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Internal Medicine↗
Biomedical subjects
M D Burton
Publications and source records attributed to M D Burton.
The use of gold reagents to quantitate antibodies eluted from nitrocellulose blots: application to electron microscopic immunocytochemistry.
An assay is described in which gold reagents were used to quantitate nanogram amounts of antibody that had been eluted from antigens immobilized on nitrocellulose paper. Standard curves were generated by the application of rabbit immunoglobulin G (IgG) to nitrocellulose sheets assembled in a dot blot matrix apparatus. Blots were stained using either colloidal gold or immunogold, enabling quantitation of IgG concentration by scanning densitometry. Linear and reproducible standard curves were obtained. As little as 1 ng IgG/dot could be quantified using either gold reagent. In contrast to colloidal gold, immunogold could be used specifically to quantitate rabbit IgG regardless of the presence of bovine serum albumin or antigen coeluted from the nitrocellulose blot. The applicability of the immunogold assay was demonstrated by fractionating a complex rabbit antiserum raised against the RIM protein of frog retinal rod outer segments. Anti-RIM antibody was affinity-purified, quantitated by the immunogold assay, and subsequently employed in immunocytochemical studies using thin sections of retina embedded in a hydrophilic plastic, LR-Gold.
Animals↗
CSF acidosis augments ventilation through cholinergic mechanisms.
Ventilation is influenced by the acid-base status of the brain extracellular fluids (ECF). CO2 may affect ventilation independent of changes in H+. Whether the acidic condition directly alters neuronal firing or indirectly alters neuronal firing through changes in endogenous neurotransmitters remains unclear. In this work, ventriculocisternal perfusion (VCP) was used in anesthetized (pentobarbital sodium, 30 mg/kg) spontaneously breathing dogs to study the ventilatory effects of acetylcholine (ACh), eucapnic acidic (pH approximately 7.0) cerebrospinal fluid (CSF), and hypercapnic acidic (pH approximately 7.1) CSF in the absence and presence of atropine (ATR). Each animal served as its own control. Base line was defined during VCP with control mock CSF (pH approximately 7.4). With ATR (4.8 mM) there was an insignificant downward trend in minute ventilation (VE). ACh (6.6 mM) increased VE 53% (n = 12, P less than 0.01), eucapnic acidic CSF increased VE 41% (n = 12, P less than 0.01), and hypercapnic acidic CSF increased VE 47% (n = 6, P less than 0.01). These positive effects on ventilation were not seen in the presence of ATR. This suggests that acidic brain ECF activates ventilatory neurons through muscarinic cholinergic mechanisms. Higher concentrations of ACh increased ventilation in a concentration-dependent manner. Higher concentrations of ATR decreased ventilation progressively, resulting in apnea. The results suggest that ACh plays a significant role in the central augmentation of ventilation when the brain ECF is made acidic by either increasing CSF PCO2 or decreasing CSF bicarbonate.
Acetylcholine↗
Sialoglycoproteins of the frog rod outer segment plasma membrane.
Photoreceptors can segregate inner segment proteins from outer segment proteins. It is uncertain whether such a sorting process occurs during disc morphogenesis in rods, thereby resulting in an outer segment plasma membrane which differs compositionally from the discs. In this study, the plasma membranes of intact, purified frog rod outer segments (ROS) have been labeled with a peanut lectin (PNA), either with or without prior neuraminidase treatment of the ROS. Neuraminidase removes terminal sialic acid residues from membrane-bound sialoglycoconjugates. PNA was demonstrated to bind to the plasma membrane of ROS only after neuraminidase treatment, as detected by fluorescence light microscopy and electron microscopy. In order to identify the sialoglycoproteins responsible for this labeling, ROS proteins were resolved by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper for incubation with PNA. A single, prominent band at 125 Kd bound PNA after neuraminidase treatment of ROS. This band represents a protein which is an integral component of the ROS plasma membrane and is not associated with the interphotoreceptor matrix or other potential contaminants. Selective degradation of ROS prior to neuraminidase treatment indicates that this sialoglycoprotein is absent from discs and, thus, can serve as a marker specific for ROS plasma membrane during fractionation studies.
Animals↗
Effects of thiamine deprivation and replacement on the mitochondrion of Polytomella agilis.
Late log-phase cells of Polytomella agilis, grown with or without thiamine, were examined by electron microscopy. The mitochondrial profiles of cells cultivated in the presence of thiamine are relatively few in number and irregular in shape. The inner membranes, randomly dispersed in a light matrix, are elongated, vesicular, or branched in appearance. In vitamin-deficient cells, numerous mitochondrial profiles are evident. They have a regular circular or ovoid appearance. The inner membranes are regularly arrayed in an electron-dense matrix and generally appear elongated. By means of partial 3-dimensional reconstruction of whole cells the appearance of mitochondrial profiles in vitamin-deficient cells can be explained by the increased branching of a single structure. Following transfer of vitamin-deficient cells to complete medium, normal mitochondrial structure is attained by similar to 3 hr. Reduced-minus-oxidized difference spectra of suspensions of normal and vitamin-deficient cells, grown with gentle aeration, were recorded. The concentrations of a- and b-type cytochromes are reduced by 80-90 per cent, and c-type cytochromes are reduced by 40 per cent in thiamine-deficient cells.
Culture Media↗
Calbindin D-28K immunoreactivity of human cone cells varies with retinal position.
Calbindin D-28K is a calcium-binding protein found in the cone but not rod photoreceptor cells in the retinas of a variety of species. Recent studies of the monkey retina indicated that calbindin D-28K may be expressed preferentially in non-foveal regions of the retina. In the current studies of human retinas, immunohistochemical experiments demonstrated that calbindin D-28K is reduced or absent in the fovea and parafovea, but prevalent in the perifovea and periphery. These findings were supported by the quantification of calbindin D-28K in 1-mm trephine punches obtained from different regions of the human retina. The specificity of the anti-calbindin D-28K antibodies used in these studies was confirmed by Western blot analysis using purified calbindin D-28K. The protein was purified from retinal tissue and its identity confirmed by partial amino-acid sequence analysis. The expression of calbindin D-28K did not correlate with the spectral properties of the cones, rather to their position in the retina. The study of spatially expressed genes, like the one encoding calbindin D-28K, may help explain the patterns of retinal degeneration seen in some human cone-rod dystrophies.
Aged↗