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Biomedical subjects

John H Miller

Publications and source records attributed to John H Miller.

At least 19 recordsLinked to original sources
PubMed2006

Differences in electrostatic potential around DNA fragments containing adenine and 8-oxo-adenine. An analysis based on regular cylindrical projection.

Changes of electrostatic potential (EP) around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through large scale density functional theory computations. Quantum mechanical structural optimizations of DNA fragments with three pairs of nucleotides and accompanying counteractions were performed with a B3LYP exchange-correlation functional and 6-31G(d,p) basis sets. The "intact" DNA fragment contained adenine in the middle layer, while the "damaged" fragment had the adenine replaced with 8-oxo-adenine. The electrostatic potential around these DNA fragments was projected on a cylindrical surface around the double helix. The two-dimensional maps of EP of the intact and damaged DNA fragments were analyzed to identify these modifications of EP that result from the occurrence of 8-oxo-adenine (8oA). It was found that distortions of a phosphate group neighboring 8oA and displacements of the accompanying countercation are clearly reflected in the EP maps.

Adenine↗
PubMed2006

Synergistic effects of peloruside A and laulimalide with taxoid site drugs, but not with each other, on tubulin assembly.

Previous studies on the drug content of pelleted tubulin polymers suggest that peloruside A binds in the laulimalide site, which is distinct from the taxoid site. In a tubulin assembly system containing microtubule-associated proteins and GTP, however, peloruside A was significantly less active than laulimalide, inducing assembly in a manner that was most similar to sarcodictyins A and B. Because peloruside A thus far seems to be the only compound that mimics the action of laulimalide, we examined combinations of microtubule-stabilizing agents for synergistic effects on tubulin assembly. We found that peloruside A and laulimalide showed no synergism but that both compounds could act synergistically with a number of taxoid site agents [paclitaxel, epothilones A/B, discodermolide, dictyostatin, eleutherobin, the steroid derivative 17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol, and cyclostreptin]. None of the taxoid site compounds showed any synergism with each other. From an initial study with peloruside A and cyclostreptin, we conclude that the synergism phenomenon derives, at least in part, from an apparent lowering of the tubulin critical concentration with drug combinations compared with single drugs. The apparent binding of peloruside A in the laulimalide site led us to attempt construction of a pharmacophore model based on superposition of an energy-minimized structure of peloruside A on the crystal structure of laulimalide. Although the different sizes of the macrocycles limited our ability to superimpose the two molecules, atom correspondences that were observed were consistent with the difficulty so far experienced in creation of fully active analogs of laulimalide.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed2006

Impedance spectroscopy of alpha-beta tubulin heterodimer suspensions.

Impedance spectroscopy is a technique that reveals information, such as macromolecular charges and related properties about protein suspensions and other materials. Here we report on impedance measurements over the frequency range of 1 Hz to 1 MHz of alpha-beta tubulin heterodimers suspended in a buffer. These and other polyelectrolyte suspensions show enormous dielectric responses at low frequencies, due both to the motion of charges suspended in the medium and to an electrical double layer that forms at each electrode-medium interface. We propose an equivalent circuit model to minimize electrode polarization effects and extract the intrinsic response of the bulk medium. At megaHertz frequencies, the conductivity increases with concentration below the critical concentration of approximately 1 mg/ml for microtubule polymerization, above which the conductivity decreases. This suggests that such measurements can be used to monitor the dynamics of microtubule polymerization. Finally, we obtain the net charge number per tubulin dimer of /Z/ = 306 in the saline buffer, which, if maintained as the dimers polymerized, would yield a linear charge density of 3.8 e/angstroms for the assembled microtubules. These results are potentially important for fundamental electrostatic processes in biomolecules and suggest the possibility of developing future bioelectronic applications.

Binding Sites↗
PubMed2006

Daily patterns of metabolic rate among New Zealand lizards (Reptilia: Lacertilia: Diplodactylidae and Scincidae).

In addition to the effects of temperature fluctuations on metabolic rate, entrained endogenous rhythms in metabolism, which are independent of temperature fluctuations, may be important in overall energy metabolism in ectotherms. Daily entrained endogenous rhythms may serve as energy-conserving mechanisms during an animal's active or inactive phase. However, because nocturnal lizards often take advantage of thermal opportunities during the photophase (light), their daily metabolic rhythms may be less pronounced than those of diurnal species. We measured the rate of oxygen consumption (VO(2)) as an index of metabolic rate of eight temperate lizard species (four nocturnal, three diurnal, and one crepuscular/diurnal; n = 7-14) over 24 h at 13 degrees C and in constant darkness to test whether daily patterns (including amplitude, magnitude, and time of peak VO(2)) of metabolic rate in lizards differ with activity period. We also tested for phylogenetic differences in metabolic rate between skinks and geckos. Three daily patterns were evident: 24-h cycle, 12-h cycle, or no daily cycle. The skink Cyclodina aenea has a 12-h crepuscular pattern of oxygen consumption. In four other species, VO(2) increased with, or in anticipation of, the active part of the day, but three species had rhythms offset from their active phase. Although not correlated with activity period or phylogeny, amplitude of VO(2) may be correlated with whether a species is temperate or tropical. In conclusion, the metabolic rate of many species does not always correlate with the recorded activity period. The dichotomy of ecology and physiology may be clarified by more in-depth studies of species behaviors and activity periods.

Animals↗
PubMed2006

Regulation of T-cadherin by hormones, glucocorticoid and EGF.

The cell adhesion molecule T-cadherin is an unusual member of the cadherin superfamily that lacks a cytoplasmic domain, binding instead to the cell membrane via a glycophosphatidyl inositol anchor. T-cadherin is a receptor for hexameric Acrp30/adiponectin and binds low-density lipoproteins in endothelial cells. T-cadherin is expressed widely in the brain and cardiovascular system, but expression is absent or decreased in several cancers. Little is known about the mechanisms and factors that control T-cadherin expression. Therefore, to investigate regulation of T-cadherin expression, we analysed 3.9 kb of the 5'-flanking region of human T-cadherin for promoter activity and identified potential transcription factor binding sites. Western blotting and a quantitative real-time RT-PCR assay developed for T-cadherin showed that estradiol, progesterone, EGF, dexamethasone and factors in serum were involved in transcriptional and post-transcriptional regulation of T-cadherin in human osteosarcoma cells; the effects observed were opposite to those described for T-cadherin's ligand, adiponectin. The data suggest that T-cadherin is regulated in a complex manner indicative of a role in hormone and drug-induced changes in bone morphology and pathology.

5' Flanking Region↗
PubMed2005

Reversal of morphine, methadone and heroin induced effects in mice by naloxone methiodide.

Opioid overdose, which is commonly associated with opioid induced respiratory depression, is a problem with both therapeutic and illicit opioid use. While the central mechanisms involved in the effects of opioids are well described, it has also been suggested that a peripheral component may contribute to the effects observed. This study aimed to further characterise the effects of the peripherally acting naloxone methiodide on the respiratory, analgesic and withdrawal effects produced by various opioid agonists. A comparison of the respiratory and analgesic effects of morphine, methadone and heroin in male Swiss-Albino mice was conducted and respiratory depressive ED(80) doses of each opioid determined. These doses (morphine 9 mg/kg i.p., methadone 7 mg/kg i.p., and heroin 17 mg/kg i.p.) were then used to show that both naloxone (3 mg/kg i.p.) and naloxone methiodide (30-100 mg/kg i.p.) could reverse the respiratory and analgesic effects of these opioid agonists, but only naloxone precipitated withdrawal. Further investigation in female C57BL/6J mice using barometric plethysmography found that both opioid antagonists could reverse methadone induced decreases in respiratory rate and increases in tidal volume. Its effects do not appear to be strain or sex dependent. It was concluded that naloxone methiodide can reverse the respiratory and analgesic actions of a variety of opioid agonists, without inducing opioid withdrawal.

Analgesics, Opioid↗
PubMed2005

Absence of an hypoxic depression of metabolism in preproenkephalin knockout mice.

Opioids inhibit breathing in mammals, especially in newborns, and are also implicated in the control of hypoxic anapyrexia. We measured breathing patterns and metabolic responses to 12% oxygen in six adult male wildtype C57B/6J mice and six preproenkephalin knockout (PPNK-/-) mice in a flow-through respirometer and barometric plethysmograph with ambient temperature maintained in the thermoneutral zone. Breathing air, there was no significant difference between the two groups of mice in ventilation ((.)V), oxygen consumption ((.)V(O(2)), convection requirement ((.)V/(.)V(O(2)), tidal volume (V(t)), frequency (f), or inspiratory time (T(i)); however, PPNK-/- mice had a significantly shorter expiratory time (T(e)). The breathing pattern response to 5% CO(2) was the same between wildtype and PPNK-/- in terms of absolute values, but the % change in V(t) was greater in the wildtype. Breathing 12% O(2), there was no significant difference in V , V(t), f, T(i), T(e) or body temperature between groups, but there was a significant difference in (.)V(O(2) (PPNK-/- 1.24+/-0.05 ml O(2)min(-1) versus 0.91+/-0.05 for wildtype, P<0.001) and % change in (.)V(O(2), (2.3+/-6.6% for PPNK-/- versus -28+/-3.8% for wildtype); in ((.)V/(.)V(O(2)), (54+/-4 versus 78+/-10, P<0.05) and the % change in (.)V/(.)V(O(2), (37+/-9 versus 131+/-28, P<0.01). These data implicate enkephalin as a signaling molecule in the control of hypoxic depression of metabolism in mice.

Animals↗
PubMed2005

Interobserver variation in sonography of the painful shoulder.

PURPOSE: This study was conducted as a prospective assessment of interobserver variability in the sonographic evaluation of the rotator cuff. METHODS: Three musculoskeletal radiologists who had different levels of scanning experience each performed shoulder sonography on 24 consecutive patients during 1 patient visit. The diagnostic criteria full-thickness rotator cuff tear, tendon calcification, tendinosis of the supraspinatus, subacromial fluid, subacromial synovial/bursal thickening, dynamic signs of impingement, and abnormality in the long head of biceps tendon were scored, with independent observers recording the data. Statistical analysis was performed using Cohen's kappa test, with significance assessed at p values of less than 0.05. RESULTS: There was good agreement (kappa >0.60, p <0.01) between the experienced operators for full-thickness rotator cuff tear, tendon calcification, dynamic signs of impingement, and abnormality of the long head of biceps tendon. There was no significant agreement between the experienced operators and the less experienced operator in several categories, including (and importantly) full-thickness rotator cuff tears (kappa=0.18-0.21). CONCLUSIONS: In experienced hands sonography of the rotator cuff is a reproducible diagnostic test, but agreement is poor when there is marked disparity between the operators' experience levels. Our findings suggest a need for a more clearly defined training curriculum for sonography of the shoulder in radiology training programs.

Adult↗
PubMed2005

Identification of shed proteins from Chinese hamster ovary cells: application of statistical confidence using human and mouse protein databases.

The shedding process releases ligands, receptors, and other proteins from the surface of the cell and is a mechanism whereby cells communicate. Even though altered regulation of this process has been implicated in several diseases, global approaches to evaluate shed proteins have not been developed. A goal of this study was to identify global changes in shed proteins in media taken from cells exposed to low-doses of radiation to develop a fundamental understanding of the bystander response. Chinese hamster ovary cells were chosen because they have been widely used for radiation studies and are reported to respond to radiation by releasing factors into the media that cause genomic instability and cytotoxicity in unexposed cells, i.e., a bystander effect. Media samples taken for irradiated cells were evaluated using a combination of tandem- and Fourier transform-ion cyclotron resonance (FT-ICR)-mass spectrometry (MS) analyses. Since the hamster genome has not been sequenced, MS data was searched against the mouse and human protein databases. Nearly 150 proteins identified by tandem mass spectrometry were confirmed by FT-ICR. When both types of MS data were evaluated, using a new confidence scoring tool based on discriminant analyses, about 500 proteins were identified. Approximately 20% of these identifications were either integral membrane proteins or membrane associated proteins, suggesting that they were derived from the cell surface and, hence were likely shed. However, estimates of quantitative changes, based on two independent MS approaches, did not identify any protein abundance changes attributable to the bystander effect. Results from this study demonstrate the feasibility of global evaluation of shed proteins using MS in conjunction with cross-species protein databases and that significant improvement in peptide/protein identifications is provided by the confidence scoring tool.

Animals↗
PubMed2005

A variable-energy electron microbeam: a unique modality for targeted low-LET radiation.

We have designed and constructed a low-cost, variable-energy low-LET electron microbeam that uses energetic electrons to mimic radiation damage produced by gamma and X rays. The microbeam can access lower regions of the LET spectrum, similar to conventional X-ray or 60Co gamma-ray sources. The device has two operating modes, as a conventional microbeam targeting single cells or subpopulations of cells or as a pseudo broad-beam source allowing for direct comparison with conventional sources. By varying the incident electron energy, the target cells can be selectively exposed to different parts of the energetic electron tracks, including the track ends.

Electrons↗
PubMed2004

Compensating bends in a 16-base-pair DNA oligomer containing a T(3)A(3) segment: A NMR study of global DNA curvature.

In-phase ligated DNA containing T(n)A(n) segments fail to exhibit the retarded polyacrylamide gel electrophoresis (PAGE) migration observed for in-phase ligated A(n)T(n) segments, a behavior thought to be correlated with macroscopic DNA curvature. The lack of macroscopic curvature in ligated T(n)A(n) segments is thought to be due to cancellation of bending in regions flanking the TpA steps. To address this issue, solution-state NMR, including residual dipolar coupling (RDC) restraints, was used to determine a high-resolution structure of [d(CGAGGTTTAAACCTCG)2], a DNA oligomer containing a T3A3 tract. The overall magnitude and direction of bending, including the regions flanking the central TpA step, was measured using a radius of curvature, Rc, analysis. The Rc for the overall molecule indicated a small magnitude of global bending (Rc = 138 +/- 23 nm) towards the major groove, whereas the Rc for the two halves (72 +/- 33 nm and 69 +/- 14 nm) indicated greater localized bending into the minor groove. The direction of bending in the regions flanking the TpA step is in partial opposition (109 degrees), contributing to cancellation of bending. The cancellation of bending did not correlate with a pattern of roll values at the TpA step, or at the 5' and 3' junctions, of the T3A3 segment, suggesting a simple junction/roll model is insufficient to predict cancellation of DNA bending in all T(n)A(n) junction sequence contexts. Importantly, Rc analysis of structures refined without RDC restraints lacked the precision and accuracy needed to reliably measure bending.

Bacterial Proteins↗
PubMed2004

Peloruside A does not bind to the taxoid site on beta-tubulin and retains its activity in multidrug-resistant cell lines.

Peloruside A (peloruside), a microtubule-stabilizing agent from a marine sponge, is less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump and is not affected by mutations that affect the taxoid binding site of beta-tubulin. In vitro studies with purified tubulin indicate that peloruside directly induces tubulin polymerization in the absence of microtubule-associated proteins. Competition for binding between peloruside, paclitaxel, and laulimalide revealed that peloruside binds to a different site on tubulin to paclitaxel. Moreover, laulimalide was able to displace peloruside, indicating that peloruside and laulimalide may compete for the same or overlapping binding sites. It was concluded that peloruside and laulimalide have binding properties that are distinct from other microtubule-stabilizing compounds currently under investigation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Mu and delta opioid receptor immunoreactivity and mu receptor regulation in brainstem cells cultured from late fetal and early postnatal rats.

Cultured cells from the rat brainstem were used to study opioid receptor (OpR) expression during late fetal and early postnatal development. Mu and delta opioid receptor (MOR and DOR) expression was investigated from embryonic day 16 (E16) to 6 days postnatal (P6). Postnatal neurons showed more intense MOR immunoreactivity (IR) than neurons cultured from fetal brainstem (P < 0.006). DOR IR showed a similar pattern, but the differences between fetal and neonatal animals were not statistically significant. Using confocal microscopy, MOR and DOR IR were shown to be present on both the cell membrane and within the cytoplasm, in a similar pattern to the IR seen in SH-SY5Y neuroblastoma cells that endogenously express both MOR and DOR. Double-labeling experiments demonstrated colocalization of MOR and DOR in the same brainstem neurons; however, not all MOR IR regions of a single neuron were also positively stained for DOR, and not all DOR IR regions were also positive for MOR. MOR was down-regulated after a 1- or 2-h treatment with 1 microM DAMGO, a potent mu opioid agonist, in both non-transfected and MOR-transfected SH-SY5Y cells and in primary cell cultures. It was concluded that many brainstem neurons express functional MOR or DOR or coexpress both receptors, although intracellular distributions of the receptors are unique for each receptor type.

Analgesics, Opioid↗
PubMed2004

Abundant expression of mu and delta opioid receptor mRNA and protein in the cerebellum of the fetal, neonatal, and adult rat.

Opioid receptor proteins and mRNAs have been localized to a variety of regions within the rat brain. It is generally accepted that within the lobes of the rat cerebellum, only delta opioid receptor (DOR) is expressed. This is in contrast to that observed in humans and rabbits which express both mu opioid receptor (MOR) and DOR. In this study, we report detection of MOR as well as DOR protein by immunohistochemical localization, and mRNA by fluorescent in situ hybridization (FISH) within Purkinje cells (PK) and the granular layer of neonatal (P6) and adult rat cerebellum. Expression of MOR mRNA was also detected within cells of the molecular layer, but at lower levels than those seen within the PK cells. Abundant expression of MOR and DOR mRNA was detected in the external germinal layer of the immature cerebellum of the fetal (E16) rat, supporting a role for MOR and DOR in regulating neurogenesis of the cerebellum. In addition, using exon-specific cRNA probes, exons 1 and 4, which are both found in the MOR-1 splice variant mRNA, were detected in PK cells in the cerebellum and also within deep cerebellar nuclei in the adult.

Animals↗
PubMed2004

Developmental expression of mu and delta opioid receptors in the rat brainstem: evidence for a postnatal switch in mu isoform expression.

Opioid receptors are expressed in the brain during fetal and postnatal development, and the expression patterns vary with developmental age. To investigate the role of opioids in brain development, immunoblotting and immunohistochemical techniques were used to determine mu (MOR) and delta (DOR) opioid receptor expression levels and regional distributions in fetal, early postnatal and adult rat brainstem. Two immunoreactive bands were seen on Western blots of brainstem lysates for both MOR (50 and 70 kDa) and DOR (30 and 60 kDa). The expression levels of the isoforms changed dramatically between 6 and 15 days after birth. Total MOR protein was expressed at low levels in fetal and early postnatal animals with the 50-kDa band predominating. MOR expression then increased in the older animals and the 70-kDa isoform became dominant. Total DOR protein showed the opposite pattern, being high in the fetal and neonatal brainstem and low in the juvenile and adult. A postnatal switch in isoform expression for DOR was not evident in our study. In general, regional brainstem distributions in developing and adult animals were comparable to those reported in the literature, and both receptors were localized in the same areas where opioid receptor expression was high. It was concluded that MOR and DOR are developmentally regulated in the brainstem of the rat, that the isoform ratio switches postnatally from a fetal-neonatal pattern to a juvenile-adult pattern and that both receptors are generally expressed in the same brainstem regions from E16 to adult.

Animals↗
PubMed2003

Characterization of the active site of DNA polymerase beta by molecular dynamics and quantum chemical calculation.

It is well established that the fully formed polymerase active site of the DNA repair enzyme, polymerase beta (pol beta), including two bound Mg2+ cations and the nucleoside triphosphate (dNTP) substrate, exists at only one point in the catalytic cycle just prior to the chemical nucleotidyl transfer step. The structure of the active conformation has been the subject of much interest as it relates to the mechanism of the chemical step and also to the question of fidelity assurance. Although crystal structures of ternary pol beta-(primer-template) DNA-dNTP complexes have provided the main structural features of the active site, they are necessarily incomplete due to intentional alterations (e.g., removal of the 3'OH groups from primer and substrate) needed to obtain a structure from midcycle. Working from the crystal structure closest to the fully formed active site [Protein Data Bank (PDB) code: 1bpy], two molecular dynamics (MD) simulations of the solvated ternary complex were performed: one with the missing 3'OHs restored, via modeling, to the primer and substrate, and the other without restoration of the 3'OHs. The results of the simulations, together with ab initio optimizations on simplified active-site models, indicate that the missing primer 3'OH in the crystal structure is responsible for a significant perturbation in the coordination sphere of the catalytic cation and allow us to suggest several corrections and additions to the active-site structure as observed by crystallography. In addition, the calculations help to resolve questions raised regarding the protonation states of coordinating ligands.

Binding Sites↗
PubMed2003

8-Oxoguanine enhances bending of DNA that favors binding to glycosylases.

Molecular dynamics (MD) simulations were carried out on the DNA oligonucleotide GGGAACAACTAG:CTAGTTGTTCCC in its native form and with guanine in the central G(19):C(6) base pair replaced by 8-oxoguanine (8oxoG). A box of explicit water molecules was used for solvation, and Na(+) counterions were added to neutralize the system. The direction and magnitude of global bending were assessed by a technique used previously to analyze simulations of DNA containing a thymine dimer. The presence of 8oxoG did not greatly affect the magnitude of DNA bending; however, bending into the major groove was significantly more probable when 8oxoG replaced G(19). Crystal structures of glycosylases bound to damaged-DNA substrates consistently show a sharp bend into the major groove at the damage site. We conclude that changes in bending dynamics that assist the formation of this kink are a part of the mechanism by which glycosylases of the base excision repair pathway recognize the presence of 8oxoG in DNA.

DNA↗