A cell fraction enriched with lipid droplet-containing cells (perisinusoidal stellate cells, vitamin A-storing cells, or fat-storing cells), and freed from contamination of parenchymal cells, was prepared by low speed centrifugation followed by Percoll density gradient centrifugation. Cells in this fraction were found to take up and store the largest amount of vitamin A per cell among the liver cells including parenchymal cells, when [3H]retinol was injected through the portal vein of rats. Analysis of collagen production by this fraction as well as another non-parenchymal cell fraction (rich in Kupffer cells and endothelial cells) in culture has shown that only the stellate cell-rich fraction produced collagen which was composed of type I (88.2%), type III (10.4%) and membranous type (1.4%). There was no indication of collagen production in other non-parenchymal cell fractions under the experimental conditions employed. These results indicate that, among non-parenchymal cells, lipid droplet-containing cells or stellate cells are primarily responsible for collagen production in the liver.
Perikarya of bovine spinal ganglion cells were dissected out from freeze-dried sections and their neurofilament proteins were analyzed by two-dimensional gel electrophoresis. Neurofilament proteins purified from the bovine spinal cord, a triplet of 76 K, 170 K and 220 K proteins, were used as reference. In the perikarya, the 220 K component was not detected, and the 170 K component was seen in only a trace amount. Instead, a 150 K protein, which was more alkaline than the 170 K component, and a group of proteins ranging from 150 K to 170 K (150-170 K proteins) were dominant in the perikarya. The 150-170 K proteins were also observed in the intraganglionic nerve fibers, in addition to the typical triplet proteins. Immunoblotting with anti-170 K antibody confirmed that the 150 K protein and 150-170 K proteins share the antigenicity with the 170 K protein.
Chick limb bud mesenchyme cells were isolated from stage 22-23 embryos and embedded within exogenous collagen gels at low cell density. They grew to form a colony of round cells in situ and differentiated into cartilage with an overt metachromatic matrix. Synthesis of sulfated glycosaminoglycan increased markedly with time and most were deposited within the collagen gel matrix. The cells primarily synthesized type I collagen followed by predominant synthesis of cartilage-specific type II collagen, during the collagen gel culture. The control cells seeded in the monolayer cultures at the same initial density never showed chondrogenic activity. The results indicate that the three-dimensionally enclosed environment of collagen gel culture is required for the limb bud mesenchyme cells to effectively accumulate synthesized matrix macromolecules and to differentiate into cartilage.
Pineal glands from albino rats, Syrian hamsters, Mongolian gerbils, normal random bred mice and Snell dwarf mice have been found to contain measurable levels of immunoreactive somatostatin (IRS). The Snell dwarf (79.24±19.18 ng IRS/mg protein) and normal mice (37.33±4.8 ng IRS/mg protein) exhibited the highest concentrations followed by hamsters (4.05±0.93 ng IRS/mg protein), gerbils (3.66±1.26 ng IRS/mg protein) and rats who had the lowest (2.9±0.56 ng IRS/mg protein). A circadian rhythm of IRS was found to exist in the albino rat pineal with a peak value at the end of the light period (14 h) and a nadir towards the end of the dark period (10 h). Neither continuous light for one week, nor bilateral superior cervical ganglionectomy was found to exert any effect content between male and female mice. Daily injections of 25 μg of melatonin or 5-methoxytryptamine (5-MT) for 8 weeks produced no significant changes in pineal IRS levels in male hamsters; however, IRS tended to be higher in the melatonin injected group (277.84±90.3 pg IRS/pineal) than in the control (159.68±53.54 pg IRS/pineal) and 5-MT-treated (91.21±24.98 pg IRS/pineal) animals. It is concluded that IRS is found in the pineal gland of 5 different rodent species and exhibits a circadian rhythm in rats.
An endogenous inhibitor for Ca2+-activated neutral protease (CANP) was purified to homogeneity from the human liver. One mole of the inhibitor inhibited 5 mol of CANPs from various sources. The purified inhibitor has a molecular weight of 107 K, which is significantly larger than the value previously reported for the CANP inhibitor.
The occurrence, distribution and ultrastructure of nerve elements containing opioid peptide-like immunoreactivity (enkephalin neurons) in the guinea pig duodenum were investigated using an antiserum (R-0171) to methionine-enkephalin-Arg6-Gly7-Leu8 (Met-Enk-Arg-Gly-Leu). Whole mount preparations of duodenal tissues perfusion-fixed Leu-like immunoreactivity. The morphology of these enkephalin neurons was different from cell to cell. Immunopositive Dogiel type 1 (characterized by short, broad buttonlike processes and a single, long axon-like process), Dogiel type 2 (characterized by smooth perikaryon and a few tapering processes) and Dogiel type 3 neurons (characterized by dendrite-like processes branching around other ganglion cells) were demonstrated. The single axon-like process of many Dogiel type 1 neurons was anally directed and ran in the primary fasciculi across the ganglia. Non-varicose processes and beaded terminals of the enkephalin neurons were demonstrated not only in the myenteric plexus but also in the longitudinal muscle layer, in the circular muscle layer, in the deep muscular plexus, in the submucous plexus, in the muscularis mucosae and around the duodenal gland. No immunopositive perikarya were found in the submucous plexus. No nerve elements in the perivascular plexus and in the mucosa showed immunoreactivity. In the electron microscopic immunocytochemistry by the protein A-colloidal gold method, Met-Enk-Arg-Gly-Leu-like immunoreactivity was demonstrated in the largecored vesicles in the nerve fiber varicosities and the perikarya. Synapse formation between enkephalin-immunoreactive nerve terminals and either immunopositive or immunonegative perikarya was found in the myenteric plexus.
In the presence of Mg2+, and [y-32P]ATP or [y-32P]GTP, casein and phosvitin were phosphorylated when incubated with coated vesicles prepared from synaptosomes from the bovine cerebral cortex (synaptosomal coated vesicles). The phosphorylation was independent of cyclic nucleotides or Ca2+ plus calmodulin, and was inhibited by heparin, suggesting the occurrence of casein kinase II activity in the synaptosomal coated vesicles. The kinase also phosphorylated a protein with a molecular weight of 52,000 (C-52) which was endogenously present in the synaptosomal coated vesicles. Another endogenous protein with a molecular weight of 48,000 (C-48) was phosphorylated in the presence of labeled ATP, but not of labeled GTP. Phosphorylation of C-48 was not suppressed by heparin. The results suggest the presence of two types of protein kinase in the synaptosomal coated vesicles; casein kinase II which phosphorylates C-52, and exogenous acidic proteins including casein and phosvitin, and another kinase capable of phosphorylating C-48.
A role of Ca2+ in electric pulse-induced fusion of mouse lymphoma cells adhered by dielectrophoresis was investigated under the direct measurement of the extracellular free Ca2+ concentration with Ca2+-selective electrodes. Ca2+ over micromolar concentrations were prerequisite for the electrofusion. A quantitative evaluation of the yield of fusion shows a sigmoidal relation with the Ca2+ concentration. In the presence of extracellular Ca2+ higher than 0.1 mM, most of the cells fused after exposure to electric pulses. It is concluded that Ca2+ is essentially involved in electrofusion of lymphoma cells.
A new clonal astrocytic cell line (F6) was isolated from a Rous sarcoma virus-induced mouse brain tumor cells. This clone expresses glial fibrillary acidic protein (GFAP) antigen and simultaneously possesses many intermediate filaments in nearly 100% ofcells. The arrangement ofthe intermediate filaments in the F6 cells is in good accordance with the fibrillar staining pattern of GFAP. These findings suggest that the F6 clone has the well-developed astrocytic cytoskeleton. The clone would be of value in studying glial intermediate filaments and other biological characteristics of astrocytes.
Na+,K+-ATPase (EC 220.127.116.11) was purified to homogeneity from the kidney of spontaneously hypertensive rats (SHR), and their normotensive control rats (Wistar Kyoto rats; WKY. Purity of the enzyme was confirmed by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis, in combination with immunoblotting using anti-dog Na+,K+-ATPase antibody. The enzyme purified from SHR and WKY did not differ in respect to the molecular weight, subunit composition, specific activity, ouabain sensitivity, requirements for Na+ and K+ (and also for Mg2+ and Ca2+), and to the extent of heat denaturation. The results indicate that the difference in Na+,K+-pump so far reported between SHR and WKY is not attributable to any difference in biochemical properties of the purified Na+,K+-ATPase.
1) An improved enzymatic microdetermination method for glutamate decarboxylase (GAD) using an enzymatic amplification reaction (NADP cycling) was developed. In the enzymatic assay, y-aminobutyric acid (GABA) formed by GAD is measured by determining NADPH in the redox reaction catalyzed by ‘GABAase’ (mixture of aminobutyrate aminotransferase and succinate-semialdehyde dehydrogenase). The GABAase is contaminated by glutamate dehydrogenase, which produces NADPH from NADP+ (substrate for GABAase) and glutamate (substrate for GAD remaining in the GABA assay mixture). In the present method, the glutamate was enzymatically eliminated before the assay reaction for GABA, resulting in a low blank value. This made it possible to determine a minute amount of GABA (300 fmol) produced by GAD in a nanogram sample, after 10,000-fold amplification using NADP cycling. 2) The GAD activity in a single Purkinje cell (1.2-2.9 ng in dry weight) of the rabbit cerebellum was 4-5-fold higher than that in the rabbit cerebellar layers. 3) Significant GAD activity was detected in the rat oviduct. The activity was 5-fold higher in the mucosa than in the muscle layer.