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Biomedical subjects

D H Williams

Publications and source records attributed to D H Williams.

At least 91 records · Page 5Linked to original sources
PubMed1993

On the stability of nucleic acid structures in solution: enthalpy-entropy compensations, internal rotations and reversibility.

The thermodynamics of self-association (stacking) of free bases and nucleotides, intramolecular stacking in dinucleotides, nearest-neighbour base pair stacking interactions in duplex DNA and RNA, and the formation of hairpin loops illustrate enthalpy/entropy compensations. Large stacking exothermicities are associated with large negative entropy changes that ensure that delta G is small, permitting readily reversible associations in solution. We rationalise enthalpy/entropy compensations with reference to residual motions and torsional vibrations which make a larger entropic contribution to binding when - delta H approximately kT (thermal energy at room temperature), than when - delta H >> kT. We present a factorisation of experimental free energies for helix formation in terms of approximate contributions from the restriction of rotations, hydrophobic interactions, electrostatic interactions due to base stacking, and contributions from hydrogen bonding, and estimate the adverse free energy cost per rotor (mainly entropy) of ordering the phosphate backbone as between 1.9 and 5.4 kJ mol-1 [averaged over 12 rotors per base pair for A-U on A-U stacking (lower limit), and G-C on C-G stacking (upper limit)]. The largest cost is associated with the most exothermic stacking interactions, while the range of values is consistent with earlier conclusions from data on the fusion of hydrocarbon chains (lower value), and with entropy changes in covalent isomerisations of small molecules involving severe restrictions (upper value).

Base Composition↗
PubMed1993

Molecular basis for methoxyamine-initiated mutagenesis: 1H nuclear magnetic resonance studies of oligonucleotide duplexes containing base-modified cytosine residues.

Methoxyamine, N4-methoxycytidine and its 2'-deoxyribo analogue are transition mutagens. The mechanism by which the latter acts after incorporation into or generation within DNA has been ascribed to the ability of the base analogue to pair effectively with both adenine and guanine. To obtain a detailed understanding of these interactions, the solution structures of the self-complementary octanucleotide d(CGGATCCG) and its analogues d(CGGATTCG), d(CGGATMCG) and d(CGGATPCG) (designated 8mer-GC, -GT, -GM and -GP, respectively) were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2] oxazin-7-one. A variable temperature study showed the order of stability as 8mer GC > GP > GT > GM. Nuclear Overhauser spectroscopy permitted the assignment of the base, anomeric and H2'/H2" protons in these 8mers. All had spectra consistent with regular B-DNA duplex structures. Imino proton spectra showed that the 8mers GC, GP and GM involved Watson-Crick base-pairing but that the G.P and to a greater extent G.M base-pairs were in slow exchange on the nuclear magnetic resonance time-scale with the wobble configuration. Indeed, the G.M pair showed an additional exchange process interpreted in terms of the presence of syn and anti conformers of the methoxy group in the wobble pair. This accounts for the destabilization of M compared with the P-containing duplex. The observations are compared with those made earlier on the corresponding AT, AP and AM octamers. It is evident that M and P can form stable base-pairs with both A and G with essentially Watson-Crick geometry. This confirms the earlier, although unsubstantiated explanation for the transition mutational propenstty of methoxyamine which, in turn, was based on the fact that methoxycytosine bases have tautomeric constants (KT) much nearer to unity than the normal bases. The same general explanation for hydroxylamine and hydrazine-induced mutations is correspondingly rendered more certain.

Base Composition↗
PubMed1993

Toward an estimation of binding constants in aqueous solution: studies of associations of vancomycin group antibiotics.

An approach toward the estimation of binding constants for organic molecules in aqueous solution is presented, based upon a partitioning of the free energy of binding. Consideration is given to polar and hydrophobic contributions and to the entropic cost of rotor restrictions and bimolecular associations. Several parameters (derived from an analysis of entropy changes upon the melting of crystals and from the binding of cell wall peptide analogues to the antibiotic ristocetin A) which may be useful guides to a crude understanding of binding phenomena are presented: (i) amide-amide hydrogen bond strengths of -(1 to 7) +/- 2 kJ.mol-1, (ii) a hydrophobic effect of -0.2 +/- 0.05 kJ.mol-1.A-2 of hydrocarbon removed from exposure to water in the binding process, and (iii) free energy costs for rotor restrictions of 3.5-5.0 kJ.mol-1. The validity of the parameters for hydrogen bond strengths is dependent on the validity of the other two parameters. The phenomenon of entropy/enthalpy compensation is considered, with the conclusion that enthalpic barriers to dissociations will result in larger losses in translational and rotational entropy in the association step. The dimerization of some vancomycin group antibiotics is strongly exothermic (-36 to -51 kJ.mol-1) and is promoted by a factor of 50-100 by a disaccharide attached to ring 4 (in vancomycin and eremomycin) and by a factor of ca. 1000 by an amino-sugar attached to the benzylic position of ring 6 in eremomycin. The dimerization process (which, as required for an exothermic association, appears to be costly in entropy) may be relevant to the mode of action of the antibiotics.

Anti-Bacterial Agents↗
PubMed1993

Protein folding in the absence of the solvent ordering contribution to the hydrophobic interaction.

Despite considerable effort there is no consensus as to what interaction, or set of interactions, provides the dominant force that drives protein folding and specifies folded protein structures. A key thermodynamic observation is that a large drop in heat capacity (delta Cp) usually accompanies folding in water. Various factors may contribute to this effect, especially changes in the structure of the solvent upon exposure of both non-polar and polar groups in the unfolded state. The unfavourable Gibbs free energy of solvating non-polar groups, in particular, is thought to provide a central driving force for folding (the hydrophobic effect) but the role of solvent ordering in this remains a matter of controversy. We report here a series of experiments that show that a protein can fold into its native conformation under conditions where solvent ordering effects are demonstrably negligible. In methanol/water mixtures ubiquitin unfolds reversibly with a delta Cp value that falls close to zero above about 30% (v/v) methanol. We are able to reason, on the basis of these data, that the net contribution to the heat capacity change arising primarily from the protein structure itself is not significant and that contributions from changes in solvent ordering are rendered negligible by the change in composition. Nuclear magnetic resonance measurements, however, indicate that non-polar side-chains do still become exposed to solvent in the denatured state under these conditions. The combination of these results and model compound studies suggests that the elimination of ordering effects is an intrinsic property of the mixed solvent. We can, therefore, conclude that the solvent ordering component of the hydrophobic effect is not an obligatory factor in determining the three-dimensional structure into which the protein will fold.

Animals↗
PubMed1993

Active immunization of heifers against a synthetic fragment of bovine inhibin.

Two experiments were conducted in cyclic beef heifers to determine whether active immunization against bovine inhibin alpha 1-26 Gly-Tyr (bINH) affected follicular dynamics, hormone concentration or ovulation rate. In Expt 1, heifers (n = 9) were actively immunized against bINH conjugated to human alpha globulins (HAG) using bis-diazotized benzidine in non-ulcerative Freund's adjuvant (NUFA; primary on day 0; booster injections on days 53, 84 and 116 using conjugated bINH and on days 176 and 366 using unconjugated bINH; ten heifers were used as controls). Ovaries were examined daily using ultrasound scanning (days 70-155 and 384-391) and corresponding blood samples were collected for bINH antibody titre, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol determinations. Four treated and four control heifers were injected with 10 micrograms gonadotrophin-releasing hormone (GnRH) on day 386 (day 2 of the oestrous cycle). Although bINH-immunized heifers had variable antibody titres ranging from 4 to 50% I125-labelled bINH bound to serum diluted 1:2000, ovulation rate was unaffected. In oestrous cycles with three dominant follicles, the ovulatory follicles grew faster (2.5 +/- 0.2 versus 1.6 +/- 0.3 mm day-1; mean +/- SEM), had shorter durations of growth (5.7 +/- 0.8 versus 9.6 +/- 1.6 days) and duration of detection (7.5 +/- 0.8 versus 12.0 +/- 2.4 days) in immunized heifers. Mean concentrations of FSH, LH and oestradiol were unaltered in most cases during oestrous cycles in bINH-immunized compared with control heifers. There was no significant difference in the percentage increase in FSH or LH, after GnRH injection, between control and immunized heifers. As ovulation rate was unaltered in the first experiment, a second similar study was designed using a different immunization protocol. In Expt 2, heifers were immunized with bINH conjugated to human serum albumin using glutaraldehyde with the following doses: 0.0 (control; n = 7), 0.33 (n = 7), 1.0 (n = 8) and 3.0 (n = 7) mg. Three booster immunizations were given 33, 66 and 209 days after primary immunization. Immunization increased the number of oestrous cycles with multiple ovulations (42 of 132 (32%) oestrous cycles examined) compared with controls (1 of 30 (3.3%) oestrous cycles examined). Neither titre nor ovulation rate was affected by dose of bINH used. In summary, following bINH immunization, ovulation rate was not increased despite changes in follicular dynamics in Expt 1, but was increased in 32% of oestrous cycles in Expt 2.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Triplicate prescriptions in Washington State.

In summary, I believe that this program, although limited in scope, has been of value to our State in controlling the inappropriate prescribing of controlled substances. We have identified several problems that have been or are being addressed in order to make the program even more effective. The program targets only those prescribers who have been identified as having drug use or prescribing problems rather than subjecting all practitioners to a program for the purpose of finding a relatively small number of violators. Thus, the intimidation factor alleged to be present in full triplicate programs is absent in the limited program. It is flexible since the drugs included in the monitoring can range from a single controlled substance schedule, all schedules, or even to all prescriptions. The program includes both prescribed drugs and those administered in the office or dispensed by the practitioner. The responsibility for compliance is placed on the prescribers, not on the pharmacists. The way the law is written, the program could be expanded to include a sample of all prescribers, providing information that could be used to determine prescribing norms. Overprescribing appears to be in the eyes of the beholder. A pharmacist may view certain prescribing habits differently than a disciplinary board. The availability of norms would assist boards in making more scientific determinations regarding alleged overprescribing practices.

Drug Prescriptions↗
PubMed1992

Aurothioglucose inhibits induced NF-kB and AP-1 activity by acting as an IL-1 functional antagonist.

We have designed a series of recombinant CAT genes to study IL-1 signal transduction in murine fibroblast NIH 3T3 cells. We demonstrate that the HSV thymidine kinase (tk) promoter does not respond to IL-1, but that IL-1 induction of this promoter is observed after insertion of either NF-kB or AP-1 binding sites upstream of the HSV tk cap-site. We have studied the effects of indomethacin, dexamethasone and aurothioglucose (which have been used in the treatment of patients affected by rheumatoid arthritis) in the IL-1 inducible CAT assay. We show that aurothioglucose or dexamethasone is able to inhibit IL-1 induced CAT activity whereas a non-steroidal anti-inflammatory drug (indomethacin) is inactive. Order of addition experiments indicate that aurothioglucose, which has disease-modifying activity in treated patients, acts as an IL-1 functional antagonist in this system.

3T3 Cells↗
PubMed1992

Why water-soluble, compact, globular proteins have similar specific enthalpies of unfolding at 110 degrees C.

The changes in free energy, enthalpy, and entropy of unfolding have been measured for many water-soluble, compact, globular proteins by a number of workers. In principle, a wide range in stability could be achieved by proteins, as measured by the free energy of unfolding; in practice, evolution only allows a narrow range in this quantity. Proteins are only marginally stable at room temperature for many possible reasons, including ensuring that folding is reversible and polypeptide chains are not trapped in incorrectly folded structures. Many of these proteins have approximately the same values of enthalpy of unfolding around 110 degrees C. We show here that this arises because the change in entropy of unfolding at room temperature and the change in heat capacity on unfolding, which governs the temperature variation of the enthalpy and entropy, both vary with the magnitude of the hydrophobic effect in the protein. As all these proteins have evolved to achieve similar stabilities at room temperature, the enthalpy of unfolding will also vary with the size of the hydrophobic effect in the protein. A consequence of this is that curves of the specific unfolding enthalpy against temperature for different proteins intersect around 110 degrees C. A similar conclusion, on the basis of similar melting points rather than similar free energies of unfolding, has been reached independently by Baldwin and Muller (R. L. Baldwin, personal communication).

Energy Transfer↗
PubMed1992

The evolutionary role of secondary metabolites--a review.

It is argued that organisms have evolved the ability to biosynthesise secondary metabolites ('natural products') due to the selectional advantages they obtain as a result of the functions of the compounds. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes in Streptomyces, is interpreted in terms of the defense roles of antibiotics. The clustering together of antibiotic biosynthesis, regulation, and resistance genes, and in particular the staggering complexity shown in the case of the gene cluster for erythromycin A biosynthesis, implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature.

Anti-Bacterial Agents↗
PubMed1992

Why are secondary metabolites biosynthesized? Sophistication in the inhibition of cell wall biosynthesis by vancomycin group antibiotics.

The evidence that secondary metabolites serve sophisticated roles in the survival strategy of the producer is briefly reviewed. This evidence stems from the common involvement of tens of kilobases of DNA in the programming of their synthesis, of up to several tens of discrete enzymic conversions in their biosynthesis, and of the existence of sophisticated mechanisms in the producers for resistance against their physiological effects. It also stems from a study of the molecular basis for these physiological effects. The molecular basis for the antibacterial action of the vancomycin group antibiotics is presented, and demonstrates that essentially every portion of these molecules appears to be finely honed to promote efficient antibacterial action.

Adaptation, Physiological↗
PubMed1992

Spinal fusion for back pain: a clinical and radiological review.

Eighty-one patients who had spinal fusions performed for back pain over a 7-year period were reviewed; 74% were satisfied with the outcome of their surgery, mainly because of the degree of pain relief obtained. Based on lateral radiographs of the fusion area in flexion and extension, there was a 34% pseudarthrosis rate in first-time fusions. However, there was no clear relationship between the integrity of fusion and clinical success, indicating that many factors other than bony fusion influence the eventual outcome of the operation.

Adult↗
PubMed1992

On the evolution of functional secondary metabolites (natural products).

It is argued that organisms have evolved the ability to biosynthesize secondary metabolites (natural products) because of the selectional advantages they obtain as a result of the functions of the compounds. The clustering together of antibiotic biosynthesis, regulation, and resistance genes implies that these genes have been selected as a group and that the antibiotics function in antagonistic capacities in nature. Pleiotropic switching, the simultaneous expression of sporulation and antibiotic biosynthesis genes, is interpreted in terms of the defence roles of antibiotics. We suggest a general mechanism for the evolution of secondary metabolite biosynthesis pathways, and argue against the hypothesis that modern antibiotics had prebiotic effector functions, on the basis that it does not account for modern biosynthetic pathways.

Anti-Bacterial Agents↗
PubMed1992

Serial MRI and neurobehavioural findings after mild to moderate closed head injury.

Fifty patients who sustained mild to moderate closed head injury (CHI) underwent a CT scan, MRI, and neurobehavioural testing. At baseline 40 patients had intracranial hyperintensities detected by MRI which predominated in the frontal and temporal regions, whereas 10 patients had lesions detected by CT. Neurobehavioural data obtained during the first admission to hospital disclosed no distinctive pattern in subgroups of patients characterised by lesions confined to the frontal, temporal, or frontotemporal regions, whereas all three groups exhibited pervasive deficits in relation to normal control subjects. The size of extraparenchymal lesion was significantly related to the initial Glasgow Coma Scale score, whereas this relation was not present in parenchymal lesions. One and three month follow up MRI findings showed substantial resolution of lesion while neuropsychological data reflected impressive recovery. The follow up data disclosed a trend from pervasive deficits to more specific impairments which were inconsistently related to the site of brain lesion. These results corroborate and extend previous findings, indicating that intracranial lesions detected by MRI are present in most patients hospitalised after mild to moderate CHI. Individual differences in the relation between site of lesion and the pattern of neuropsychological findings, which persist over one to three months after mild to moderate CHI, remain unexplained.

Adult↗
PubMed1991

Molecular basis for methoxyamine initiated mutagenesis. 1H nuclear magnetic resonance studies of base-modified oligodeoxynucleotides.

In order to reach a more detailed understanding of the mechanism of the mutagenic action of methoxyamine and of N4-methoxycytidine and its 2'-deoxyribo-analogue, the solution structures of the self-complementary octanucleotide, d(CGAATTCG) and its analogues, d(CGAATCCG), d(CGAATMCG) and d(CGAATPCG) (designated 8mer-AT, 8mer-AC, 8mer-AM, and 8mer-AP, respectively), were investigated by 1H nuclear magnetic resonance spectroscopy; M is N4-methoxycytosine (mo4C) and P is an analogue, the bicyclic dihydropyrimido[4,5-c][1,2]oxazin-7-one, in which the N-O bond is held in the anti configuration with respect to N3 of the cytosine ring. Correlated spectroscopy and nuclear Overhauser spectroscopy allowed assignment of the base, anomeric and H2'/H2" protons in 8mers-AT, -AM and -AP, and showed that all three had features consistent with a regular B-DNA duplex structure. Duplex-to-coil transition temperatures were determined to be 52(+/- 2) degrees C (8mer-AT), 51(+/- 2) degrees C (8mer-AP), 32(+/- 2) degrees C (8mer-AM); on the chemical shift timescale, the melting transition was fast for 8mer-AT and 8mer-AP, but slow for 8mer-AM. Imino proton spectra were indicative of Watson-Crick base-pairing in 8mers-AT, -AP and -AM. The 8mer-AP duplex had a structure and melting characteristics virtually identical with those of the 8mer-AT duplex. The preferred syn configuration of the methoxyl group in M had a destabilising effect on the 8mer-AM duplex. At low temperatures, the A.M base-pair was in fast equilibrium between Watson-Crick and wobble configurations, with the methoxyl function anti-oriented, but the melting transition was accompanied by isomerization of the methoxyl group to the syn conformation. This syn-anti isomerization was the rate-determining step in the duplex-to-coil transition. The 8mer-AC oligomer did not form a stable duplex.

Cytidine↗
PubMed1991

Conserved positioning of proline residues in membrane-spanning helices of ion-channel proteins.

Proline residues are a common feature of known and putative transmembrane helices of transport proteins. We find considerable consistency in the positioning of these residues within the structures. The proline residues are usually found on the hydrophilic (interior) faces of the pore-forming helices. This general observation adds considerable support to hypotheses concerning the structure of the ion-channels formed by alamethicin and melittin. As proline kinks helices, our observation suggests that the pores formed in ion-channel proteins tend to be funnel-shaped having a constriction near their center. Such a structure can aid in the capture of ions by the channel (an entropic effect) and should help in the gating mechanism of the channel. The observation will aid identification of putative transmembrane helices of ion-channels.

Animals↗
PubMed1991

Characterization of a partially denatured state of a protein by two-dimensional NMR: reduction of the hydrophobic interactions in ubiquitin.

A stable, partially structured state of ubiquitin, the A-state, is formed at pH 2.0 in 60% methanol/40% water at 298 K. Detailed characterization of the structure of this state has been carried out by 2D NMR spectroscopy. Assignment of slowly exchanging amide resonances protected from the solvent in the native and A-state shows that gross structural reorganization of the protein has not occurred and that the A-state contains a subset of the interactions present in the native state (N-state). Vicinal coupling constants and NOESY data show the presence of the first two strands of the five-strand beta-sheet that is present in the native protein and part of the third beta-strand. The hydrophobic face of the beta-sheet in the A-state is covered by a partially structured alpha-helix, tentatively assigned to residues 24-34, that is considerably more flexible than the alpha-helix in the N-state. There is evidence for some fixed side-chain--side-chain interactions between these two units of structure. The turn-rich area of the protein, which contains seven reverse turns and a short piece of 3(10) helix, does not appear to be structured in the A-state and is approaching random coil.

Amino Acid Sequence↗
PubMed1991

Left handed alpha-helix formation by a bacterial peptide.

The alpha-helix is a common element of secondary structure in proteins and peptides. In eukaryotic organisms, which exclusively incorporate L-amino acids into such molecules, stereochemical interactions make such alpha-helices, invariably right-handed. Pseudomonas tolaasii Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach. P. Tolaasii proceduces an extracellular lipodepsipeptide toxin, tolaasin, which causes the brown pitted lesions on the mushroom cap. Circular dichroism studies on tolaasin in a membrane-like environment indicate the presence of a left-handed alpha-helix, probably formed by a sequence of 7 D-amino acids in the peptide. P. tolaasii represents the first reported example of an organism which has evolved the ability to biosynthesize a left-handed alpha-helix.

Amino Acid Sequence↗