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M A Reynolds

Publications and source records attributed to M A Reynolds.

At least 37 records · Page 2Linked to original sources
PubMed1997

High-resolution NMR of an antisense DNA x RNA hybrid containing alternating chirally pure Rp methylphosphonates in the DNA backbone.

A high-resolution proton NMR study has been performed on a hybrid duplex formed by a methylphosphonate (MP) oligodeoxyribonucleotide (MPO) and its target oligoribonucleotide, d(T(MP)CC(MP)-TT(MP)AG(MP)CT(MP)CC(MP)TG) x r(CAGGAGCUAAGGA), where MP corresponds to positions of methylphosphonate linkages in the pure R(p) stereoconfiguration. MP-containing analogs of DNA are reported to be effective antisense agents capable of specifically inhibiting protein synthesis with the R(p) chiral MPOs exhibiting greater affinity for the target mRNA than their S(p) counterparts. Nearly complete proton resonance assignments of the hybrid duplex have been made using two-dimensional nuclear Overhauser effect (2D NOE) spectra, at three different mixing times, and double quantum-filtered COSY (2QF-COSY) spectra. The 2QF-COSY cross-peak patterns which are resolved have been analyzed qualitatively to suggest sugar conformations. Distance restraints have been obtained from the 2D NOE spectra of the duplex in D(2)O. These interproton distance restraints were determined using a complete relaxation matrix method to improve accuracy. Specifically, a new approach termed RANDMARDI has been utilized to calculate these distance restraints, accounting for spectral noise and errors in 2D NOE peak volume integration. The calculated interproton distances and sugar puckers have been analyzed to assess the solution conformation of the hybrid. The hybrid duplex appears to have an overall solution structure which is distinct from standard B- and A-forms, but the RNA strand exhibits features of the A-form. The absence of H1'-H2' cross-peaks in the 2QF-COSY spectrum indicates a C3'-endo type of conformation for ribose sugars in the RNA strand. The deoxyriboses in the antisense DNA strand exhibit a mixed behavior with almost equal scalar coupling constant values for H1'-H2' and H1'-H2" and a strong H3'-H4' 2QF-COSY peak pattern. Variations in calculated values of interproton distances and sixth-root R factor analysis of experimental intensities indicate that the hybrid duplex may have a DNA strand with significant conformational plasticity.

Base Composition↗
PubMed1997

Cellular uptake properties of a 2'-amino/2'-O-methyl-modified chimeric hammerhead ribozyme targeted to the epidermal growth factor receptor mRNA.

Catalytic RNA or ribozymes have important potential applications as molecular biological tools in the study of gene expression and as therapeutic inhibitors of disease-causing genes. Very little is known, however, about the cellular uptake mechanisms of exogenously delivered synthetic ribozymes. In this study, we have characterized the uptake properties of a synthetic, 2'-O-methyl-modified ribozyme containing U4/U7 amino groups within the catalytic core of the hammerhead motif. The cellular uptake of the internally [32P]-radiolabeled hammerhead ribozyme in U87-MG glioma cells was temperature, energy, and pH dependent and involved an active process that could be competed with cold ribozyme of the same chemistry and sequence, an all 2'-O-methyl-modified ribozyme of the same sequence, antisense PS-ODNs, and a variety of other polyanions (salmon sperm DNA, spermidine, dextran sulfate, and heparin). Subcellular distribution studies of fluorescently labeled ribozymes confirmed an extranuclear, punctate localization similar to that observed for an endosomal marker, dextran. Our study highlights that hammerhead ribozymes, despite exhibiting a defined secondary structure, enter cells by an endocytic mechanism that appears to be similar to that reported for a variety of antisense ODNs. These observations should facilitate the development of more efficient delivery systems.

Base Sequence↗
PubMed1997

Arbitrarily primed polymerase chain reaction fingerprinting and clonal analysis of oral Fusobacterium nucleatum isolates.

F. nucleatum is the most commonly isolated microorganism from subgingival plaque, but the role of this microorganism in periodontal diseases remains undefined. Arbitrarily primed polymerase chain reaction (AP-PCR) was evaluated as a method for fingerprinting F. nucleatum isolates and for use in clonal analysis. Pulsed field gel electrophoresis was used to further differentiate F. nucleatum isolates, with identical AP-PCR patterns. Extremely heterogeneous AP-PCR fingerprints were observed among the 98 F. nucleatum isolates, with 36 different genotypes observed with primer C1 and 30 different genotype detected with primer C2. Combining the results of the AP-PCR genotype analysis from C1 and C2 primer amplifications revealed that up to 7 different genotypes could be distinguished from isolates from the same oral cavity and that up to 4 different genotypes were observed within a single site. An intense amplicon at approximately 450 bp generated in AP-PCR amplification with primer C2 was associated with F. nucleatum subsp. nucleatum (ATCC 25586) and with 15 F. nucleatum isolates from diseased sites and 2 isolates from healthy sites. Pulsed field gel electrophoresis confirmed the AP-PCR genotypes and demonstrated increased discriminatory power over AP-PCR. The results indicated that AP-PCR and pulsed field gel electrophoresis provide a simple and sensitive means for differentiating oral F. nucleatum isolates and further demonstrate the heterogeneity of this species. These techniques may serve as useful tools in the clonal and epidemiological analysis of F. nucleatum isolates, which may help define the role of these microorganisms in periodontal diseases.

Bacterial Typing Techniques↗
PubMed1996

Synthesis and thermodynamics of oligonucleotides containing chirally pure R(P) methylphosphonate linkages.

Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.

Chromatography, High Pressure Liquid↗
PubMed1996

Interstrand complex formation of purine oligonucleotides and their nonionic analogs: the model system of d(AG)8 and its complement, d(CT)8.

We have investigated the role of purines in interstrand complex formation with regard to substitution of the negatively-charged, phosphodiester backbone by a nonionic, internucleoside linkage. Using the purine oligomer, d(AG)8, its methylphosphonate analog, d(AG)8, and the complementary pyrimidine oligomer, d(CT)8, as a model system, the stoichiometry, conformation, and stability of complexes formed at pH 8 were studied by spectroscopic and electrophoretic methods. When there is only one oligomer species in solution, d(AG)8 behaves as a single-stranded molecule. In contrast, the d(AG)8 oligomer readily forms an intermolecular self-complex, particularly in the presence of magnesium ion. Using either purine oligomer, duplexes can form with the d(CT)8 strand which differ in terms of their conformation and in the dependence of their thermal stability on sodium and magnesium ions. All studies show that a stable triplex forms with a 1:2 d(CT)8:d(AG)8 stoichiometry which does not require high concentrations of sodium or magnesium ions. Triplex formation between the d(CT)8 strand and two d(AG)8 strands was not observed. Native gel electrophoresis suggests that a 1:1:1 d(CT)8:d(AG)8:d(AG)8 complex may be formed. In regard to triplex formation, the advantage of the methylphosphonate backbone on the purine strand is clearly demonstrated.

Circular Dichroism↗
PubMed1996

Antisense oligonucleotide containing an internal, non-nucleotide-based linker promote site-specific cleavage of RNA.

We have designed and synthesized a series of novel antisense methylphosphonate oligonucleotide (MPO) cleaving agents that promote site-specific cleavage on a complementary RNA target. These MPOs contain a non- nucleotide-based linking moiety near the middle of the sequence in place of one of the nucleotide bases. The region surrounding the unpaired base on the RNA strand (i.e. the one directly opposite the non-nucleotide-linker) is sensitive to hydrolytic cleavage catalyzed by ethylenediamine hydrochloride. Furthermore, the regions of the RNA comprising hydrogen bonded domains are resistant to cleavage compared with single-stranded RNA alone. Several catalytic moieties capable of supporting acid/base hydrolysis were coupled to the non-nucleotide-based linker via simple aqueous coupling chemistries. When tethered to the MPO in this manner these moieties are shown to catalyze site-specific cleavage on the RNA target without any additional catalyst.

Base Sequence↗
PubMed1996

Influence of smoking on long-term clinical results of intrabony defects treated with regenerative therapy.

This retrospective study compares the short-term (1 year) and long-term (2 to 5 year) clinical results of regenerative therapy in clinical private practice using a bone allograft for the treatment of intrabony defects in smokers and non-smokers. A total of 110 intrabony lesions were treated with demineralized freeze-dried bone allograft (DFDBA) following thorough defect debridement and root preparation in 53 patients (15 cigarette smokers and 38 non-smokers). Assessments of clinical attachment level (CAL) and probing depth (PD) were recorded at pre-treatment, 1 year post-treatment, and 2 to 5 years post-treatment. At 1 year post-treatment, significant gains in mean CAL were maintained for both smokers (2.7 mm) and non-smokers (3.4 mm). Similarly, significant reductions in mean PD were observed for smokers (3.0 mm) and non-smokers (3.8 mm) at the 1-year follow-up. However, when comparing relative improvements in clinical measures, smokers were found to exhibit significantly poorer treatment results (i.e., sites exhibited less CAL gain) at 1 year and 2 to 5 years follow-up. Relative to pre-treatment scores, differences in improvements observed for CAL at the 1-year evaluation (29.2% for smokers and 42.5% for non-smokers) were sustained in the subgroup of patients at 2 to 5 years follow-up (31.3% for smokers and 41.8% for non-smokers). Similar but non-significant trends were observed for relative reductions in probing depth for smokers and non-smokers at 1 year (41.9% for smokers and 49.3% for non-smokers) and 2 to 5 years follow-up (43.9% for smokers and 48.3% for non-smokers) for the subgroup of patients followed beyond 1 year. These results suggest that smoking adversely affects treatment outcome, as measured by gains in clinical attachment levels of intrabony defects treated by regenerative therapy using DFDBA.

Adult↗
PubMed1996

Fate of demineralized freeze-dried bone allografts in human intrabony defects.

Demineralized freeze-dried bone (DFDBA) is the most widely used allograft in periodontics. Little information exists, however, on the fate of DFDBA matrix or on the effects of residual particles within grafted defects. The purpose of this study was to histologically examine the fate of DFDBA used for regeneration in intrabony defects. A secondary objective was to compare the amount of new attachment apparatus formation, including component tissues, in relation to the presence or absence of residual graft material. Histologic data were obtained from earlier studies in which intrabony defects grafted with DFDBA were removed at 6 months en bloc and submitted for histologic examination. Histologic sections (1,120) from 12 patients with 32 grafted defects revealed that 72% of the grafted defects exhibited residual DFDBA particles. When present, DFDBA appeared amalgamated within the new viable bone. Data from 5 patients with 14 grafted sites permitted a within-subject comparison of the amount of regeneration in relation to the presence or absence of residual graft material. Defects harboring residual graft particles exhibited significantly greater amounts of new attachment apparatus formation (1.72 mm vs. 0.20 mm), including new bone (2.33 mm vs. 0.23 mm), cementum (1.74 mm vs. 0.23 mm), and associated periodontal ligament than sites without evidence of graft matrix (P < or = 0.05). No apparent differences were seen in the nature of the new attachment apparatus or component tissues, other than in amount of formation. Inflammation and graft containment appear to be important factors influencing the fate of DFDBA and the regenerative response.

Alveolar Bone Loss↗
PubMed1996

Periodontal regeneration following surgical treatment.

Predictable and complete regeneration of lost periodontium remains an elusive goal, despite advances in surgical procedures and materials. Nevertheless, studies clearly demonstrate the potential for significant clinical improvements after regenerative therapy. Collectively, studies support the use of bone grafts and guided tissue regeneration (GTR) for the correction of intrabony and furcation defects. Results of several studies suggest the possibility of enhanced periodontal regeneration and enhanced stability following the use of combination techniques, such as GTR procedures with osseous grafts. Demineralized freeze-dried bone allograft (DFDBA) remains the most widely used allogeneic graft material in periodontics. Recent evidence suggests that substantial variations exist in the osteoinductive potentials of available DFDBA material. The predictability and extent of periodontal regeneration are associated with defect morphology, compliance, plaque control, inflammation, bacterial colonization, and smoking. Long-term (3- to 5-year) studies suggest that improvements following periodontal regeneration remain stable provided that patients comply with oral hygiene regimens and regular supportive periodontal treatment.

Alveolar Bone Loss↗
PubMed1995

Structure and dynamics of a DNA.RNA hybrid duplex with a chiral phosphorothioate moiety: NMR and molecular dynamics with conventional and time-averaged restraints.

The three-dimensional structure of two thiophosphate-modified DNA.RNA hybrid duplexes d(GCTATAApsTGG).r(CCAUUAUAGC), one with R-thiophosphate chirality and one with S-thiophosphate chirality, have been determined by restrained molecular dynamics simulations (rMD). As the two yielded almost identical results, a description of results can be presented in the singular. The conformational flexibility of this hybrid has been investigated by employing time-averaged constraints during the molecular dynamics simulations (MD-tar). A set of structural restraints, comprising 322 precise interproton distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities as well as J coupling constants obtained from quantitative simulations of DQF-COSY cross-peaks in deoxyriboses, was reported in our previous paper [González, C., Stec, W., Kobylanska, A., Hogrefe, R. I., Reynolds, M., & James, T. L. (1994) Biochemistry 33, 11062-11072]. Multiple conformations of the deoxyribose moieties were evident from the scalar coupling constant analysis. Accurate distance constraints, obtained from complete relaxation matrix analysis, yielded a time-averaged solution structure via conventional restrained molecular dynamics which is not compatible with the experimental J coupling constants (root-mean-square deviation in J value approximately 2 Hz). However, vicinal coupling constant information can be reproduced when time-averaged constraints are used during the molecular dynamics calculations instead of the conventional restraints (Jrms approximately 0.6 Hz). MD-tar simulations also improve the NMR R factors. This improvement is more evident in the DNA than in the RNA strand, where no indication of conformational flexibility had been obtained. Analysis of the MD-tar trajectories confirms that deoxyriboses undergo pucker transitions between the S and N domain, with the major conformer in the S domain. The ribose moieties in the RNA strand, however, remain in the N domain during the entire simulation. Conformations of deoxyriboses in the intermediate domain near O4'-endo are obtained when the average structure is calculated with conventional NMR restraints. Since these conformations cannot account for the experimental J coupling information, and they only appear in a very low population in the MD-tar ensemble, we conclude that intermediate E sugar puckers are artifacts produced by the attempt to fit all the structural constraints simultaneously when in reality more than one conformer is present. Most structural features of the duplex remain the same in the average structure and in the MD-tar ensemble, e.g., the minor groove width, exhibiting an intermediate value compared with those of canonical A- and B-like structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Is the timing of fracture fixation important for the patient with multiple trauma?

OBJECTIVE: The effect of timing of femur fracture fixation for patients with multiple trauma was studied to determine the effect of operative timing on eventual outcome. METHODS: The relationship between timing of intramedullary rod (IMR) placement, degree of injury, and pulmonary complications was studied in 424 consecutive patients. The authors focused on 105 patients undergoing IMR placement with an Injury Severity score (ISS) of greater than or equal to 18. The effects of timing of IMR placement on various pulmonary complications, organ failure, intensive care unit (ICU) admission, and ventilatory assistance were studied for various time intervals. RESULTS: Of the 424 patients, pulmonary complications increased slightly in the more seriously injured group (ISS > 18) but were not influenced by the timing of IMR placement. Of the 105 patients undergoing IMR placement with an ISS > or = 18, only 2 patients died. Both patients had an IMR placed in less than 24 hours and died later of head injury and delayed hemorrhage. The incidence of organ failure, number of ventilator days, and length of ICU stay did not differ between the groups based on timing of fracture fixation. The incidence of severe head injuries was higher in the group undergoing delayed IMR placement (> 48 hours). CONCLUSIONS: Modest delays in IMR placement did not adversely affect patient outcome. Pulmonary complications were related to the severity of injury rather than to timing of fracture fixation. In a well-integrated trauma system, clinical judgment regarding the timing of IMR placement was the most important determinant of outcome. Delays that were made to stabilize the patient, treat associated injuries, and plan orthopedic reconstruction did not adversely affect patient outcome.

Adult↗
PubMed1995

Transpyloric passage of feeding tubes in patients with head injuries does not decrease complications.

Early enteral nutrition is reported to improve outcome of patients with severe closed head injuries (CHI). The efficacy and safety of nasoenteric tube (NET) feeds, however, has been questioned; the risk of aspiration is the major concern. Our purpose was to determine the rate of transpyloric migration, the efficacy of adjunctive measures to promote passage, and the effect on pulmonary complications. Seventy-four consecutive patients with moderate to severe CHI received enteral nutrition. Glasgow Coma Scale (GSC) score was 5.2 on admission and 6.9 at 48 hours. NETs were placed an average of 5.6 days after admission; an average of three abdominal films per patient were used to assess tube position. No patients had endoscopic NET placement during this period. Ten patients required fluoroscopic placement after failure to pass spontaneously by 5 days. Overall, transpyloric passage was achieved in 32 patients (43%), whereas 42 (57%) remained intragastric. There were no differences between the postpyloric and intragastric groups in days to full feeding (5 vs. 7 days), ventilator days (11.9 vs. 12.5), intensive care unit length of stay (15.5 vs. 15.1), or incidence of pneumonia (81 vs. 69%) or aspiration (6 vs 7%). Sixty-two patients (83%) were transferred to extended care facilities and 50 (68%) were still receiving NET feedings. Spontaneous transpyloric passage of NET occurred in less than one-half of patients with severe CHI. The routine use of adjunctive measures to promote transpyloric passage was not particularly successful, had no obvious benefit, and therefore may not be necessary.

Adolescent↗
PubMed1995

The role of bile acid secretion in the hepatic response to operation and infection.

Hepatic function is altered in many surgery-related diseases. Bile acid secretion is the major determinant of bile formation and an important indicator of overall hepatic function. To investigate the cause of intrahepatic cholestasis, which is frequently associated with sepsis, we studied the effects of cecal ligation and puncture (CLP) and surgical stress on bile acid secretion and composition. CLP or a sham operation was performed on 20 male Sprague-Dawley rats. Bile was collected from each rat by cannulation of the common bile duct for 10-minute intervals, at 5 and 20 hours after the initial procedure. Bile acid analysis was then performed by high performance liquid chromatography (HPLC). In CLP rats, there was a significant (P < 0.05) cholestatic effect. Bile flow was reduced to 70 +/- 13 per cent at 5 hours, and to 55 +/- 16 per cent at 20 hours (per cent of the sham mean value). In the sham-operated rats, there was a significant choleresis at 20 hours. Bile flow was increased to 146 +/- 13 per cent; bile acid secretion to 245 +/- 24 per cent; and total bile acid concentration to 175 +/- 19 per cent of the sham 5-hour value (P < 0.05). This increased secretion was significantly greater in the metabolites of chenodeoxycholate. However, these surgical stress-associated changes in bile acid secretion and composition did not occur in CLP rats. These findings are consistent with surgical stress-induced induction of 7 alpha-hydroxylase, which was not found in the septic animals. These observations may provide useful insights into the early stages of the pathogenesis of sepsis-related hepatic dysfunction and failure.

Animals↗
PubMed1994

Triple-strand-forming methylphosphonate oligodeoxynucleotides targeted to mRNA efficiently block protein synthesis.

Antisense oligonucleotides are ordinarily targeted to mRNA by double-stranded (Watson-Crick) base recognition but are seldom targeted by triple-stranded recognition. We report that certain all-purine methylphosphonate oligodeoxyribonucleotides (MPOs) form stable triple-stranded complexes with complementary (all-pyrimidine) RNA targets. Modified chloramphenicol acetyltransferase mRNA targets were prepared with complementary all-pyrimidine inserts (18-20 bp) located immediately 3' of the initiation codon. These modified chloramphenicol acetyltransferase mRNAs were used together with internal control (nontarget) mRNAs in a cell-free translation-arrest assay. Our data show that triple-strand-forming MPOs specifically inhibit protein synthesis in a concentration-dependent manner (> 90% at 1 microM). In addition, these MPOs specifically block reverse transcription in the region of their complementary polypyrimidine target sites.

Base Sequence↗
PubMed1993

Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.

Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-O6-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 mumole scales. These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.

Ammonia↗
PubMed1993

An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides.

The methylphosphonate oligonucleotide synthesis methods described here give the desired products in good yield. Superior amounts of product are achieved by modifying both the DNA synthesis program and the reagent to compensate for the unstable methylphosphonite intermediate. Deprotection conditions have also been altered to maximize the recovery of oligonucleotide from DNA synthesis supports and to minimize the amount of base modification. Mass-spectrometry analysis of our oligonucleotides has verified their purity and confirmed the absence of modified bases. When compared to standard DNA synthesis methods, this procedure uses only about one-third the usual amount of monomer. Using these procedures, it should be possible to synthesize reliably methylphosphonate oligonucleotides at 1- and 15-mumol scales.

Base Sequence↗