THE OCCURRENCE OF INTRACELLULAR CHONDROITIN SULFATE

Suspensions of chondrocytes were prepared by treatment with trypsin of the epiphyses of tibias and femurs of 13-day-old chick embryos. After washing to remove the matrix, such suspensions readily incorporate radioactive sulfate into both intracellular and extracellular chondroitin sulfate. Following disruption of the cells, the cell constituents were fractionated by centrifugation. Fractions obtained from cells incubated for 10 minutes showed a concentration of radioactivity in the material which sediments at 10,000 to 20,000 g. At this time the radioactivity of the extracellular chondroitin sulfate is low, but at 1 hour the radioactivity of the intracellular material is relatively unchanged, while that of the extracellular polysaccharide is markedly increased. Following incubation of the chondrocyte suspensions in a tissue culture medium, the intracellular chondroitin sulfate was isolated. This was compared with chondroitin sulfate isolated from the cartilage matrix. Chemical analysis and infrared spectroscopy indicated that both the intracellular and extracellular polysaccharides consist of a mixture of chondroitin sulfuric acids A and C. A portion of the chondroitin sulfate is not sulfated.     

STUDIES IN THE ANTAGONISM BETWEEN BLASTOMYCES LUTEUS AND SPECIES OF VERTICILLIUM

Blastomyces luteus has been shown to be antagonistic in culture to both Verticillium albo-atrum and V. dahliae over a wide range of temperature and irrespective of the pH value of the medium. When B. luteus is grown on Dox's and potato-dextrose solutions it produces an exudate which, added to agar media, has an inhibiting effect upon the growth of these two species of Verticillium. The effectiveness of this inhibiting material is slightly reduced by boiling for 15 min.
When 'spent' Dox's liquid medium, which has supported the growth of B. luteus , is injected into tomato and antirrhinum seedlings inoculated with Verticillium no control of the disease is effected, but when B. luteus mycelium is added to soil infected with Verticillium , marked reduction in the incidence of disease results when the antagonistic organism and the pathogens have co-existed in the soil a sufficient length of time for the exudate of the former to be effective upon the development of the latter.     

Cholesterol-rich Fluid Membranes Solubilize Ceramide Domains: IMPLICATIONS FOR THE STRUCTURE AND DYNAMICS OF MAMMALIAN INTRACELLULAR AND PLASMA MEMBRANES*

A uniquely sensitive method for ceramide domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide concentrations, ranging from low or no cholesterol (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high cholesterol (similar to mammalian plasma membrane). Diverse fluorescence spectroscopy and microscopy experiments were conducted showing that for low cholesterol amounts ceramide segregates into gel domains that disappear upon increasing cholesterol levels. This was observed in different raft (sphingomyelin/cholesterol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Cholesterol-ceramide interactions have been described mainly as raft sphingomyelin-dependent. Here sphingomyelin independence is demonstrated. In addition, ceramide-rich domains re-appear when either cholesterol is converted by cholesterol oxidase to cholestenone or the temperature is decreased. Ceramide is more soluble in cholesterol-rich fluid membranes than in cholesterol-poor ones, thereby increasing the chemical potential of cholesterol. Ceramide solubility depends on the average gel-fluid transition temperature of the remaining membrane lipids. The inability of cholestenone-rich membranes to dissolve ceramide gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. We also show that the solubility of cholesterol in ceramide domains is low. The results are rationalized by a ternary phospholipid/ceramide/cholesterol phase diagram, providing the framework for the better understanding of biochemical phenomena modulated by cholesterol-ceramide interactions such as cholesterol oxidase activity, lipoprotein metabolism, and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed unless a stress or pathological situation occurs.Cholesterol (Chol)³ is the most abundant sterol in mammalian plasma membrane and has unique biophysical properties (1, 2). Chol interacts with the high melting temperature (T_m) sphingolipids (SL) in the membrane, leading to the formation of SL/Chol-enriched microdomains (so-called lipid rafts). These domains are in a more ordered state (usually referred to as liquid-ordered (l_o) phase) than the bulk membrane (liquid-disordered phase (l_d)) (3, 4). Ceramide (Cer) is an SL formed in stress situations either from sphingomyelin (SM) in rafts or synthesized de novo by serine palmitoyltransferase and ceramide synthase. Both of these processes can be induced by diverse stimuli (5). Cer-induced membrane alterations (e.g. raft fusion into large signaling platforms (6)) were proposed to be the mechanism by which this lipid mediates diverse cellular processes, namely apoptosis (7–10). Cer presents an unusually small polar headgroup and in general very high gel-fluid T_m (e.g. for palmitoyl-Cer (PCer) it is ∼90 °C) (11). Membrane Cer levels are usually very low, although in cells undergoing apoptosis it can reach values up to 12 mol % total lipid (7), a percentage that in model membranes leads to Cer-rich gel domain formation (12–17). It was suggested that the formation of these domains might also be involved in Cer biological action (8, 18, 19).However, Cer effects on membrane properties are extremely dependent on membrane lipid composition, especially on Chol amounts (13, 20–23). For instance, in raft-forming model membranes (i.e. ternary mixtures of phosphocholines (PC), sphingomyelin (SM), and Chol), Cer-rich gel domains are formed at low but not at high Chol content (23). This result was explained by the competition between the two small headgroup molecules, Chol and Cer, for the bulkier headgroup, SM, to minimize acyl chain exposure to water. In fact, it is suggested that Cer selectively displaces Chol molecules from rafts, both in model (24–27) and in cell membranes (28, 29). However, a recent study showed that Cer-generated from SM hydrolysis leads to the formation of gel domains in these ternary mixtures only when Chol levels are low, suggesting that even for SM-depleted mixtures Chol is still able to modulate Cer effects (30). Therefore, to fully disclose the conditions that lead to the activation/regulation of Cer-mediated processes, further knowledge about Cer effects on membrane properties and their modulation by Chol is required.It is important to clarify the relation between Cer threshold for gel formation, cholesterol amount, and the properties of the remaining lipids (namely their propensity to form gel phases, which depends mainly on their gel-fluid transition temperature). This is because of the fact that each organelle membrane has its own specific composition. For example, there is a gradient of cholesterol concentration from the endoplasmic reticulum (ER) to the plasma membrane (PM) (31). In addition, there is a close relation between intracellular Cer levels, Ca²⁺ release from the ER, and Cer-induced permeability increase of the mitochondrial outer membrane (but not the inner membrane) (32, 33).The application of a uniquely sensitive method for Cer-rich gel detection allowed us to study for the first time Chol-Cer interactions in detail for high Chol and low Cer concentrations, i.e. a composition similar to mammalian plasma membranes. In addition, low Chol membranes were also studied. Our results clearly show that in a fluid matrix of representative mammalian membranes lipids, Cer-rich gel domains are destroyed by high amounts of Chol in the absence of SM and even in the absence of an l_o phase. We show that this outcome is a consequence of the higher solubility of Cer in Chol-rich membranes than in poor ones, the low solubility of Chol in Cer domains, and that it depends on the average T_m of the remaining lipids. These solubility differences offer a unified rationale for all Cer-Chol biophysical studies that can be translated into a ternary phase diagram, and the biological implications of the results are discussed.     

MITOCHONDRIA AND THE CONTROL OF INTRACELLULAR CALCIUM

1.Because the calcium (Ca) ion is intimately associated with so many biochemical and physiological phenomena, it is fundamental to understand how intracellular Ca is maintained and controlled. This review draws attention to the vital role played by mitochondria in controlling intracellular Ca and describes how transport of the ion into and out of mitochondria may itself be controlled. 2.The heterogeneous distribution of Ca is a property of most, if not all cells. This arises because the ion binds strongly to a variety of biological compounds, especially those containing oxyanions, which themselves have a heterogeneous distribution in cells, but mostly because of the existence in the cell of specific Ca-ion transport systems. 3.Although the concentration of total Ca in the cell may be quite high, a very large proportion of it is bound and non-diffusible; a small fraction is diffusible but unionized. The proportion of Ca that is ionized is probably much less than I% of the total 4.The mechanisms by which Ca is transported into and out of the mitochondrial matrix are discussed. Inward movement of the ion occurs in response to the membrane potential (negative inside) generated by respiration. The process is carrier-mediated and exhibits characteristics such as substrate specificity, high affinity for Ca, satur-ability, cooperativity, stimulation by permeant anions and is specifically inhibited by low concentrations of Ruthenium Red and lanthanum. The properties of the Ca carrier are geared therefore to facilitate rapid inward movement of Ca into the mitochondria. Such a carrier system is found in mitochondria isolated from a wide variety of tissues and species. 5.Ionized Ca appears not to be distributed across the inner mitochondrial membrane according to the Nernst equation, so the possibility exists that the ion is transported as Ca/H⁺ antiport or as Ca/anion symport. Alternatively, an efflux system coupled to inward movement of a cation may serve to prevent the [Ca ion]_in/[Ca ion]_out from attaining equilibrium. These components together contribute to a Ca-translocation cycle that permits Considerable flexibility in the overall control of Ca flux. 6.Evidence for Ca cycling in mitochondria is presented and the influence of physiological agents such as Mg, phosphoenolpyruvate, inorganic phosphate and adenine nucleotides, on the influx and efflux components are discussed in some detail. Moreover, various hormones administered in vivo are able to induce changes in mitochondrial Ca cycling. One important feature that emerges from this collection of data is that the ability of mitochondria to retain Ca is associated with their ability to retain also their adenine-nucleotide complement. 7.Various lines of research provide convincing evidence in support of the view that mitochondria play a major role in controlling cell Ca in vivo. Especially significant are the observations that the ‘activity’ of mitochondrial Ca transport can change during development in both insect and mammalian tissue, can depend on the hormonal status of the tissue and undergoes a permanent change in certain tumour cells. 8.Finally, consideration is given as to how the mitochondrial Ca transport system is able to modify Ca-sensitive enzyme activities by regulating the Ca concentration in specific environments. Some biological activities that might be susceptible to such control are discussed.     

THE INTRACELLULAR DISTRIBUTION OF FERRITIN FOLLOWING MICROINJECTION

Ferritin solutions were microinjected into the ground cytoplasm of intact amebae. At several time-intervals after injection the cells were fixed and the distribution of the protein in various organelles was studied with the aid of the electron microscope. Individual molecules of ferritin were found randomly dispersed throughout the ground cytoplasm and the ground nucleoplasm. Within the mitochondria, the ferritin was localized between the outer and inner membranes. Aggregates of ferritin were found in vacuoles, some of which could be identified as food vacuoles. The findings, which provide evidence for a rapid penetration of large molecules into the nucleus, the outer compartment of the mitochondria, and the digestive vacuoles, are discussed in relation to other reports on intracellular permeability.     

THE MOLECULAR ORGANIZATION OF CHLOROPLAST MEMBRANES

The presence of subunits in chloroplast membranes is suggested by polarization, fluorescence, and X-ray studies. Subunits (quantasomes) may be observed in the electron microscope on dried shadowed membranes and in replicas of membranes produced by the freeze-etching technique. Regular subunits are also observed with the electron microscope in thin sections of chloroplast membranes. Chemical considerations suggest that many membranes are composed of lipoprotein subunits. Thin sections reveal two types of chloroplast membranes, the fret membranes composed of one layer of subunits, and the partitions composed of two layers of subunits. Chloroplast membranes consist of about 45% protein and 55% lipid. Some 80% of the lipids are the highly surfactant glycolipids. In this paper the subunits are visualized as assymetric lipoproteins, probably having a protein core surrounded by components determined by the nature and environment of the membrane. Since the stroma, fret channels, and loculi contain aqueous materials, it is further postulated that the membranes bordering these spaces bind the highly surfactant glycolipids. The region between the two rows of subunits in the partition appears to be highly hydrophobic, rich in chlorophyll, and low in glycolipids. Some chlorophyll also may occur within the subunits both in the partitions and in the fret membranes. Since four subunits appear to comprise a quantasome, at least two types of forces, inter- and intra-quantasome forces, bind the subunits together in sheets. Chloroplast membranes thus differ from a “unit membrane” in two important respects: (1) they must be an aggregate of globular subunits, and (2) the lipoprotein subunits consist of a protein matrix which binds the chlorophylls and lipids by hydrophobic association with their hydrocarbon moieties.     

THE INTRACELLULAR LOCALIZATION OF PITUITARY THYROTROPIC HORMONE

The intracellular localization of a bovine anterior pituitary preparation of thyroid-stimulating hormone (TSH) was studied in guinea pigs and dogs. The preparation was administered intravascularly or applied directly to tissue sections. TSH was detected by an indirect technique utilizing bovine TSH antiserum and fluorescein-labeled anti-rabbit globulin; the presence of TSH in the tissue was indicated by fluorescence when the tissue was examined under the microscope with an ultraviolet light source. After either intravascular administration or direct application of the TSH preparation, striking fluorescence was found in the nuclei of the thyroid cells and to a lesser degree in the nuclei of retro-orbital fat tissue and kidney tubules in both species studied. A little fluorescence was also seen in spleen tissue. No fluorescence was noted in comparable tissues removed from control animals injected with bovine albumin or globulin or when the tissues were treated with the fluorescein-labeled globulin alone. Fluorescence was also noted in the nuclei of adrenal cells treated with unabsorbed antiserum, but this was greatly diminished when antiserum absorbed with crystalline ACTH was used. The positive reactions were all markedly decreased when the tissues were treated with antisera absorbed with the original TSH preparation. Fluorescence was noted in the cytoplasm of pituitary tissue from both treated and control animals, suggesting a cross-reaction between the bovine pituitary antisera and guinea pig or dog hypophysis. The indirect technique seems to be highly satisfactory for demonstration of the pitiutary hormone within the cell. In addition, the demonstration of immunologically active anterior pituitary TSH bound to cell nuclei offers a clue to the site of action of this hormone.     

层粘连蛋白与膜受体结合下膜受体构象与膜脂有序性的研究

配体蛋白与细胞膜受体蛋白结合后,可引起膜受体的构象与膜脂的有序性变化.本文研究外源性层粘连蛋白与腹水肝癌细胞膜受体结合后膜热量变化,膜序参数改变和膜电荷及细胞迁移率的变更.就膜蛋白构象与膜脂有序性以及膜电荷等方面改变的生理意义与层粘连蛋白抗癌细胞脱落转移寻找理论关系.本文应用微量量热法、顺磁共振和细胞电泳等技术,得知层粘连蛋白与癌细胞膜作用后细胞膜有放热效应,膜流动性增大,细胞电泳动变慢.癌细胞膜的这些变化对于限制癌的恶性生长与脱落均起重要作用.     

INTRACELLULAR LOCALIZATION OF KYNURENINE IN THE FATBODY OF DROSOPHILA

Light blue fluorescent globules accumulate in the cells of the anterior region of the fatbody of Drosophila larvae near the time of pupation. This fluorescent material appears in the Ore-R wild type strain as well as mutant strains in which the synthesis of both the red and brown eye pigments is affected. The vermilion mutant, which is characterized by the absence of the brown pigment component in the eye, was the only strain among those examined which did not develop the light blue fluorescent globules. Utilizing chromatographic techniques together with the information gained by examination of the mutant strains, the fluorescent material has been identified as kynurenine. Of particular interest is the manner of appearance of the fluorescent material in the vicinity of the nuclear membrane of the fat cells.     

THE REVERSAL OF THE SIGN OF THE CHARGE OF MEMBRANES BY HYDROGEN IONS

1. It had been shown in previous papers that when a collodion membrane has been treated with a protein the membrane assumes a positive charge when the hydrogen ion concentration of the solution with which it is in contact exceeds a certain limit. It is pointed out in this paper that by treating the collodion membrane with a protein (e.g. oxyhemoglobin) a thin film of protein adheres to the membrane and that the positive charge of the membrane must therefore be localized in this protein film. 2. It is further shown in this paper that the hydrogen ion concentration, at which the reversal in the sign of the charge of a collodion membrane treated with a protein occurs, varies in the same sense as the isoelectric point of the protein, with which the membrane has been treated, and is always slightly higher than that of the isoelectric point of the protein used. 3. The critical hydrogen ion concentration required for the reversal seems to be, therefore, that concentration where enough of the protein lining of the membrane is converted into a protein-acid salt (e.g. gelatin nitrate) capable of ionizing into a positive protein ion (e.g. gelatin) and the anion of the acid used (e.g. NO₃).     

THE ULTRASTRUCTURE OF LIPID-DEPLETED ROD PHOTORECEPTOR MEMBRANES

The structure of lipid-depleted retinal rod photoreceptor membranes was studied by means of electron microscopy. Aldehyde-fixed retinas were exhaustively extracted with acetone, chloroform-methanol, and acidified chloroform-methanol. The effect of prefixation on the extractability of lipids was evaluated by means of thin-layer chromatography and fatty acid analysis. Prefixation with glutaraldehyde rendered 38% of the phospholipids unextractable, while only 7% were unextractable after formaldehyde fixation. Embedding the retina in a lipid-retaining, polymerizable glutaraldehyde-urea mixture allows a comparison of the interaction of OsO₄ with lipid-depleted membranes and rod disk membranes which contain all their lipids. A decrease in electron density and a deterioration of membrane fine structure in lipid-depleted tissue are correlated with the extent of lipid extraction. These observations are indicative of the role of the lipid bilayer in the ultrastructural visualization of membrane structure with OsO₄. Negatively stained thin sections of extracted tissue reveal substructures in the lipid-depleted rod membranes. These substructures are probably the opsin molecules which are the major protein component of retinal rod photoreceptor membranes.