Progenitors of oligodendrocytes: Limiting dilution analysis in fetal rat brain culture

In this paper, we describe the use of a combination of cell culture techniques and limiting dilution analysis to determine the number of oligodendrocyte progenitor cells and the oligodendrocyte clone size in primary dispersed cultures of 20- to 21-day-old fetal rat brain. Single-cell suspensions (1,2,3 × 10⁶ cells/ml) were plated in either microwell or 100 mm dishes. After 22 days in culture the number of differentiated oligodendrocytes was ascertained by determining the amount of myelin basic protein by radioimmunoassay. The total amount of myelin basic protein was the same in the two types of dish, indicating that proliferation and differentiation were unaffected when oligodendrocytes were grown in microwells. The fraction (F₀) of microwells containing no oligodendrocytes was determined at each cell dilution. F₀ decreased exponentially with increasing total cell concentration. The linearity of the plot of ln F₀ versus cell number indicates that the number of oligodendrocyte progenitor cells is limiting. From the equation describing the Poisson distribution of progenitor cells in microwells we calculated that, at the time of plating, primary cultures of fetal rat brain contain one oligodendrocyte progenitor cell per 1.3 × 10⁵ brain cells, or a total population of 300–500 progenitor cells per brain. The mean oligodendrocyte clone size was determined to be approximately 825 at 22 days and close to 2000 by 35 days in culture. Therefore, each progenitor cell must undergo approximately 11 divisions, on the average, during postnatal development.     

The effects of hydrogen ions on the kinetics of calcium transport by rat kidney mitochondria

The influence of pH on the kinetics of the initial rate of calcium uptake by isolated kidney mitochondria was studied using the ruthenium red-ethylene glycol bis(β-aminoethyl ether) N,N-tetraacetic acid quench method (K. Reed and F. Bygrave, 1975, Eur. J. Biochem.55, 497–504). In the absence of phosphate, the K_m is increased 50% and the V is decreased 57% when the pH is decreased from pH 7.4 to 7.0. Conversely, when the pH is increased to 7.8, the K_m is decreased 25% while the V is not affected. The presence of 0.1 or 0.4 mm phosphate in the incubation medium abolishes the change in K_m at a low pH while the V remains depressed by 36 and 25%, respectively. The presence of phosphate does not affect the decrease in the K_m seen with an increased medium pH. Mitochondria incubated in steady-state conditions with a medium free calcium of 0.7 μm also show significant changes in calcium exchange and distribution with pH. Two kinetic calcium pools are found in isolated mitochondria. Decreasing pH from 7.4 to 7.1 decreases mitochondria total calcium 32%, decreases the rapidly exchanging pool 28%, and depresses both the mitochondrial membrane and an intramitochondrial calcium exchange by 54 and 22%, respectively. Raising the pH to 7.7 increases both exchangeable pools (63 and 46%), and increases the mitochondrial membrane calcium exchange 44%. These results are consistent with previous studies on the influence of intracellular pH on calcium metabolism of kidney cells in which the mitochondrial pool was markedly affected by pH (R. Studer, and A. Borle, 1979, J. Membrane Biol.48, 325–341). Alterations in cellular pH may modify mitochondrial calcium transport and cellular calcium metabolism and thus affect cell functions which are calcium dependent.     

Cell free synthesis of rat brain myelin proteolipids and their identification by immunoprecipitation

Rat-brain poly(A)-containing RNAs were prepared by affinity chromatography on Poly(U)-Sepharose and translated in the rabbit reticulocyte lysate system in the presence of [³⁵S]-methionine. By using antimyelin proteolipids antibodies, immunoreactive polypeptides of 23.5 K and 19 K were isolated from the translation products : they had electrophoretic mobilities similar to those of the rat brain myelin proteolipids apoproteins.     

Special features of Pi transport in pig heart and rat liver mitochondria as revealed by 6,6'-dithiodinicotinic acid (CPDS).

CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.     

Simultaneously entry of palmitic acid into proteolipid protein in different myelin subfractions of rat brain

Rats of 20-days of age were injected intracranially with radioactive palmitic acid to study its incorporation into proteolipid protein (PLP) of myelin and myelin subfractions. At short times (120 min), the radioactivity present in PLP was shown to be due to palmitic acid bound to the protein by ester linkages. The specific radioactivity of palmitic acid labeled PLP was identical in all the myelin subfractions except the myelin-like fraction, in which it was lower, suggesting that the entry of the fatty acid into PLP of the different subfractions occurs simultaneously.Experiments using time staggered injections of ¹⁴C- and ³H-labeled palmitic acid also showed that entry of the fatty acid into PLP of the various subfractions was simultaneous. These results seem to indicate that the acylation of PLP occurs in the myelin membrane and that synthesis and transport of this protein are events unrelated to the acylation process.     

Tissue fractionation in rat brain, kidney and liver. I. Intracellular localization of a 5-methyltetrahydrofolic acid requiring enzyme.

The intracellular distribution of N-methyl-transferase requiring 5-methyl-tetrahydrofolic acid (5 MT-NMT) was studied in brain, kidney and liver of rats. Among these different tissues, the kidney displayed the highest enzyme activity, more than 20 times the activity detected in the brain. As the striatum and, to a lesser extent the hypothalamus, were found to contain slightly higher 5 MT-NMT than other cerebral regions, they were also selected for the study of the subcellular localization. Tissue fractionation was performed by differential centrifugation yielding five different fractions which were analyzed for their enzymatic content not only of 5 MT-NMT but also of marker enzymes, such as cytochrome oxidase, acid phosphatase and inosine diphosphatase. In all the tissues studied, 5 MT-NMT was recovered in the supernatant fraction. Therefore one may consider this enzyme to belong to the cytosol. Although a neuronal localization cannot be excluded, it is beyond doubt that the enzyme is contained in other cellular types. In the brain fractionation, the five fraction procedure seems to be very useful especially when the subcellular distribution of a given enzyme is compared to that obtained in other tissues like liver or kidney. Finally 5 MT-NMT may be considered a good marker enzyme for the supernatant fraction.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Fatty acid distribution of the normal sciatic nerve lipids in the rat

We isolated and purified the main lipids of the rat sciatic nerve. After methanolysis, fatty acids were isolated and purified by thin layer chromatography, and studied by gas chromatography. C 16 and C 18 fatty acids are the most abundant. Among the long-chain fatty acids, arachidonic acid (20:4) is present in some lipids; highly desaturated fatty acids in C 22 and C 24 are also present. In general, the fatty acids are highly saturated; cholesterol esters and ethanolamine phosphoglyceride fatty acids are highly unsaturated.     

A characterization of alpha-bungarotoxin binding in the brain of the horseshoe crab, Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin to membrane fragments prepared from Limulus brain tissue has been investigated. Toxin binding approaches saturation in the range of 30 to 40 nm, with maximum binding of 2 to 6 pmol/mg of protein. The saturation kinetics and the rate of displacement of bound toxin are consistent with multiple toxin binding sites. Pharmacological studies show that binding is inhibited by both cholinergic agonists and antagonists, I₅₀′s for inhibition by d-tubocurarine, nicotine, decamethonium, carbachol, and atropine are 2 × 10^?6, 7 × 10^?6, 2 × 10^?5, 6 × 10^?4, and 3 × 10^?4m, respectively. Nicotinic ligands inhibited binding much more effectively than muscarinic ligands. Toxin binding activity was solubilized with Triton X-100. Velocity sedimentation analysis of the solubilized activity revealed three separate components. Seventy to eighty percent of the binding activity had a sedimentation coefficient of 8.6 S. The remaining activity was composed of two components with sedimentation coefficients of 15.1 and 17 S.     

Muciliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models

High voltage and conventional electron microscopy were used to investigate the ultrastructure of the ciliary apparatus in intact and in Triton-extracted, reactivated sheets of mucociliary epithelium isolated from newt lung. Each long (about 13 microns) ciliary axoneme terminates on a barrel-shaped basal body which is anchored in the apical cytoplasm by a variety of accessory structures. A basal foot is associated with the midpoint of each basal body and acts as a focal point for numerous microtubules (MTs). In many cases MTs can be seen to interconnect the feet of neighbouring basal bodies. Attached to the proximal end of each basal body and extending in a direction opposite the basal foot is a large 'ciliary root'. Each ciliary root is associated with a distinct bundle of 6-7 nm microfilaments which appear to stain with the specific F-actin probe NBD-phallacidin. A single 3-4 microns long striated rootlet inserts into each ciliary root and extends toward the cell nucleus through an extensive network of microfilaments. At the level of the basal plate 'Y-shaped' structures appear to connect each axonemal outer doublet MT to the plasma membrane. All of these ciliary accessory structures are present in the same relationship in Triton-extracted models. Their morphology and distribution indicates that they serve to anchor the cilia in the apical cytoplasm. In addition some of these structures appear to be responsible for maintaining the structural and functional integrity of the ciliary field in the demembranated and reactivated models.     

Cardiac carnitine deficiency and altered carnitine transport in cardiomyopathic hamsters

The Bio 14.6 hamster has a well-documented cardiomyopathy which leads to congestive heart failure. Previous work demonstrated that hearts from these hamsters have depressed fatty acid oxidation and depressed carnitine concentrations compared to those of normal hamsters. Analyses of tissue carnitine concentrations from 40 to 464 days of age demonstrate that the cardiomyopathic hamsters have a cardiac carnitine deficiency throughout life. Therefore, the carnitine deficiency is not a secondary effect of an advanced stage of the cardiomyopathy. Both the observation that other tissues of the cardiomyopathic hamster have normal or markedly elevated carnitine concentrations and the observation that oral carnitine treatment could not increase the cardiac carnitine concentrations to those of normal hamsters are consistent with the hypothesis that the cardiac carnitine deficiency is the result of a defective cardiac transport mechanism. Cardiac carnitine-binding protein (which may function in the cardiac carnitine transport mechanism) prepared from hearts of cardiomyopathic hamsters had a lower maximal carnitine binding and an increased dissociation constant for carnitine compared to the cardiac carnitine-binding protein prepared from normal hamsters. Thus, several types of data indicate that the cardiomyopathic hamster has an altered cardiac carnitine transport mechanism.     

Kinin-forming activity in rat brain

The present study shows that rat brain contains a kinin-forming activity which is distinguishable from plasma kallikrein. Kinin-forming activity was found in an acetone powder of frozen brain tissue (between 27 and 175.5 ng generated bradykinin/g fresh brain tissue/h). Analysis by high pressure liquid chromatography (HPLC) indicated that the kinin formed chromatographed like true bradykinin (BK). After subcellular fractionation using differential centrifugation of homogenized fresh brain tissue the kinin-forming activity was found mainly in a microsomal (P-3) fraction after preincubation with 2 μM melittin. Further fractionation of P-3 fraction using discontinuous sucrose gradient centrifugation identified activity in both the 1 M sucrose layer (5.8 ± 3.1 ng kinin/mg protein/h) and at the interface between the 0.8 and 0.3 sucrose layers (9.4 ± 4 ng kinin/mg protein/h). Melittin pretreatment did not change these values. The distribution pattern of the kallikrein-like activity was different from that of cathepsin d-like acid protease. The two kinin-forming activities were equally sensitive to treatment with various trypsin inhibitors but were clearly distinguishable from plasma kallikrein: brain activity was inhibited completely by Trasylol but not by soybean trypsin inhibitor (SBTI) or ovomucoid while plasma kallikrein was completely inhibited by SBTI and partially by ovomucoid and Trasilol. Our results clearly distinguish between plasma kallikrein, brain cathepsin d-like acid protease activity and an apparent brain kinin-forming activity, but do not by themselves establish a central biosynthetic pathway for kinin generation.     

Desensitization of ornithine decarboxylase activity to norepinephrine in the testis of rat

Injection of norepinephrine (NE) at a dose of 10 micrograms per testis caused the testis refractory in terms of ornithine decarboxylase (ODC) activity at 24 h. This desensitization was found to be both time and dose dependent. Injection with follicle stimulating hormone, luteinizing hormone, prostaglandin F2 alpha, cyclic AMP or epinephrine to norepinephrine desensitized testis caused stimulation of ODC activity. This indicates that the refractoriness caused by norepinephrine is specific to this agent alone.     

Possible interrelationship of multiple alpha-bungarotoxin binding components in the brain of the horseshoe crab, Limulus polyphemus.

Three $\text{[}{}^{125}\text{I]α-bungarotoxin}$ binding components have been detected in solubilized extracts of $\text{Limulus}$ brain tissue. These components have sedimentation coefficients of 9.0S, 15.4S and 17.4S. All three components were degraded by α-chymotrypsin. The addition of butyl alcohol to brain extract suggests an interrelationship of the toxin binding proteins by promoting a simultaneous decrease in the 9S component and increase in the 15.4S and 17.4S components. This transition was also demonstrated by the readdition of isolated fractions of each component to brain extract.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Changes of pyruvate dehydrogenase in brain during alloxan diabetes

Alloxan diabetes causes a decrease in the active form of pyruvate dehydrogenase in rat brain. The effect is severe in the cerebellum and brain stem compared to cerebral hemispheres. The changes observed in the total form are not as significant as those found in the active (dephosphorylated) form. The effects are reversed after administration of insulin to diabetic animals. The severity of diabetes was also found to affect the activity of pyruvate dehydrogenase with inverse correlation. There was a gradual increase in the proportion of active (dephosphorylated) form with increase of time after the onset of diabetes.     

Effects of progesterone implants in the habenula and midbrain on proceptive and receptive behavior in the female rat

Two brain areas behaviorally responsive to progesterone (P) were examined to determine their possible involvement in the control of rat preceptive behavior, i.e., solicitation behavior directed at the male. Progesterone implants were placed in the habenular nuclei and the interpeduncular nucleus-ventral tegmental area of the midbrain reticular formation (MRF). Different testing procedures and levels of priming with estradiol benzoate (EB) were used in order to distinguish the effects of P in either region on proceptive and receptive behavior during exposure to 10 mounts by stimulus males. To test for receptivity, sexually experienced 60-day-old ovariectomized (ovx) rats bearing stereotaxically placed guide cannulas extending to the habenula or MRF were given 10 μg EB subcutaneously. Forty-eight hours later, lordosis quotient (LQ) was determined. Immediately following this test, each animal was implanted with cholesterol (C) or P and was retested 2 hr later. Treatments for the proceptivity test were similar except that the animals received 2.5 μg EB/100 g body wt sc for 7 days before testing on the eighth day; LQ as well as hopping, darting, and ear wiggling were scored. In the receptivity test, P implantation in both the medial portions of the habenula and the MRF significantly increased lordosis above the levels found both in their preimplantation tests and following control implantation of C. Little proceptivity was observed. In the proceptivity test, P implants in both regions also significantly increased proceptive behavior above both types of control tests. All animals were highly receptive, and there was no difference in LQ among the groups. There was no increase of plasma P levels in similarly implanted animals during a 24-hr monitoring period, indicating that systemic leakage of the hormone was not responsible for the observed behavior. The data indicate that both the habenula and MRF are P-sensitive regions. Progesterone's action on the two areas facilitates expression of both proceptive and receptive components of female sexual behavior, indicating that the neural regulation of the two kinds of behavior is integrated at these levels.     

Effects of atractyloside and palmitoyl coenzyme A on calcium transport in cardiac mitochondria.

Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.