High-resolution scanning electron microscopy of bacteriophages 3C and T4.

An account is presented of the design and operation of a new scanning electron microscopic, and its first application to the study of biological samples. Bacteriophages were chosen because much of their ultrastructure is beyond the resolution of the conventional scanning electron microscope. The new instrument permits examination of bulk samples with a resolution that exceeds, by at least a factor of 2.5, the resolution obtained in the best secondary electron scanning electron microscopes using high brightness guns, and exceeds by an order of magnitude the resolution of standard scanning electron microscopes using tungsten filament guns. It also permits examination of biological samples in scanning transmission mode at resolutions similar to conventional transmission electron microscopes.

Transporting renal epithelium: culture in hormonally defined serum-free medium.

A hormonally defined medium was used to isolate a homogeneous epithelioid cell population from canine kidney. Monolayers of these cells form domes, an indication of active ion transport, and this process is inhibited by ouabain. This technique allows the isolation of primary cultures of renal epithelial cells, free of fibroblasts, for the characterization of biochemical and physiological properties related to renal function.

Damage to the high-affinity ?-aminobutyric acid (GABA) uptake system in mouse brain by horseradish peroxidase (HRP).

Presynaptic nerve terminals when depolarized are sensitive to morphological and functional alteration by horseradish peroxidase. Mouse brain slices, 0.1 mm, depolarized by a K(+)-HEPES buffer and exposed to horseradish peroxidase exhibited alterations in both synaptic vesicle membrane structure and in high-affinity [(14)C]?-aminobutyric acid uptake. The post stimulatory retrieval of synaptic vesicles from the nerve terminal plasma membrane in the presence of horseradish peroxidase resulted in a decrease in the synaptic vesicle population with a concurrent increase in non-synaptic vesicle membrane structures. High-affinity [(14)C]?-aminobutyric acid uptake into 0.1-mm slices of mouse cerebral cortex and ponsmedulla-spinal cord was inhibited by 31% and 24%, respectively, after incubation for 60 min in K(+)-HEPES buffer containing horseradish peroxidase. Superoxide dismutase protected both the synaptic vesicle membrane and the high-affinity uptake system from the deleterious effects of horseradish peroxidase, pointing to the possible involvement of superoxide anion radicals in the horseradish peroxidase-related effects. These horseradish peroxidase induced alterations appear to be directed towards the exposed synaptic vesicle membrane, since non-stimulated brain slices exposed to horseradish peroxidase do not exhibit a reduction in either high- or low-affinity [(14)C]?-aminobutyric acid uptake. Low-affinity uptake of [(14)C]?-aminobutyric acid and [(14)C]?-aminoisobutyric acid into cortical slices was not affected after incubation in K(+)-HEPES with horseradish peroxidase. Low-affinity uptake, however, is reduced by the high-K(+)/Na(+)-free stimulatory incubation prior to uptake. It appears, thus, that high- and low-affinity uptake are distinct and different systems, with the high-affinity transport system structurally associated with synaptic vesicle membrane.

Ultrastructural localization in the mouse lung of venom from the western diamondback (Crotalus atrox) rattlesnake.

Procedures which make immune complexes between venom antigens and their complementary antibodies visible have been applied to detect the site of deposition of rattlesnake venom in the lung tissue of mice after in vivo envenomation. Lung tissue, from mice envenomated with reconstituted but otherwise unmodified Crotalus atrox venom, was incubated in commercially available polyvalent antiserum (against North American crotalid snakes) which had been conjugated to the enzyme horseradish peroxidase. The enzyme reaction was developed for visualization by transmission electron microscopy. The enzyme reaction products were located along alveolar surfaces and were associated with multilamellar bodies in cytosomes of type II pulmonary epithelial cells. It was concluded that the venom has a specific affinity towards the extracellular surfactant in the lung and towards intracellular sites of surfactant synthesis.

The influence of the buffer on maintenance of tissue lipid in specimens for scanning electron microscopy.

Preparations of tissues and cells for scanning electron microscopy (SEM) fixed with glutaraldehyde (G CHO) buffered in either Na-cacodylate or Piperazine-N-N' bis (20-ethanol sulfonic acid) (PIPES) differ in their morphology. The influence of the nature of these buffers in the tissue fixing and washing solutions on appearance in scanning electron microscopy is described. Biochemical determinations of lipid retention were performed on frog and chick embryos fixed for 24 hours with 3% G CHO in either 0.03 and 0.1 M PIPES (ph. 7.3) or 0.1 M Na-cacodylate (ph. 7.3). Embryonic tissue was chosen for its relatively high lipid content and delicacy which may be expected to enhance the sensitivity at which buffer effects become apparent. The comparable small and uniform sizes of the embryos minimize differences in fixation quality due to penetration. Lipid, recovered from homogenized tissue after treatment with chloroform/methanol/water (1:2.1:1, v/v) was considered as retained. The extraction results, which showed a significant reduction of lipid losses when PIPES buffer was used, are taken to account for the morphological differences observed in SEM and transmission electron microscopy (TEM).

Glomerular filtration and fluid balance in genetically hypertensive mice.

The Schlager genetically hypertensive mouse has been shown to be a valuable animal model with which to study human essential hypertension. Previous studies have characterized renal morphology, juxtaglomerular index, hematocrit, prostaglandin levels, brain catecholamines, social behavior, and patterns of inheritance. The present study continues the phenotypic characterization of this animal model. Using desiccation, isotope dilution, and clearance, the total body water, extracellular fluid volume, and glomerular filtration rate (GFR) in hypertensive and normotensive animals during normal postnatal development were measured. Additionally, using an electron microscopic tracer, the relative permeabilities of the glomerular filter in these animals were assessed. The data indicate a volume expansion in the young hypertensive animals along with a reduction in GFR. As the animals mature the volume expansion in the hypertensives subsides and is eventually reversed resulting in a lower than normal fluid volume level. The significance of the reduced GFR in the hypertensives is also diminished with age although not to the same degree as that of the fluid volume. The indication of a reduced glomerular permeability may account for the above in light of Guyton's cascade hypothesis.

A morphometric analysis of the glomerular capillary wall of a new strain of genetically hypertensive mice.

An investigation was performed on a new strain of genetically hypertensive mice to study those aspects of the renal glomerulus which have in the past been implicated in the etiology of renal parenchymal hypertension. Morphometric analyses were carried out utilizing a computerized graphic data analysing system on information obtained through transmission electron microscopy. Chronically hypertensive animals exhibited thinner basement membranes with numerous sub-epithelial focal thickenings, which were largely absent from the normotensive controls. No difference was noted in the width of the epithelial slit pores (interpedicelar spaces). The glomerular capillary loops of the hypertensive animals appeared otherwise unremarkable, as did the urinary space and parietal epithelium of Bowman's capsule. No evidence of renal parenchymal pathologies implicated in the etiology of systemic hypertension was observed, therefore, these animals would seem to be suitable models for human essential hypertension.

Reversible depletion of synaptic vesicles induced by application of high external potassium to the frog neuromuscular junction.

1. Reversible depletion of synaptic vesicles from frog cutaneous pectoris neuromuscular junctions was studied by application of a Ringer solution containing 115 mM-K propionate.2. During the release of transmitter, the synaptic vesicle membrane is added to the axolemmal membrane. Under the conditions of high K(+)-induced release, the synaptic vesicle membrane accumulates as folds formed in the region of the axolemmal membrane between the active zones. In depleted terminals, large vesicular structures appear and the evidence shows that some of them (possibly all) are formed as axolemmal infoldings. During formation of such infoldings the active zones remain fixed in position with respect to the post-junctional membrane.3. During recovery in normal Ringer solution, which followed 30 min depolarization in high K(+) Ringer solution, spontaneous m.e.p.p.s were detected as early as 9 min after the start of the recovery period and the average time for their reappearance was 17 min.4. At the end of a 20 min recovery period which followed K(+) depolarization, small accumulations of synaptic vesicles were again found within the terminal close to the active zones. At this time coated vesicles and coated pits were seen associated with the prejunctional axolemma and its infoldings. It appears that synaptic vesicles are re-formed directly from these coated vesicles.5. After 60 min recovery from K(+) depolarization, at which time stimulation of the motor nerve induced a muscle twitch, the structure of the terminals closely resembled that of control preparations.6. The entire synaptic vesicle recycling process can take place in the absence of the neurone soma.

Quantitative structural aspects of the renal glomeruli of hypertensive mice.

Kidneys of genetically hypertensive and normotensive mice were studied with respect to kidney weight, number and volume of glomeruli, and filtration surface area. The hypertensive animals have a larger kidney weight to body weight ratio and possess fewer glomeruli per kidney. The superficial cortical glomeruli of the hypertensives are slightly smaller in volume than those of the normotensives although the filtration surface area is similar. The juxtamedullary glomeruli of the hypertensives are both smaller in volume and have less filtration surface area. The possible implications of these findings in the development and/or maintenance of hypertension are discussed.

Superoxide radical-mediated alteration of synaptosome membrane structure and high-affinity gamma-[14C]aminobutyric acid uptake.

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.