) was more stable than the C(m)/T and G/T triplexes by factors of 300 and 12, respectively.

Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.

Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization.

The ability of conjugated minor groove binding (MGB) residues to stabilize nucleic acid duplexes was investigated by synthesis of oligonucleotides bearing a tethered dihydropyrroloindole tripeptide (CDPI3). Duplexes bearing one or more of these conjugated MGBs were varied by base composition (AT- or GC-rich oligonucleotides), backbone modifications (phosphodiester DNA, 2'-O-methyl phosphodiester RNA or phosphorothioate DNA) and site of attachment of the MGB moiety (5'- or 3'-end of either duplex strand). Melting temperatures of the duplexes were determined. The conjugated CDPI3 residue enhanced the stability of virtually all duplexes studied. The extent of stabilization was backbone and sequence dependent and reached a maximum value of 40-49 degrees C for d(pT)8. d(pA)8. Duplexes with a phosphorothioate DNA backbone responded similarly on CDPI3 conjugation, although they were less stable than analogous phosphodiesters. Modest stabilization was obtained for duplexes with a 2'-O-methyl RNA backbone. The conjugated CDPI3 residue stabilized GC-rich DNA duplexes, albeit to a lesser extent than for AT-rich duplexes of the same length.

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.

Minor groove DNA alkylation directed by major groove triplex forming oligodeoxyribonucleotides.

We describe sequence-specific alkylation in the minor groove of double-stranded DNA by a hybridization-triggered reactive group conjugated to a triplex forming oligodeoxyribonucleotide (TFO) that binds in the major groove. The 24 nt TFOs (G/A motif) were designed to form triplexes with a homopurine tract within a 65 bp target duplex. They were conjugated to an N 5-methyl-cyclopropapyrroloindole (MCPI) residue, a structural analog of cyclopropapyrroloindole (CPI), the reactive subunit of the potent antibiotic CC-1065. These moieties react in the DNA minor groove, alkylating adenines at their N3 position. In order to optimize alkylation efficiency, linkers between the TFO and the MCPI were varied both in length and composition. Quantitative alkylation of target DNA was achieved when the dihydropyrroloindole (DPI) subunit of CC-1065 was incorporated between an octa(propylene phosphate) linker and MCPI. The required long linker traversed one strand of the target duplex from the major groove-bound TFO to deliver the reactive group to the minor groove. Alkylation was directed by relative positioning of the TFOs. Sites in the minor groove within 4-8 nt from the end of the TFO bearing the reactive group were selectively alkylated.

Direct, solid phase assembly of dihydropyrroloindole peptides with conjugated oligonucleotides.

A new controlled pore glass (CPG) support is described that allows for the direct synthesis of oligonucleotide derivatives carrying a minor groove binding (MGB) agent at the 3'-terminus. The MGB consisted of three repeating 1,2-dihydro-3H-pyrrolo[2,3-e]indole-7-carboxylate (DPI) subunits. The DPI trimer (DPI3) was prepared directly on the CPG support using repeated addition of the DPI subunit. The subunit was protected at the N-3-position with tert-butyloxycarbonyl residue and activated at the 7-carboxy residue by esterification with the 2,3,5,6-tetrafluorophenyl group. A linker, which provided the starting point for oligonucleotide synthesis, was introduced by reaction of the terminal N-3 with p-nitrophenyl 4-[bis(4-methoxyphenyl)phenylmethoxy]butyrate. When used as a support for oligonucleotide synthesis, this modified CPG gave the desired 3'-DPI3-octathymidylate [(dTp)8-DPI3] conjugate in good yield. This conjugate formed hyperstabilized complexes with complementary polyribo- (Tmax = 35 degrees C) and polydeoxyriboadenylic (Tmax = 69 degrees C) acids. In contrast to the N-carbamoyl derivative reported earlier by us, it demonstrated higher cooperativity of melting transitions.

Oligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agents.

A pair of complementary oligodeoxynucleotides (ODNs) uniformly substituted with 2-amino-adenine (A') in place of adenine and 2-thiothymine (T') in place of thymine did not hybridize to each other but did form very stable hybrids with unmodified complementary ODNs. These unusual properties were a consequence of the hydrogen-bonding properties of the two base analogs. Thermal denaturation studies of short duplexes which contained these bases demonstrated that the A'-T and A-T' doublets formed stable base pairs whereas the A'-T' doublet acted like a mismatch. Complementary ODNs substituted with these base analogs are referred to as SBC or selectively binding complementary ODNs. When used as a pair, these single-stranded ODNs invaded the ends of homologous duplexes and formed stable three-arm junctions under conditions where unmodified ODNs failed to give a product. SBC ODNs have a fundamental thermodynamic advantage in hybridizing to short segments of double-stranded nucleic acid and represent a new approach for the design of oligomeric probes and antisense agents. Many secondary structure features present in long single-stranded nucleic acids should be accessible to these reagents.