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B Rayner

Publications and source records attributed to B Rayner.

70 records · Page 4Linked to original sources
PubMed1987

Structure and conformation of the duplex consensus 5'-splice site d[(CpApGpGpTpApApGpT).(ApCpTpTpApCpCpTpG)] deduced from high field 1H-NMR of the non-exchangeable and imino protons.

The complementary consensus donor exon intron junction d(ApCpTpTpApCpCpTpG) has been synthesized by a solid phase procedure. The non-exchangeable proton assignments were obtained using one- and two-dimensional NMR techniques including NOE, COSY, NOESY and 1H-1H-INADEQUATE. The non self-complementary nonamer exists as a random coil form in aqueous buffer at 21 degrees C as evidenced by the temperature variable 1H-NMR and NOE measurements. The nonamer was annealed to the primary consensus donor junction d(CpApGpGpTpApApGpT) and confirmation of complete annealing was obtained by detection and assignment of base pair imino protons in D2O/H2O mixtures. Application of one- and two-dimensional NMR techniques permitted the complete assignment of all the non-exchangeable protons in the duplex nonamer. These data, together with determination of vicinal coupling constants in the individual deoxyribose moieties, permits the following conclusions on the structure and conformation of the consensus donor junction: (i) it exists in aqueous solution in a conformation that belongs to the B family (ii) the sugar-base orientations are anti (iii) the deoxyribose units exist predominantly in the S conformation (2'-endo-3'-exo) (iv) the contiguous A.T base pairs d[T(5)-A(6)-A(7)].d[T(12)-T(13)-A(14)], two positions removed downstream from the splice site (5'-CAG decreases GTAAGT-3'), are uniquely propeller twisted. The propeller twisting occurs in the region in which there is partial complementarity with the branch site splice signal TACTAAC. The cross-correlation rates were used to derive the interproton distances between adjacent AH2 protons of 4.00 A in the T(5)-A(6).T(13)-A(14) step and of 3.87 A in the A(6)-A(7).T(12)-T(13) step. This structural and conformational feature if carried over into the primary RNA transcripts may serve as a recognition signal for this critical site in the genome.

DNA↗
PubMed1986

alpha-DNA. I. Synthesis, characterization by high field 1H-NMR, and base-pairing properties of the unnatural hexadeoxyribonucleotide alpha-[d(CpCpTpTpCpC)] with its complement beta-[d(GpGpApApGpG)].

The novel deoxyribonucleotide alpha-[d(CpCpTpTpCpC)] and its complement beta-[d(GpGpApApGpG)] were synthesized by the phosphotriester method. 1H-NMR-NOE examination of the alpha-hexamer revealed that the cytosine and thymine bases appear to adopt anti conformations in this strand. In addition the deoxyribose of the thymidine moieties may adopt average conformations approximating to C3'-endo while the cytidine furanose groups are close to C2'-endo conformations. Both hyperchromicity in thermal melting and detection of base paired imino protons in 1H-NMR studies in H2O provide evidence for the annealing of alpha-d[CCTTCC] with its complement beta-d[GGAAGG] in potassium phosphate buffer pH 7.1 containing 10 mM magnesium chloride. Under these conditions thermal melting begins at 38 degrees C and its complete at approximately 45 degrees C. NOE experiments do not permit a decision on the polarity of annealing (predicted to be parallel) for this particular pair of sequences.

Chemical Phenomena↗
PubMed1986

Preparation of a short synthetic apurinic oligonucleotide.

The synthesis of the model apurinic oligonucleotide Tp(AP)pT is reported. Furthermore during the course of purification of this compound we have shown that the adduct formed upon reaction with methoxyamine has a Schiff base structure.

Apurinic Acid↗
PubMed1986

Structure and conformation of the duplex consensus acceptor exon:intron junction d[(CpTpApCpApGpGpT). (ApCpCpTpGpTpApG)] deduced from high-field 1H-NMR of non-exchangeable and imino protons.

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and 1H-1H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D2O:H2O mixtures. Assignment of the non-exchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5' regions exist predominantly in the S form (2'endo-3'exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2'exo-3'endo). The contiguous bases A5-G6 (adjacent to the junction) and A15-G16 are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.

Base Composition↗
PubMed1986

1H and 31P-NMR assignments of the non-exchangeable protons of the consensus acceptor exon:intron junction d(CpTpApCpApGpGpT).

The consensus acceptor exon:intron junction d(CpTpApCpApGpGpT) has been synthesized by a modified phosphotriester method. The non-self complementary octamer exists in the single strand form in aqueous buffer at 20 degrees C as evidenced by temperature variable 1H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and the double quantum technique 1H-1H-INADEQUATE as well as inversion recovery T1 experiments. The new technique of 31P-1H shift correlation is particularly valuable in removing certain ambiguities in the sugar proton assignments. Characteristic chemical shifts for the base protons which are determined by their immediate molecular environments are also useful in assignments. The consensus acceptor exon:intron junction adopts a random coil conformation in solution under the experimental conditions employed.

Exons↗
PubMed1985

1H-NMR assignments of the non-exchangeable protons of the consensus donor exon:intron junctioń d(CpApGpGpTpApApGpT).

The consensus donor exon:intron junction d(CpApGpGpTpApApGpT) has been synthesized by a modified phosphotriester method. The non-self-complementary nonamer has, in principle, only two G,C or four A,T points of self-recognition. The inference that it exists in the single strand form at 20 degrees C was confirmed by temperature variable 1H-NMR and NOE measurements. The proton assignments were secured using two-dimensional COSY which provided intra-nucleotide correlations, then NOE difference measurements as well as inversion recovery T1 experiments. Systematic procedures were developed for the assignment of the individual bases and their component protons based on the effects of molecular environment on chemical shifts. These latter procedures should be useful for the assignment of other random-coil single strand oligodeoxyribonucleotides.

Base Sequence↗
PubMed1984

Synthesis, complete 1H assignments and conformations of the self-complementary hexadeoxyribonucleotide [d(CpGpApTpCpG)]2 and its fragments by high field NMR.

The two deoxyribonucleotides [d(CpGpApTpCpG)]2 and [d(CpGpCpG)]2 were synthesized by the phosphotriester method. Their duplex form under the conditions of the 1H-nmr experiments was proven by end 32P labeling with T4 polynucleotide kinase followed by butt end joining employing the absolute specificity of T4 ligase for double stranded DNA and analysis using gel electrophoresis and autoradiography. Complete nmr assignment of the 1H chemical shifts and coupling constants was achieved. The assignments were secured using sequential decoupling, NOE difference measurements, and two-dimensional COSY and SECSY experiments. Spectrum simulation confirmed the experimental values of chemical shifts and coupling constants. The techniques for the assignment outlined together with 31P and 2-D heteronuclear shift correlation permit an approach to a systematic analysis of more complex single-strand and duplex oligodeoxyribonucleotides.

Base Sequence↗
PubMed1983

Use of inter-proton nuclear Overhauser effects to assign the nuclear magnetic resonance spectra of oligodeoxynucleotide and hybrid duplexes in aqueous solution.

Inter-proton nuclear Overhauser enhancements (NOEs) have been used to assign the aromatic, anomeric and 2' resonances in the 1H nuclear magnetic resonance spectrum of the duplex formed between d(T-C-A-C-A-T) and d(A-T-G-T-G-A). The same techniques have been applied to assignments in the hybrid duplex formed by d(T-C-A-C-A-T) with r(A-U-G-U-G-A). Comparison of intra-residue with inter-residue NOEs yields structural information which suggests that the conformations of both duplexes are similar. The NOEs are consistent with inter-proton distances measured from models of B-form DNA. Circular dichroic results confirm these deductions.

Chemical Phenomena↗
PubMedDate not supplied

Selective mRNA degradation by antisense oligonucleotide-2,5A chimeras: involvement of RNase H and RNase L.

Antisense oligonucleotides (ON) allow the specific control of gene expression and phosphorothioate derivatives are currently being evaluated for possible clinical applications. Numerous second generation ON analogues with improved pharmacological properties have been described. Most of them, however, do not recruit RNase H, which is known to increase ON potency by eliciting the specific degradation of the target RNA. Silverman, Torrence and colleagues have conjugated 2,5A to natural antisense ON and demonstrated the preferential cleavage of a target RNA in cell-free and intact cell experiments. We have established for the first time that RNase H-incompetent ON, viz. alpha-anomeric ON analogues, can be converted into sequence-specific nucleases upon conjugation to 2,5A. The use of alpha-ON- and beta-ON-2,5A chimeras has allowed us to delineate the part played by RNase H and RNase L in target RNA degradation and translation arrest. Finally, the present studies have revealed limitations which are encountered in the choice of a suitable target for such ON-2,5A chimeras.

Animals↗
PubMedDate not supplied

Esterase-triggered fluorescence of fluorogenic oligonucleotides.

In the prooligonucleotide approach, a step of activation by cellular esterases is necessary for the removal of internucleoside phosphate masking groups and subsequent intracellular delivery of active antisense oligonucleotides. The efficacy of this approach implies that prooligonucleotides, once they are taken up by cells, are demasked by esterases during their course to their nucleic acid targets. In this regard, a method for labeling oligomers with esterase-activable fluorogenic tag was designed. The two phenolic functions of carboxyfluorescein were protected by pivaloyl groups, yielding a nonfluorescent lactone which was further activated as a N-hydroxysuccinimide ester. Two nuclease-resistant phosphorothioate 18-mer and methylphosphonate 19-mer oligodeoxynucleosides were attached to this biprotected fluorescein derivative via an amino linker at the 5'-end of the oligomers. The two conjugates were assayed for their carboxyesterase substrate ability in different biological media. In the presence of purified esterases or when incubated in serum or cell extracts, both oligonucleotide conjugates became fluorescent. In addition, the phosphorothioate oligoconjugate was microinjected into the cytoplasm of human fibroblasts, and a fast cytoplasmic release of fluorescence was observed with a rapid translocation of the fluorescent oligomer into the nucleus.

Ammonia↗
PubMedDate not supplied

Prooligonucleotides exhibit less serum-protein binding than phosphodiester and phosphorothioate oligonucleotides.

The protein-binding properties of dodecathymidine derivatives (prooligos) bearing either methyl- or tert-butyl-S-acylthioethyl (Me- or tBuSATE) protecting groups were evaluated. The dissociation constants (Kd) were estimated for complexes of prooligos with serum blood proteins and lactoferrin using prooligos to compete the binding of the radiolabeled, alkylating probe oligonucleotide CIRp(T)12 with the proteins. tBuSATE and MeSATE prooligos have decreased affinity of binding with serum proteins and lactoferrin compared with their parent oligos. These data suggest that prooligos should cause less side effects which combined with their stability to nucleases and enhanced permeability into cells make them potentially useful for design of therapeutics.

Animals↗
PubMedDate not supplied

Potential renoprotective effects of the angiotensin receptor blocker eprosartan: a review of preliminary renal studies.

The importance of the renin-angiotensin-aldosterone system (RAAS) in the pathogenesis of hypertension and in renal disease, particularly in patients with diabetes, has become increasingly evident. Pharmacological blockade of the RAAS offers potential for the therapeutic management of these pathologies. Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) receptor blockers have been shown to exhibit effectiveness in the treatment of hypertension. AII receptor blockers have a renal protective effect owing to their ability to reduce systemic blood and intraglomerular pressures. Eprosartan is a chemically distinct AII blocker, which displays a dual mode of action whereby it blocks both pre- and postsynaptic AT(1) receptors, potentially benefiting patients with hypertension and renal disease. In addition, evidence suggests that eprosartan is well tolerated by both healthy subjects and patients with varying degree of renal impairment, such that the dose does not need to be modified in patients with mild to moderate renal impairment. Results from preliminary studies demonstrates that eprosartan doses will below those required for blood pressure control have a pronounced effect on the kidney and do not compromise renal autoregulatory mechanisms. Therefore, eprosartan may have a benefit in the prevention or delay of renal damage in hypertensive patients with renal impairment, although this remains to be determined in a clinical setting.

Acrylates↗
PubMedDate not supplied

The prevalence of microalbuminuria and ECG left ventricular hypertrophy in hypertensive patients in private practices in South Africa.

INTRODUCTION: In South Africa a cluster of cardiovascular diseases accounts for 17% of all deaths. It is also predicted that South Africa will face an epidemic of chronic kidney disease due to obesity, type 2 diabetes and hypertension. It is important to estimate the burden of underlying cardiovascular and renal disease in South Africa. OBJECTIVES: The primary objective of the study was to establish the prevalence of left ventricular hypertrophy by ECG criteria, and micro- and macroalbuminuria in mild, moderate and severe hypertensive groups. The secondary objective was to establish the prevalence of left ventricular hypertrophy and micro- and macroalbuminuria in hypertensive patients with underlying type 2 diabetes. METHODS: Patients > or = 35 years with essential hypertension with or without type 2 diabetes were recruited from 100 general practices throughout South Africa. BP, weight, height, waist circumference and urinary albumin/creatinine ratio were measured, and an ECG was performed. The Sokolow-Lyon and Cornell criteria were used for estimation of left ventricular hypertrophy. An overall prevalence rate was determined using weights from the severity category distribution inside the study population. RESULTS: One thousand and ninety-one patients were available for analysis. There were 530 (48.5%) males and 561 (51.5%) females, 691 (63.3%) whites, 162 (14.8%) blacks, 150 (13.7%) Asians and 88 (8.1%) Coloureds. Of the patients, 10.9% had newly diagnosed hypertension, 20% had type 2 diabetes, and 38.1% mild, 32.1% moderate and 30.6% severe hypertension. The prevalence of left ventricular hypertrophy in the sample weighted to the hypertensive population over the age of 35 years with access to medical aids was 18.9%. Relative to white ethnicity, black ethnicity (OR 2, p = 0.03), and relative to mild hypertension, moderate (OR 2.2, p = 0.05) and severe hypertension (OR 4.9, p < 0.0001) were independent predictors of left ventricular hypertrophy. The overall prevalence of micro- and macroalbuminuria in the weighted sample was 21.3 and 4.1%, respectively. In the diabetics the prevalence of microalbuminuria was 32.3% and macroalbuminuria 10.4%, respectively. The independent predictors of microalbuminuria or worse were severe hypertension (OR 2.9, p < 0.0001), type 2 diabetes (OR 2.5, p < 0.002), and Asian ethnic group (OR 2, p = 0.02). CONCLUSIONS: The study, based on a convenience sample of hypertensives from private practices in South Africa showed that the prevalence of left ventricular hypertrophy and microalbuminuria or worse was 18.9 and 25.4%, respectively.

Adult↗