Biomedical subjects

P D Ellis

Publications and source records attributed to P D Ellis.

At least 37 records · Page 2Linked to original sources

PubMed1995

The effects of sequence context on base dynamics at TpA steps in DNA studied by NMR.

Base dynamics, heretofore observed only at TpA steps in DNA, were investigated as a function of sequence context by NMR spectroscopy. The large amplitude conformational dynamics have been previously observed in TnAn segments where n > or = 2. In order to determine whether the dynamic characteristics occur in more general sequence contexts, we examined four self-complementary DNA sequences, [d(CTTTA-NATNTAAAG)2] (where N = A, C, T, G and N = complement of N). The anomalous broadening of the TpA adenine H2 resonance which is indicative of large amplitude base motion was observed in all nine unique four nucleotide contexts. Furthermore, all the adenine H2 resonances experienced a linewidth maximum as a function of temperature, which is a characteristic of the dynamic process. Interestingly, the temperature of the linewidth maximum varied with sequence indicating that the thermodynamics of TpA base dynamics are also sequence dependent. In one example, neither a T preceding nor an A trailing the TpA step was required for base dynamics. These results show that base dynamics, heretofore observed in only a few isolated sequences, occurs at all TpA steps which are either preceded or followed by a thymine or adenine, respectively, and may be characteristic of all TpA steps in DNA notwithstanding sequence context.

Adenine↗

PubMed1995

Biomechanics of snoring.

A large proportion of the population either snores or suffers the snoring of others. Recent advances with the use of fibre-optic endoscopes have enabled surgeons to observe the inside of the pharynx while a patient is asleep and snoring. In this article we look at the underlying structure of the upper airway and explain, with the use of simple mechanical models, the aerodynamic events occurring inside the upper airway during snoring.

Adult↗

PubMed1995

Observation of the mechanism of snoring using sleep nasendoscopy.

We have performed sleep nasendoscopy on 54 adult snorers in whom obstructive sleep apnoea had been excluded by an overnight sleep study. The purpose of the study was to identify the site or sites of noise production in each case. This was successfully achieved in 50 of the 54 and 70% showed palatal flutter snoring only. In a further 20%, palatal flutter snoring was combined with evidence of noise generation at another site. The second site was supraglottic in 10%, tonsillar in 8% and tongue base in 2%. The tongue base was also the sole site of noise generation in 8% and the epiglottis was the sole site in 2%. This study suggests that sleep nasendoscopy can identify different mechanisms of snoring in individual patients. This information is likely to be of use in formulating a logical surgical treatment plan.

Adult↗

PubMed1994

Phosphorus-31 nuclear magnetic resonance studies of complexes of thymidylate synthase.

The interactions of thymidylate synthase (TS) with deoxyuridylate (dUMP), deoxythymidylate (dTMP) and 5-fluorodeoxyuridylate (FdUMP) were examined by 31P-NMR. Single 31P resonances appeared at 3.3 ppm, 3.2 ppm and 3.0 ppm from the standard, 85% phosphoric acid, for unbound dUMP, dTMP, and FdUMP, respectively. Incubation of the enzyme with either dUMP or dTMP, alone, resulted in new resonances at 3.9 and 3.6 ppm, respectively, which were assigned to noncovalent complexes with the enzyme. The same experiment employing FdUMP as the ligand gave two new resonances appearing at 3.6 and 4.6 ppm, which were attributed to noncovalent and covalent binary complexes, respectively. When the cofactor, CH2H4 folate, was present in the solution with enzyme and FdUMP, a new resonance appeared at 5.1 ppm, corresponding to the covalent inhibitory ternary complex. The ternary complex comprised of the enzyme, dUMP and the quinazoline folate CB 3731 produced a resonance at 5.0 ppm at the expense of the resonance due to the enzyme-dUMP binary complex at 3.9 ppm. Similarly, the ternary complex consisting of TS with dTMP and CB 3731 showed a deshielding of the resonance at 3.6 ppm by 0.8 ppm. A maximum binding of 1.5 nucleotides per enzyme dimer was found for dUMP and dTMP in both the presence and the absence of the quinazoline folate. The deshielding observed was attributed to changes in the interaction of the phosphate group with the nearby residues of the active site of the enzyme.

Deoxyuracil Nucleotides↗

PubMed1994

Snoring.

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Humans↗

PubMed1994

Laser palatoplasty for snoring due to palatal flutter: a further report.

The purpose of this paper is to report the longer term results of laser palatoplasty which 1 developed in the early part of 1992. The early results of this procedure were published in the Annals of the Royal College of Surgeons of England and showed that 14 of 16 patients had good snoring control while two did not. Since then a further three patients have relapsed so that five patients out of 16 (34%) must be considered failures. Only a larger series and longer follow-up can determine whether or not laser palatoplasty has a place in the management of snoring.

Adult↗

PubMed1993

Fluorine-19 nuclear magnetic resonance studies of binary and ternary complexes of thymidylate synthase utilizing a fluorine-labeled folate analogue.

Traditional fluorine-19 nuclear magnetic resonance (19F NMR) studies of thymidylate synthase (TS) have utilized the fluorine substituent of 5-fluorodeoxyuridine 5'-monophosphate (FdUMP), a mechanism-based inhibitor of the enzyme, in complexes with various folate and folate analogues in order to establish a paradigm for the formation of binary and ternary complexes. In order to extent and confirm this paradigm, complexes of thymidylate synthase (TS) and the N-10-(fluoroethyl)quinazolinylfolate analogue CB3731 with either deoxyuridine 5'-monophosphate (dUMP), deoxythymidine 5'-monophosphate (dTMP), or FdUMP were examined from the perspective of the folate analogue using 19F NMR. The spectrum of the free folate analogue gave rise to a multiplet centered at -57.0 ppm, which was broadened by approximately 50% upon incubation with the enzyme. The use of FdUMP with CB3731 afforded us the opportunity to compare the spectrum obtained for the folate with that of the nucleotide. This comparison led to the assignment of the resonance at -53.5 ppm as representing the noncovalent TS:FdUMP:CB3731 ternary complex, while a new resonance at -52.0 ppm corresponded to the species in which the nucleotide is covalently attached to the enzyme and the folate is noncovalently associated. Ternary complexes consisting of TS, CB3731, and either dUMP or dTMP displayed a resonance at -53.5 ppm which represented the noncovalent TS-nucleotide adduct. None of the TS:nucleotide:CB3731 ternary complexes, however, was stable to native polyacrylamide gel electrophoresis.

Fluorine↗

PubMed1993

Surgical relief of snoring due to palatal flutter: a preliminary report.

We describe our experience in the surgical treatment of 32 patients with severe snoring. In the first 16 patients we used the standard surgical procedure of uvulopalatopharyngoplasty (UPP) but found this unsatisfactory because of its radical nature, complications and uncertain outcome. We therefore investigated the mechanics of snoring in the laboratory as a help in devising a more effective operation. These studies have shown that there are several methods by which snoring can be generated but that palatal flutter is probably the most important. The dominant parameters in the generation of flutter of the palate are its length and stiffness. Any removal of tissue to shorten the palate as in UPP inevitably risks impairing its function, so we chose the stiffening alternative. Using the laser, a central longitudinal strip of mucosa was removed from the surface of the soft palate which healed by fibrosis, producing the required stiffening. The early results of this new procedure show that snoring has been eliminated or greatly reduced in 14 of 16 patients and that apart from local pain there have been no complications.

Adult↗

PubMed1992

The relief of snoring by nasal surgery.

Patients with nasal obstruction may also complain of snoring. It is uncertain whether surgery which relieves the nasal obstruction will also relieve the snoring. We have reviewed 126 patients who complained of both nasal obstruction and snoring and who underwent nasal surgery. Snoring was completely relieved in 39 patients (31%), was less loud in a further 72 patients (57%), unchanged in 11 and louder in 4. It occurred on fewer nights post-operatively in 61, on the same number in 24 and more frequently in 2. Patients who had nasal polypectomy as part of their nasal surgery obtained the greatest snoring relief. This study suggests that when snoring and nasal obstruction coexist nasal surgery should be considered as the first line of surgical treatment.

Humans↗

PubMed1989

113Cd NMR of Cd2+-substituted carboxypeptidase. Support for a hexa-coordinate metal ion in the presence of inhibitors.

The liquid-state 113Cd NMR data of carboxypeptidase A in the presence and absence of inhibitors obtained by Gettins (Gettins, P. (1986) J. Biol. Chem. 261, 15513-15518) are analyzed in terms of whether the inhibitors displace water from Cd2+ upon binding to the protein. This question is addressed by applying the single crystal data and the methods introduced by Honkonen and Ellis (Honkonen, R. S., and Ellis, P. D. (1984) J. Am. Chem. Soc. 106, 5488-5497). Calculations based upon these data demonstrate that displacement of water by a carboxyl group should lead to significant shielding of a 113Cd resonance by approximately 100 ppm. Since the observed 113Cd chemical shifts for carboxypeptidase A are modest and deshielding (12-17 ppm), it is argued that the chemical shifts imply that water is not displaced from the Cd2+ center upon binding of inhibitors to carboxypeptidase A. Rather, the Cd2+ ion increases its coordination number from five to six upon binding of the inhibitor.

Cadmium↗

PubMed1989

19F nuclear magnetic resonance as a probe of the spatial relationship between the heme iron of cytochrome P-450 and its substrate.

The distance between the heme iron of ferrous cytochrome P-450-CAM and a fluorine label attached to the 9-methyl carbon of its substrate, (1R)-(+)-camphor, has been determined using 19F NMR. This investigation uses the Solomon-Bloembergen equation to measure the distance from a paramagnetic heme iron to a fluorine probe incorporated into a substrate that is not in fast exchange. The structural identity of the substrate analogue, 9-fluorocamphor, has been established using one- and two-dimensional NMR methods and mass spectrometry. The relaxation rate of 9-fluorocamphor bound to high-spin paramagnetic ferrous P-450-CAM has been studied at 188, 282, and 376 MHz, and the correlation time has been directly determined from the frequency dependence of the relaxation rate. When the substrate analogue was bound to the low-spin diamagnetic ferrous-CO derivative of the enzyme, the relaxation rate was found to be 100 times slower and was therefore neglected in the distance calculation. The relaxation data for the paramagnetic system and the correlation time have been used to calculate a distance of 3.8 A between the heme iron and the C-9 fluoride. A fit of the distance and the chemical shift data to the pseudocontact shift equation predicts an angle of approximately 52 degrees between the heme normal and the Fe-F vector. The solution state Fe-F distance is somewhat shorter and the angle between the heme normal and the Fe-F vector slightly larger for the substrate-bound ferrous enzyme reported herein than the analogous values for the substrate-bound ferric enzyme determined in the solid state by x-ray crystallography. These differences may reflect a structural change at the substrate-binding site upon reduction of the iron.

Camphor↗

PubMed1988

Cadmium-substituted skeletal troponin C metal binding investigations and sequence assignment of the cadmium-113 resonances.

The binding of cadmium to the calcium binding subunit of skeletal troponin (STnC) has been reinvestigated using direct binding methods and fluorescent derivatives. These data provide straightforward explanations of the observed titration behavior in the 113Cd NMR (Ellis, P.D., Strang, P., and Potter, J.D. (1984) J. Biol. Chem. 259, 10348-10356). Further, fluorescent derivatives of skeletal troponin C provide an excellent means of establishing a sequence assignment for the resonances observed in the 113Cd NMR. The results of these experiments demonstrate that sites I and II, the Ca2+ regulatory sites, can be assigned to resonances at -108.5 and -101.5 ppm, respectively. Sites III and IV, the structural sites, are assigned to resonances -112.8 and -106.8 ppm, respectively. These data are discussed in terms of recent structural findings and speculations.

Animals↗

PubMed1988

Synaptic protein tyrosine kinase: partial characterization and identification of endogenous substrates.

The subcellular distribution of protein tyrosine kinase in rat forebrain was determined using [Val5]-angiotensin II as exogenous substrate. Enzyme activity was present in each of the fractions analyzed and was enriched in synaptic membranes (SMs) and the synaptosomal soluble fraction (2.2- and 2.5-fold over the homogenate, respectively). SMs also phosphorylated polyglutamyltyrosine (pGT; molar ratio of 4:1), the Vmax for angiotensin and pGT phosphorylation being 26.3 +/- 1.6 and 142 +/- 4 pmol/min/mg, respectively. Extraction of SMs with several different detergents resulted in enhanced enzyme activity and the solubilization of 33-37% of the angiotensin and 43-70% of the pGT-phosphorylating activity. Isolated postsynaptic densities (PSDs) contained tyrosine kinase and phosphorylated angiotensin and pGT. The Vmax values for angiotensin and pGT phosphorylation by PSDs were 17 +/- 5 and 23 +/- 1 pmol/min/mg, respectively. Six putative endogenous substrates for SM tyrosine kinase, with molecular weights of 205K, 180K, 76K, 60K, 50K, and 45K, were identified. Each of these proteins, except p76, was phosphorylated in the detergent-insoluble residue obtained following the extraction of SMs with Triton X-100 as well as in PSDs, indicating that the postsynaptic apparatus is an active site of tyrosine phosphorylation. The phosphorylation of p76 was localized to the Triton X-100 extract and also occurred in the synaptosomal soluble fraction. The results indicate that tyrosine kinase and its substrates are located in both pre- and postsynaptic compartments and suggest a role for this enzyme in synaptic function.

Angiotensin II↗

PubMed1987

Nuclear magnetic resonance investigation of cadmium 113 substituted pea and lentil lectins.

The lentil (LcH) and pea (PSA) lectins, which are members of the class of D-glucose/D-mannose binding lectins, are Ca2+ X Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. We have prepared a variety of Cd2+ derivatives of PSA and LcH, with Cd2+ in either the transition metal (S1) or calcium (S2) sites, or in both. Thus, Cd2+ X Zn2+, Cd2+ X Mn2+, and Ca2+ X Cd2+ derivatives were prepared, in addition to the Cd2+ X Cd2+ derivatives which we have recently reported. This is the first report of stable mixed metal Cd2+ complexes of lectins. The physical and saccharide binding properties of the Cd2+ derivatives of both lectins were characterized by a variety of physiochemical techniques and found to be the same as those of the corresponding native proteins. 113Cd NMR spectra of mono- and disubstituted 113Cd2+ complexes of LcH and PSA were recorded and compared with 113Cd NMR data for concanavalin A (ConA) (Palmer, A.R., Bailey, D.B., Behnke, W.D., Cardin, A.D., Yang, P.P., and Ellis, P.D. (1980) Biochemistry 19, 5063-5070). The data for the PSA and LcH derivatives were found to be very similar, indicating close homology of their metal ion binding sites. 113Cd resonances at 44.6 ppm and -129.4 ppm for 113Cd2+ X 113Cd2+ X LcH, and at 46.6 and -130.4 for the corresponding PSA derivative, are chemical shifts very similar to those observed for 113Cd2+ X 113Cd2+ X ConA. Assignment of the resonances to the transition metal (S1) and calcium (S2) sites were unambiguous since the Ca2+ X 113Cd2+ and 113Cd2+ X Zn2+ derivatives of both lectins showed single resonances characteristic of the S1 and S2 sites, respectively. The results indicate that, unlike ConA, 113Cd2+ binds tightly to PSA and LcH. Binding of monosaccharide to both lectins induce small (2 ppm) upfield shifts in their S2 113Cd resonances, in contrast to the larger shift (8 ppm) observed in ConA. The 113Cd2+ X Mn2+ complexes of PSA and LcH fail to show a 113Cd resonance characteristic of these derivatives, which provides evidence for the close proximity of the metal ions in the two proteins. The present findings indicate that the coordinating ligand atoms to the metal ions at the S1 and S2 sites in LcH, PSA, and ConA are the same.

Cadmium↗

PubMed1985

Cadmium(II)-113 NMR studies of the mechanism of metal ion activation of yeast enolase.

Yeast enolase binds one mole of 113Cd2+ per subunit at a site that consists of all oxyligands in a distorted octahedral environment. This "conformational" metal ion's environment undergoes further distortion on addition of substrate/product or analogs. At pH's below the optimum value the shifted resonance tends to break up into several, suggesting the existence of several slowly exchanging intermediate forms. At acid pH's, on addition of one additional mole/subunit of 113Cd2+, which greatly increases catalysis, "conformational" resonance(s) further broadens, suggesting that the second, "catalytic" metal ion increases the rates of interconversion between "conformational" species. At more alkaline pH's, near the optimum pH, the "conformational" peak is sharpened, which suggests that very fast interconversion is occurring. The position of the "catalytic" metal ion resonance also suggests all oxyligands in a distorted octahedral geometry. The "catalytic" resonance is often broadened to the point where it cannot be seen, suggesting rapid changes in its geometry due to interconversion of substrate and product.

Binding Sites↗

PubMed1984

Cadmium-substituted skeletal troponin C. Cadmium-113 NMR spectroscopy and metal binding investigations.

The binding of cadmium to skeletal troponin C (STnC) has been measured by equilibrium binding and by 113Cd NMR spectroscopy. The equilibrium binding experiments have shown that there are two cadmium binding sites on STnC with a high affinity for Cd2+ (KCd congruent to 10(7) M-1) and two with a lower affinity for Cd2+ (KCd congruent to 10(3) M-1). The former binding constant is comparable to Ca2+ binding to the Ca2+-Mg2+ (structural) sites of STnC and the latter is about a factor of one hundred less than Ca2+ binding to the Ca2+-specific (regulatory) sites of STnC. In the presence of Mg2+ the affinity of Cd2+ for the higher affinity sites was lowered, yielding a KMg of approximately 10(3) M-1. These data clearly suggest that the two sites with high affinity for Cd2+ are the same as the Ca2+-Mg2+ sites (Zot, H., and Potter, J. D. (1982) J. Biol. Chem. 257, 7678-7683). The 113Cd NMR is shown to be temperature-dependent. The room temperature spectrum consists of two resonances at -107.8 and -112.7 ppm with respect to a 0.1 M solution of Cd(ClO4)2. Lowering the temperature to 4 degrees C alters the cadmium exchange dynamics, and results in a four line 113Cd spectrum. The two new resonances at -103.1 and -109.8 ppm probably arise from cadmium binding to the Ca2+-specific (regulatory) sites on STnC; whereas, the resonances at -107.8 and -112.7 ppm correspond to cadmium binding at the Ca2+-Mg2+ (structural) sites, respectively. When the 113Cd2+-substituted protein was titrated with Ca2+, the two resonances corresponding to the high affinity sites were reduced in intensity, followed by a reduction in intensity of the lower affinity Cd2+ sites. Based on the assignments made here and the known binding constants of STnC for Ca2+ (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633) and the Cd2+ affinities reported here, one would not predict these results. Ca2+ should have first bound to the sites with the lower affinity Cd2+. Since the direct binding experiments clearly demonstrate that the high affinity Cd2+ sites are the Ca2+-Mg2+ sites, we can only conclude that Cd2+ binding to the protein (probably to the lower affinity Ca2+-specific sites) dramatically alters the affinity of the Ca2+-Mg2+ sites for Ca2+. It is suggested that an allosteric coupling network exists between all classes of binding sites.

Animals↗

PubMed1983

13C NMR investigation of nonenzymatic glucosylation of protein. Model studies using RNase A.

Nonenzymatic glucosylation of protein is initiated by the reversible condensation of glucose in its open chain form with the amino groups on the protein. The initial product is an aldimine (Schiff base) which cyclizes to the glycosylamine derivative. The aldimine can undergo a slow Amadori rearrangement to yield the relatively stable ketoamine adduct which is structurally analogous to fructose. 13C NMR has been used to characterize these early products of nonenzymatic glucosylation, using RNase A as a model protein. C-1 of the beta-pyranose anomer of the glycosylamine was identified at 88.8 ppm in the spectrum of RNase glucosylated approximately 1:1 with D-[1-13C]glucose. C-1 of the Amadori product was also apparent in this spectrum, resonating as a pair of intense peaks at 52.7 and 53.1 ppm. The anomeric (C-2) resonances of the Amadori adduct were seen in the spectrum of RNase glucosylated approximately 1:1 with [U-13C]glucose. This spectrum was interpreted by comparison to the spectra of reference compounds: D-fructose, fructose-glycine, N alpha-formyl-N epsilon-fructose-lysine, and glucosylated poly-L-lysine. In the protein spectrum, the most intense of the C-2 resonances was that of the beta-fructopyranose anomer at 95.8 ppm. The alpha- and beta-fructofuranose anomers were also observed at 101.7 and 99.2 ppm, respectively. One unidentified signal in the anomeric region was observed in the spectra of poly-L-lysine and RNase, both glucosylated with [U-13C]glucose; no comparable resonances were observed in the spectra of the model compounds.

Fructose↗

PubMed1983

Manganese (II) electron spin resonance and cadmium-113 nuclear magnetic resonance evidence for the nature of the calcium binding site in alpha-lactalbumins.

Bovine and goat alpha-lactalbumins were substituted with 113Cd(II) or Mn(II) at the strong calcium site [Murakami, K., Andree, P.J., & Berliner, L.J. (1982) Biochemistry 21, 5488-5494] and studied by 113 Cd NMR and electron spin resonance. The 113Cd chemical shifts were in the -80 to -85 ppm range vs. Cd(ClO4)2, which was almost identical with that found for several nearly octahedral (oxygen-coordinated) calcium binding proteins such as calmodulin, parvalbumin, and troponin C. The electron spin resonance spectra of bound Mn(II)-alpha-lactalbumin complexes at 9 or 35 GHz were also confirmatory of a highly symmetric (cubic) environment around the Mn(II) with only slight distortions. The near identity of this site in alpha-lactalbumin to those of calcium binding proteins containing an "EF hand domain" was remarkable despite the absence of such a domain sequence in the alpha-lactalbumin structure.

Animals↗