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Biomedical subjects

D J Miller

Publications and source records attributed to D J Miller.

At least 181 records · Page 10Linked to original sources
PubMed1989

The modulation of force in isolated rat EGTA-treated anococcygeus muscle by phosphate, cyclic AMP and noradrenaline.

Saponin treatment is commonly used to permeabilize the surface membrane of muscle cells. Incubation of isolated rat anococcygeus for 4 h in a low Ca2+ and low Mg2+ solution resulted in the muscle responding to Ca2+, phosphate and cyclic AMP in a similar way to a saponin-treated muscle. However, unlike saponin-treated preparations, the maximal calcium-activated force was stable for many hours and the addition of noradrenaline caused contracture. These properties would permit this preparation to be used to study aspects of receptor-mediated excitation-contraction coupling.

Animals↗
PubMed1989

Binding of bovine follicular fluid glycosaminoglycans to fibronectin, laminin and low-density lipoproteins.

Interactions of bovine follicular fluid glycosaminoglycans (GAGs) with extracellular matrix (ECM) components fibronectin and laminin and with low-density lipoproteins (LDL) were examined using affinity chromatography. Glycosaminoglycans from small (diameter less than 5 mm) and large (diameter 11-20 mm) follicles were isolated from follicular fluid. The dermatan sulphate or heparan sulphate from small or large follicles was applied to Fn-, Lm- or LDL-Sepharose columns. Portions of each fraction of the bound or unbound GAG were then subjected to gel filtration h.p.l.c. for quantification. The binding interaction between dermatan sulphate and fibronectin was significantly greater than between heparan sulphate and fibronectin (P less than 0.05); the binding interaction between GAGs from small follicles and fibronectin was significantly greater than between GAGs from large follicles (P less than 0.05). The binding interaction between GAGs from small follicles and laminin was significantly greater than for GAGs from large follicles (P less than 0.05). Dermatan sulphate from small follicles bound to fibronectin (42%), laminin (36%) and LDL (14%) and that from large follicles bound to fibronectin (14%), laminin (23%) and LDL (14%). Heparan sulphate from small follicles bound to fibronectin (17%), laminin (15%) and that from large follicles bound to fibronectin (13%), laminin (10%) and LDL (6%). These results suggest that dermatan sulphate, but not heparan sulphate, from follicles at different stages of development exhibit a varied ability to interact with components of the ECM. Both substances bound to LDL comparably in small amounts.

Animals↗
PubMed1988

Detection, characterisation, and quantification of carnosine and other histidyl derivatives in cardiac and skeletal muscle.

Isocratic reverse phase analytical high performance liquid chromatography (HPLC) has been used to examine naturally occurring imidazoles of cardiac and skeletal muscles. Elution of muscle extracts with a phosphate buffer mobile phase from columns packed with hypersil ODS (5 micron) resulted in good separation of the skeletal muscle imidazole-containing dipeptides carnosine and anserine. Measured concentrations corresponded to published values. N-Acetyl forms that were not commercially available were prepared from their parent compounds and their identities verified by NMR-spectroscopy. Examination of frog cardiac muscle confirmed the presence of N-acetylhistidine and also indicated the presence of its 1-methyl derivative. Extracts of mammalian cardiac muscle were examined by HPLC which indicated the presence of low concentrations of carnosine but substantial amounts of N-acetyl forms of histidine, 1-methylhistidine, carnosine and anserine. Fractions corresponding to the numerous peaks were examined using staining systems specific for certain chemical features and compared to results obtained for commercial or synthetic standards. Results of these tests supported the chromatographic data. The total concentrations in cardiac muscle of these imidazole-containing substances (approx. 10 mM) is sufficient to alter significantly the sensitivity of their contractile apparatus to calcium ions.

Animals↗
PubMed1988

Characterization of dodecylphosphocholine/myelin basic protein complexes.

The stoichiometry of myelin basic protein (MBP)/dodecylphosphocholine (DPC) complexes and the location of protein segments in the micelle have been investigated by electron paramagnetic resonance (EPR), ultracentrifugation, photon correlation light scattering, 31P, 13C, and 1H nuclear magnetic resonance (NMR), and electron microscopy. Ultracentrifugation measurements indicate that MBP forms stoichiometrically well-defined complexes consisting of 1 protein molecule and approximately 140 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into DPC/MBP aggregates. EPR spectral parameters and 13C and 1H NMR relaxation times indicate that the addition of MBP does not affect the environment and location of the labels or the organization of the micelles except for a slight increase in size. Previous results indicating that the protein lies primarily near the surface of the micelle have been confirmed by comparing 13C NMR spectra of the detergent with and without protein with spectra of protein/detergent aggregates containing spin-labels. Electron micrographs of the complexes taken by using the freeze-fracture technique confirm the estimated size obtained by light-scattering measurements. Overall, these results indicate that mixtures of MBP and DPC can form highly porous particles with well-defined protein and lipid stoichiometry. The structural integrity of these particles appears to be based on protein-lipid interactions. In addition, electron micrographs of aqueous DPC/MBP suspensions show the formation of a small amount of material consisting of large arrays of detergent micelles, suggesting that MBP is capable of inducing large changes in the overall organization of the detergent.

Animals↗
PubMed1988

Hysteresis and the length dependence of calcium sensitivity in chemically skinned rat cardiac muscle.

1. The relationship between pCa (-log10[Ca2+]) and steady-state isometric tension has been investigated in saponin- or Triton-treated (chemically 'skinned') cardiac muscle of rat. 2. Hysteresis exists in the relationship such that the muscle is less sensitive to Ca2+ during increasing activation (as [Ca2+] is stepped upward) than during reducing activation (as [Ca2+] is stepped downward). 3. The extent of the hysteresis is insensitive to interventions that increase overall calcium sensitivity by chemical means, such as caffeine, carnosine or increased pH. 4. The extent of the hysteresis is sensitive to sarcomere length. The phenomenon is virtually absent above sarcomere lengths of about 2.2-2.3 microns but becomes progressively greater at shorter sarcomere lengths. 5. The effect of sarcomere length on calcium sensitivity is restricted to the upward-going (increasing activation) part of the pCa-tension loop below 2.2 microns. The downward-going (decreasing activation) part of the hysteretic relationship is virtually unaffected by sarcomere length up to 2.2 microns. 6. Significant alterations in sarcomere length do not occur during tension development in the experiments described here: the phenomenon is not attributable to experimental artifacts of this kind. 7. Hysteresis develops sufficiently rapidly to be consistent with a physiological relevance during the normal heart beat. 8. The effects of sarcomere length show that the phenomenon is not due to force per se since, for example, greater peak force produces less hysteresis as sarcomere length is increased towards 2.2 microns. 9. Tonicity increase (by high-molecular-weight dextran), which shrinks the myofilament lattice, increases calcium sensitivity but reduces the effect of sarcomere length on calcium sensitivity. 10. The results suggest that lattice shrinkage is the mechanism which accounts for hysteresis in, and the sarcomere length dependence of, calcium sensitivity in cardiac muscle.

Animals↗
PubMed1988

An improved assay for measuring heparin binding to bull sperm.

The binding of heparin to sperm has been used to study capacitation and to rank relative fertility of bulls. Previous binding assays were laborious, used 10(7) sperm per assay point, and required large amounts of radiolabeled heparin. A modified heparin-binding assay is described that used only 5 X 10(4) cells per incubation well and required reduced amounts of [3H] heparin. The assay was performed in 96-well Millititer plates, enabling easy incubation and filtering. Dissociation constants and concentrations of binding sites did not differ if analyzed by Scatchard plots, Woolf plots, or by log-logit transformed weighted nonlinear least squares regression, except in the case of outliers. In such cases, Scatchard analysis was more sensitive to outliers. Nonspecific binding was insignificant using nonlinear logistic fit regression and a proportion graph. The effects were tested of multiple freeze-thawing of sperm in either a commercial egg yolk extender, 40 mM Tris buffer with 8% glycerol, or 40 mM Tris buffer without glycerol. Freeze-thawing in extender did not affect the dissociation constant or the concentration of binding sites. However, freeze-thawing three times in 40 mM Tris reduced the concentration of binding sites and lowered the dissociation constant (raised the affinity). The inclusion of glycerol in the 40 mM Tris did not significantly affect the estimated dissociation constant or the concentration of binding sites as compared to 40 mM Tris without glycerol.

Animals↗
PubMed1988

Relationship of heparin binding with computer-analyzed physical traits of human sperm.

The objective of this study was to determine whether heparin affinity or the concentration of binding sites was related to sperm traits analyzed by a routine computerized semen analysis in human males requesting infertility evaluations. Saturation of heparin binding sites on sperm was achieved in 36 of 50 samples analyzed so that accurate estimates of dissociation constants (Kd) and binding site concentrations could be made. A broad range in Kds (18.2 to 284.5 nM/micrograms deoxyribonucleic acid [DNA]) and binding site concentrations (2.70 to 44.77 nM/micrograms DNA) was found. The binding affinity of sperm for heparin was significantly correlated with concentrations of cells in the ejaculate (r = 0.56), concentrations of motile sperm (r = 0.51), percentage of motile sperm (r = 0.33), and total numbers of ejaculated sperm (r = 0.37). The concentration of heparin binding sites also was correlated with concentration of cells in the ejaculate (r = 0.60), concentration of motile sperm (r = 0.50), percentage of motile sperm (r = 0.40), and total number of ejaculated sperm (r = 0.49).

Binding Sites↗
PubMed1987

Directly coupled supercritical fluid extraction-gas chromatographic analysis of polycyclic aromatic hydrocarbons and polychlorinated biphenyls from environmental solids.

A method has been developed for the direct coupling of supercritical fluid extractions with gas chromatography (SFE-GC) that yields good chromatographic peak shapes and quantitative recovery of analytes from environmental solids with a total extraction and analysis time of less than one hour. Maximum sensitivity is achieved and analyte degradation or loss is minimized since the extracted species are quantitatively transferred into a fused-silica capillary gas chromatographic column for cryogenic focusing followed by normal GC analysis using flame ionization, electron-capture, or mass spectrometric (MS) detection. Coupled SFE-GC-MS determinations of polycyclic aromatic hydrocarbons from National Bureau of Standards urban dust (SRM 1649) gave excellent agreement with certified values.

Chromatography, Gas↗
PubMed1987

Isolation and characterization of seminal fluid proteins that bind heparin.

Heparin facilities the acrosome reaction in bull and rabbit sperm. It binds to sperm saturably, and the binding affinity and the susceptibility to heparin are related to fertility. Isolation of heparin-binding proteins from washed ejaculated sperm reveals proteins mostly in the 14-18 kilodalton (kD) range. Seminal plasma inhibits heparin-induced capacitation, possibly by binding and inactivating heparin. These experiments focused on isolating and characterizing heparin-binding proteins in seminal plasma. Semen samples from 6 bulls were pooled, centrifuged twice for 10 min at 500 xg and finally at 2000 xg for 10 min. The supernatant was ultrafiltered on an Amicon YM-5 (5 kD cutoff) membrane against 40 mM Tris with 2 mM Ca2+ plus protease inhibitors. Five mg of protein were applied to a 1.5 x 10 cm column of heparin-Sepharose. Bound proteins were eluted with 200 ml of a 0 to 2 M NaCl gradient at 0.33 ml/min collecting 3 ml fractions. Two major peaks eluted at 0.12 and 0.46 M NaCl. Those were subjected to SDS-PAGE under reducing conditions. The 0.12 M peak was composed of small (mol wt 15.5 and 16.5 kD) polypeptides which were similar to those found on sperm. The 0.46 M peak included those polypeptides plus a 19.5 kD polypeptide and its trailing edge had a 29.5 kD polypeptide. These polypeptides may regulate capacitation by viture of their ability to bind heparin.

Animals↗
PubMed1987

Localization of two L-glutamate dehydrogenases in the coral Acropora latistella.

The coral Acropora latistella has been shown to contain two distinct L-glutamate dehydrogenases. An NADPH-specific GDH, closely resembling the enzyme from other coelenterates, is located in the cytosol; a distinct NADH-specific GDH is localized within the mitochondria. This is in contrast to earlier reports, where the NADPH-specific enzyme was thought to be the only GDH present and was assumed to be mitochondrial. Preliminary kinetic and stability data are presented for the two GDHs.

Animals↗
PubMed1987

The effect of changes in facial muscle tension on respiratory resistance.

The relationship between changes in facial electromyographic (EMG) levels and respiratory resistance was examined. Thirty adult males were assigned equally to 1 of 3 groups; a facial muscle tension increase group (EMG increase), a facial tension decrease group (EMG decrease), and a noncontingent group (EMG noncontingent). Biofeedback procedures, based on EMG recordings made form electrodes positioned over the frontalis muscles, were used to effect muscle tension changes. Individuals in the EMG increase group exhibited increases in facial tension, but individuals in the EMG decrease group failed to show muscle tension changes. Respiratory resistance increased in individuals in the EMG increase group but did not change in individuals in the EMG decrease group. Like the EMG decrease group subjects, EMG noncontingent group subjects changed neither in facial tension nor in respiratory resistance. The results provided some support for a hypothesis linking facial muscle tension and respiratory resistance.

Adolescent↗
PubMed1987

Survivors of imprisonment in the Pacific theater during World War II.

Data were obtained from 41 survivors of imprisonment by the Japanese during World War II. Interview data suggested that these individuals, despite the 40 years that had passed since their prisoner of war experiences, showed manifestations of posttraumatic stress disorder, notably a sleep disturbance marked by recurrent nightmares. MMPI data suggested significant pathology, characterized as an anxiety state, in this group. Half of the subjects met the full set of DSM-III criteria for posttraumatic stress disorder.

Acting Out↗
PubMed1987

Individual variation for in vitro fertilization success in dairy bulls.

Individual semen samples from 29 bulls in routine artificial breeding service were tested for their capability to achieve in vitro fertilization. Ejaculated semen was diluted, .1 ml of semen in 2 ml of a modified Tyrode's medium with an osmolality of 340 mOsmol/kg, washed thrice, incubated 3 h at 37 degrees C before being used for in vitro fertilization, or incubated 4 h and 8 h before assessment of motility, capacitation, and acrosome integrity. The degree of variability in percentage of oocytes fertilized was assessed along with several factors that might contribute to this variation. Variation among bulls was not significantly different. Variation from one replicate to another was high. Variation was found in motility, capacitation, and frequency of acrosome reaction, but these variables were not significantly correlated to fertilization rate in vitro.

Acrosome↗