Acidic glycosaminoglycans in developing sterno-costal cartilage of the hydrocephalic (ch+/ch+) mouse

The sterno-costal cartilage of the hydrocephalic mouse carrying the autosomal recessive gene (ch+/ch+) has 40 ± 3% of the acidic glycosaminoglycan concentration of the normal control containing the satin marker (+sa/+sa). The acidic glycosaminoglycan concentration of the sterno-costal cartilage in the heterozygous mouse (ch+/+sa) is significantly higher (114 ± 8%) than the normal control. The distribution of the acidic glycosaminoglycans in the sterno-costal cartilage is similar in the normal, heterozygous and homozygous mice at all stages of development studied, (prenatal, newborn and postnatal) being 78 ± 4% chondroitin 4(6)-sulfate and 22% hyaluronic acid and/or keratan sulfate. The concentration of acidic glycosaminoglycans in the sterno-costal cartilage decreases as development progresses in all three gene types of mice. The reduced level of acidic glycosaminoglycans in the sterno-costal cartilage of the autosomal recessive mouse, ch+/ch+, is associated with a defect in the formation of the sternum. The higher than normal acidic glycosaminoglycan concentration in the sterno-costal cartilage of the heterozygous mouse ch+/+sa is associated with delayed calcification of the sternum. This study characterizes the molecular locus of a defect in the extra-cellular matrix of a mouse carrying a lethal gene and may help in understanding proteoglycan disorders (mucopolysaccharidosis) in the human.     

Chemical determination of cationized ferritin bound to synaptosomes

Surface charge of synaptosomes was studied with cationized ferritin (CF) binding at pH between 4 and 7. The characteristic of binding resembles that of the electrophoretic mobility and suggests an electrostatic interaction between CF and membranes. When sialic acid was partially removed from synaptosomes, a reduction in binding was detected. This suggests that sialic acid is one determinant of surface charge of synaptosomes. The present method differs from other CF studies in that the amount of bound CF was measured chemically instead of by electron microscopy. This method could be useful in other cell systems.     

Demonstration of an intercellular substance in mouse cerebral cortex

Summary Specimens of mouse cerebral cortex were preserved by freezing and drying and treated in vacuo with osmium tetroxide vapors. In the neuropil of the molecular layer dense, osmiophilic laminae, presumably plasma membranes of immediately adjacent cell processes, were found to be separated by an intercellular space, which appeared relatively electron lucent in unstained sections. In sections stained with uranyl acetate an intercellular substance was demonstrated, the visualized density of which was reduced when staining was accomplished at low pH. These reactions suggest the presence of a non-lipid, anionic intercellular substance which may contain acid mucopolysaccharide.This work was supported by U.S.P.H.S. Research Grants NB 05175, NB 07044 and Career Development Award 1K3 NB 4929.     

Submicroscopic morphology of granular vesicles in sympathetic nerves of rat pineal body

Summary In the rat pineal body sympathetic nerve endings are characterized by numerous agranular and granular vesicles. The latter mainly include vesicles of about 450 Å diameter, each of which is bounded by a trilaminar membrane of about 75 Å total thickness. The cores of these granular vesicles appear structurally similar to the bounding membranes and are formed by a less dense, globular or vacuolar component around which a more dense component is distributed. The relative distributions of these two structural elements result in a variety of submicroscopic configurations which suggest a morphological continuum that may be related to the cyclical uptake, storage and release of biogenic amines. A tripartite classification of granular vesicles, such as proposed previously, is not supported by these data.This investigation was supported by United States Public Health Service Research Grants NB 05175-01 and AM-0699802. — The capable technical assistance of Mrs. Caroline Wilner is gratefully acknowledged.     

Uptake of norepinephrine by isolated neurons of rat pontine reticular formation

Summary Neurons isolated from the nucleus reticularis pontis caudalis and the lateral vestibular nucleus of the rat brain stem have been incubated in buffered sucrose containing norepinephrine. Norepinephrine accumulated intracellularly was then visualized by fluorescence microscopy after formaldehyde condensation.Incubation in sucrose containing 1.0 g/ml norepinephrine resulted in a 2-fold increase in fluorescence that was blocked by 1×10^–5M cocaine. Maximal enhancement of fluorescence, accomplished by incubation of isolated neurons with 40 g/ml norepinephrine, was unaffected by cocaine but effectively blocked by 1×10^–3M metanephrine. Accumulation of norepinephrine was unaffected by pretreatment of animals with reserpine and nialamide.These properties of norepinephrine uptake suggest a neuronal membrane transport system for norepinephrine in isolated neurons similar to Uptake 2 which occurs extraneuronally. Although the intracellular localization of exogenous norepinephrine accumulated is not known, some concentration of norepinephrine in the perikaryal cytoplasm is suggested.This work was supported by U.S. Public Health Service Research Grant NB 07044. The competent technical assistance of Mrs. Gisela Griffith and Miss Susan J. Decker is acknowledged with gratitude.     

Electron microscopic evidence for the existence of an intercellular substance in rat cerebral cortex

Summary The intercellular spaces of rat cerebral cortex are filled with a dense material, demonstrable by electron microscopy. This intercellular substance is in part preserved by chemical fixation with formaldehyde and osmium tetroxide but is solubilized and largely lost during subsequent dehydration with ethyl alcohol. Dehydration with acetone or Durcupan favors the preservation of the intercellular substance, which is preserved also by freezing and drying. Whether the intercellular substance demonstrated here is part of the outer leaflets of apposing plasma membranes (glycocalyx) or truly an intercellular substance similar to connective tissue ground substance is not known. The probability of the latter is discussed with regard to proposed physiological mechanisms.This work was supported by USPHS Research Grants NB 05175 and AM 06998.     

Cortical cytoplasm and configurational changes of RGC-6 cells exposed to cytochalasin B.

RGC-6 cells, grown to confluency in monolayer cultures, are typically fusiform. In sections of these cells, fixed in situ and examined in the electron microscope, a discontinuous zone of cortical cytoplasm (5-4 nm wide), was found subjacent to the plasma membrane of the free surface. It was composed largely of 5-7 nm microfilaments distributed in an apparently irregular network and appeared to comprise a discrete zone separating the plasma membrane from the underlying endoplasm. Following incubation with cytodhalasin B (5 mug/ml), the configuration of the cells changed markedly. The perikaryal cytoplasm appeared to swell, while the cellular processes became attenuated and often terminated in flower-like tips. Although more discontinuous and somewhat less dense, the fine structure of the cortical cytoplasm appeared little affected by incubation with cytochalasin B for one hour. By 48 hours no zone of cortical cytoplasm was found. Subplasmalemmal microfilaments were no longer identifiable and endoplasmic organelles were found in direct contact with the plasma membrane of the free surface, which appeared to have collapsed onto the underlying endoplasm. The change in cell configuration resulting from exposure to cytochalasin B occurred at 37 degrees C but not at 4 degrees C, suggesting a dependency on metabolic activity. It did not, however, appeared related directly to protein synthesis. Whereas incorporation of 3H-leucine was decreased to 46.9% and 62.9% of control values after one hour or two hours exposure to cytochalasin B, respectively, no change was found after 24 or 48 hours. It is suggested that the effect of cytochalasin B on cell form, which appeared not to be a direct effect on either protein synthesis or subplasmalemmal microfilaments, may represent a primary response of the cortical cytoplasmic matrix.