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PubMed1978

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.

Adenosine Triphosphatases↗

PubMed1983

Preparation of multivesicular liposomes.

A novel type of liposome, named here multivesicular liposomes, was prepared by evaporation of organic solvents from chloroform-ether spherules suspended in water. Within each spherule were numerous water droplets that contained solutes to be trapped in liposomes upon solvent evaporation. Liposome preparations of different average diameters were made, varying from 29 +/- 10 microns to 5.6 +/- 1.7 microns. The liposomes were morphologically characterized by light microscopy and transmission electron microscopy. Materials successfully trapped within the liposomes ranged in molecular size from glucose to nucleic acids. Extremely high percentages of encapsulation (up to 89%) were achieved.

Chemical Phenomena↗

PubMed1985

Preparation of multilamellar vesicles of defined size-distribution by solvent-spherule evaporation.

A novel method of preparing multilamellar vesicles is described. The process involves dispersing in aqueous solutions small spherules of volatile hydrophobic solvents in which amphipathic lipids are dissolved. The lipids form vesicles when the solvents are evaporated in the proper manner. The resulting vesicles have been characterized morphologically with microscopy and electron microscopy. The method yields multilamellar vesicles with a defined size distribution which can be adjusted by varying the duration of mechanical agitation of the spherules and by varying the concentration of amphipathic lipids in the solvents. This is the first fundamentally new method of multilamellar vesicle preparation since Bangham's report in 1965 (Bangham, A.D., Standish, M.M. and Watkins, J.C. (1965) J. Mol. Biol. 13, 238-252).

Liposomes↗

PubMed1986

Physicochemical and immunological properties of albumin-associated dialkyl-ether phosphatidylcholine liposomes.

Multilamellar and unilamellar liposomes were prepared from sn-3-dihexadecylphosphatidylcholine/cholesterol/dicetylphosphate (7:2:1) in the presence of bovine serum albumin. Liposome-associated bovine serum albumin was separated from free bovine serum albumin by Blue Sepharose CL-6B affinity column chromatography. The chromatographed fractions were analyzed for their protein and liposomal phosphorus contents. The recovered albumin-containing liposomes were characterized morphologically by electron microscopy on negatively stained preparations. These preparations showed vesicular organizations of multilamellar or unilamellar phospholipid bilayers depending on the method of preparation used in each case. An analysis of the particle size distribution indicated that the mean radius was 280 +/- 50 nm for the multilamellar bovine serum albumin-liposomes and 150 +/- 50 nm for the unilamellar preparations. The efficacy of unilamellar and multilamellar dialkyl-ether phosphatidylcholine liposomes in eliciting antibody formation was examined. Mice were injected with liposome-entrapped bovine serum albumin and the albumin-specific plaque-forming cell response was evaluated. The unilamellar vesicles were found to be more effective than their multilamellar counterparts in promoting the elicitation of the anti-bovine serum albumin plaque-forming cell response. Within each category of lamellar structure, i.e., unilamellar or multilamellar bilayers, liposomes composed of dialkyl-ether phosphatidylcholines are less efficient than those of diacyl-ester phosphatidylcholines in potentiating the humoral immune response. These results demonstrate that liposome-mediated enhancement of the antibody response is determined, at least in part, by the lamellar arrangement of the vesicles and by the characteristic chemical structures of the phospholipids used.

Animals↗

PubMed1982

Immunosuppressive agents prevent guanethidine-induced destruction of rat sympathetic neurons.

Chronic administration of guanethidine to rats causes destruction of peripheral sympathetic neurons. Neuronal destruction, characterized morphologically by small cell infiltration and the reduction in the number of neurons within sympathetic ganglia, and biochemically by a marked reduction in tyrosine hydroxylase activity, occurred reproducibly by day 7 of treatment following 5 daily injections of 50 mg/kg guanethidine sulfate. Several observations in the literature suggested that guanethidine-induced destruction may occur by an immunologically mediated mechanism. Experiments were therefore designed to test the effects of immunosuppressive agents on guanethidine sympathectomy. A single exposure to either gamma-irradiation or cyclophosphamide, administered 8 h prior to the initiation of guanethidine treatment, protected against guanethidine-induced destruction in a dose-related manner and was virtually complete with either 900 rads of irradiation or with 100 or 150 mg/kg of cyclophosphamide. Cyclophosphamide afforded complete protection only if administered immediately prior to guanethidine treatment suggesting that it was acting during the proliferative phase of an immune response rather than non-specifically. Pretreatment with either irradiation or cyclophosphamide had no effect on the sympathectomy produced by treatment with either 6-hydroxydopamine or antibodies to nerve growth factor, nor did it prevent the accumulation of guanethidine within the sympathetic ganglia. Concurrent treatment with either azathioprine or dexamethazone also provided partial protection against guanethidine sympathectomy. These results strongly suggest that the destruction of sympathetic neurons induced by guanethidine occurs by an immunologically mediated mechanism.

Animals↗

PubMed1985

Altered patterns of evoked synaptic activity in cortical pyramidal neurons in feline ganglioside storage disease.

Postsynaptic potentials evoked by ventrolateral thalamic stimulation were recorded intracellularly from neurons in the precruciate cortex of GM1 mutants with HRP- or LY-loaded microelectrodes. Ganglioside-laden pyramidal neurons exhibiting somal distention and/or meganeurite formation were found to respond to thalamic stimulation with short duration IPSPs. Evoked EPSPs were recorded from two morphologically characterized large basket intrinsic neurons which deployed extensive intracortical axonal arborizations. These findings point to the preservation of intracortical inhibitory networks in the feline model of GM1 gangliosidosis, and to the possibility of abnormal integration of somadendritic inputs in ganglioside-laden pyramidal neurons.

Animals↗

PubMed1989

Neuropeptide Y-like immunoreactive amacrine cells in the retina of Bufo marinus.

Neuropeptide Y-like immunoreactive (NPY-LI) amacrine cells of the Bufo marinus retina were morphologically characterized, and their retinal distribution was established using immunohistochemistry on retinal wholemount preparations and sectioned material. The somas of NPY-LI amacrine cells were situated in the innermost part of the inner nuclear layer and their dendrites branched primarily in the scleral sublamina of the inner plexiform layer. A subgroup of the NPY-LI cells had dendrites in both the scleral and vitreal sublamina. All immunoreactive cells had large dendritic fields (average 0.5 mm2) that resulted in a high dendritic overlap across the retina. NPY-LI amacrine cells were evenly distributed across the retina, with an average density of 30 cells/mm2, although higher densities were observed at regions adjacent to the ciliary margin. The dendritic field size of the NPY-LI cells, together with the previously characterized substance P-like immunoreactive (SP-LI) amacrine cells, indicates that they belong to the class of wide-field amacrine cells. However, unlike the SP-LI neurons whose dendrites branch in the vitreal sublamina of the inner plexiform layer, the dendrites of the majority of the NPY-LI neurons branch in the scleral sublamina.

Animals↗

PubMed1989

Morphological analysis of single astrocytes of the adult cat central nervous system visualized by HRP microinjection.

The morphology of individual astrocytes of the adult cat was analyzed by HRP microinjection and light microscopy. The astrocytes had generally two types of processes: (1) thread-like processes of relatively constant width with few ramifications and few lamellar appendages and (2) the sinuous processes with clusters of lamellar appendages. The former processes were morphologically characterized as follows: (1) Those of fibrous astrocytes were frequently remarkably long, ranging from 600 to 1500 microns. They were much longer than any astrocytic processes hitherto reported in the literature. In contrast, those of protoplasmic astrocytes were usually short (30-400 microns), and were sometimes decorated with lamellae. (2) The processes often terminated in endfeet on the subpial surface of the brain tissue or on blood vessel walls. The number of endfeet per cell varied from 1-11 with a tendency to split into two populations close to each extreme number. Another type of endfoot was also found, i.e. swellings along the trunk of the processes which made side-to-side contact with the vessel wall. The sinuous processes rich in lamellae were predominant in protoplasmic astrocytes, and clearly corresponded to the sheet- or veil-like processes of Golgi-impregnated astrocytes. They formed an ellipsoidal field (100 microns for the longer, and 50-60 microns for the shorter, diameter) around the nucleus.

Animals↗

PubMed1994

Observation of the highly organized development of granule cells in rat cerebellar organotypic cultures.

Cerebellar slices of 9-day-old rats were cultured for a week at the interface between air and a culture medium containing horse serum and hormone cocktail, and the development of granule cells was characterized morphologically. The typical layered structure of the cerebellar cortex was well preserved during the cultivation. Many granule cells in the external granular layer (EGL) proliferated actively within the early culture period. They were migrating downward at 3 days in vitro (DIV) and almost completed the migration to the internal granular layer (IGL) after 6 DIV. In the middle and upper molecular layer (ML), parallel fibers were elongated horizontally, which is the orientation 90 degrees rotated compared to that in vivo. They formed synapses with Purkinje cell dendrites. Regional differences in synapse density and maturity existed which might reflect a gradient in progressive synapse formation comparable to that in vivo. Thus, a serial process of highly organized development of granule cells was realized for the first time in vitro, although some spatial or temporal modifications existed. Such a culture system could be a useful experimental model for the study of cellular and molecular mechanisms of spatiotemporally organized neuronal development.

Animals↗

PubMed1994

Action of vasopressin on hypoglossal motoneurones of the rat: presynaptic and postsynaptic effects.

The distribution of vasopressin binding sites in the hypoglossal nucleus of newborn rats was determined using autoradiography on film and a radioiodinated vasopressor antagonist. These sites predominated in the ventromedial and dorsal divisions of the nucleus. The effect of vasopressin on hypoglossal neurones was studied in brainstem slices of newborn animals, using the single-electrode voltage-clamp technique. Vasopressin, at 0.1-0.5 microM, generated a sustained inward current in a majority of neurones, an action which was mediated by V1-type receptors. Antidromic activation or morphological characterization of biocytin-labelled neurones indicate that part of the vasopressin-sensitive cells were motoneurones. When synaptic transmission was blocked by perfusing the preparation with a low-calcium/high-magnesium solution, the average vasopressin current decreased by 65%; and following TTX treatment, the peptide current decreased by 55%. In contrast, in a low-calcium solution, i.e., under conditions of reduced synaptic transmission but of increased neuronal excitability, the vasopressin current was not significantly altered. These results may be interpreted by assuming that the action of vasopressin is in part postsynaptic and in part presynaptic, the latter effect probably depending upon action potential propagation. Current-voltage relations suggest that the postsynaptic effect of vasopressin was due to the induction of a non-inactivating inward current, reversing in polarity at around -15 mV. The data raise the possibility that, in young animals, endogenous vasopressin may modulate the activity of hypoglossal motoneurones.

Afferent Pathways↗

PubMed1995

Microglial cell reaction in the gray and white matter in spinal cords from jimpy mice. An enzyme histochemical study at the light and electron microscope level.

Jimpy is a genetic disorder which results in a severe hypomyelination in the central nervous system associated with a variety of astroglial and oligodendroglial abnormalities. In this study, we examined the morphology and distribution of microglial cells in spinal cord sections from jimpy and normal mice at 10-12 and 20-22 days postnatal using a specific microglial marker, the nucleoside diphosphatase staining. Compared to those of normal littermates, the spinal cords of jimpy mice showed an intense microglial cell reaction in white and gray matter, as revealed by quantitative analysis and light and electron microscope study. Microglial reactivity was apparent in all spinal cord areas, although it was more pronounced in white than in gray matter. The mean microglial densities in the jimpy white matter were about threefold (10-12 days) and fivefold (20-22 days) higher than in the normal, whereas in the gray matter, microglial density in jimpy was about 60% higher than in normal at both ages. Morphologically, microglial cells in the normal spinal cord showed a ramified appearance, similar in size and ramification pattern to those reported in other normal CNS areas. In contrast, microglial cells in the jimpy spinal cord showed a reactive morphology, characterized by a shortening and coarsening of their cell processes, swelling of their cell body and accumulation of lipid inclusions. Reactive microglial cells were found in close association with axons and oligodendroglial cells. The possible role of microglial cells in hypomyelination is discussed.

Acid Anhydride Hydrolases↗

PubMed1996

Comparative, in vitro, studies of hippocampal tissue from homing and non-homing pigeon.

The purpose of this research was to characterize morphologically and electrophysiologically tissue slices obtained from the hippocampus of homing and non-homing pigeons. When hippocampal slices from the brain of homing and non-homing pigeons are observed under the dissecting microscope, diffuse fiber paths can be seen. These fiber pathways appeared to be identical with the medial fiber tract (VM) previously described histologically in the hippocampus of homing pigeon. Visualization of these tracts in living slices allowed placement of stimulating and recording electrodes in corresponding locations in these slices in both homing and non-homing pigeons. Extracellular potentials recorded from VM regions of the brains of both homing and non-homing pigeons were sensitive to CNQX indicating that glutamate may be a neurotransmitter in this area of pigeon hippocampus. These potentials could undergo long-term potentiation (LTP) following high frequency stimulation. This LTP was blocked by NMDA receptor antagonist APV in the hippocampus of homing pigeon, but was APV-resistant in the hippocampus of non-homing pigeon. Extracellular potentials from the hippocampus of homing pigeons were increased in amplitude when slices were perfused with Mg(2+)-free Ringer, while potential recorded from hippocampal slices from non-homing pigeons wre unaffected by Mg(2+)-free solutions. Intracellular recordings from the hippocampal slices of homing pigeons revealed that about half the cells demonstrated excitatory synaptic potentials evoked by extracellular stimulation. The EPSP was sometimes large enough to trigger an action potential. Neurons filled with the fluorescent dye, Lucifer Yellow, in the hippocampus of homing pigeons showed multipolar structure. The response of these cells to extracellular stimulation provides the activity responsible for the extracellular potentials which can undergo LTP.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1992

Induction of manganese superoxide dismutase in cultured human trophoblast during in vitro differentiation.

The antioxidant responses of human cell differentiation and membrane fusion are not known and may be important in understanding cellular response to injury in the human placenta. We studied the regulation of antioxidant enzymes in human trophoblasts which differentiate from mononucleated cellular trophoblasts to synctium in vivo and in culture. We characterized morphological and biochemical differentiation of cultured trophoblasts from term placenta in the presence or absence of serum, on different growth surfaces, and with a range of plating densities. Culture of cellular trophoblasts consistently and transiently induced the mRNAs of the mitochondrial antioxidant manganese superoxide dismutase (Mn SOD) but not the mRNAs for the antioxidant enzymes copper zinc SOD or catalase. Fibrin and type I collagen substrates modulated only the expression of the placental specific proteins, human chorionic gonadotropin, and human placental lactogen. Both Mn SOD induction and terminal differentiation, as reflected by human chorionic gonadotropin expression, were dependent on trophoblastic plating density. Increased levels of a smaller Mn SOD mRNA species correlated temporally with an increase in Mn SOD enzyme activity in cultured trophoblasts. These results demonstrate that Mn SOD gene expression and enzyme activity precede or are coordinately regulated with morphological and biochemical trophoblastic differentiation.

Antioxidants↗

PubMed1991

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents.

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxyellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants, ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.

Animals↗

PubMed1988

Biochemical characterization of retinal protein and phospholipid synthesis in mice exposed transplacentally to N-methyl-N-nitrosourea.

Transplacental exposure to the DNA alkylating agent N-methyl-N-nitrosourea on day 16 of gestation in CD-1 albino mice induces a degeneration of the retina, the severity of which depends upon the dosage level of the drug. A 1 mg kg-1 dose provokes a progressive retinal degeneration in the offspring which begins at about 4-6 weeks of age and is characterized by gradual thinning of the retinal layers. A 15 mg kg-1 dosage of MNU provokes severe retinal dysplasia characterized morphologically by rosettes in the outer nuclear layer and loss of rod outer segments (ROS). In the present biochemical experiments, retinal protein synthesis was examined in mice 2-, 4-, and 6 weeks of age exposed to 1 mg kg-1 MNU and 2- and 5 weeks of age exposed to 15 mg kg-1 MNU. Phospholipid synthesis was examined in mice 2-, 4-, 6- and 12 weeks of age exposed to 1 mg kg-1 MNU and at 2 weeks in mice exposed to 15 mg kg-1 MNU. Retinas were incubated for 2 hr at 37 degrees C in media supplemented with either [3H]leucine for protein synthesis studies or [3H]glycerol for phospholipid synthesis experiments. Aliquots of crude ROS and the retinal debris were taken for protein determination, scintillation counting, SDS-PAGE separation of labeled opsin, phosphorus determination and TLC separation of phospholipids. Results indicated that mice exposed to 1 mg kg-1 MNU did not differ significantly from age-matched controls in these measurements, whereas mice exposed to 15 mg kg-1 MNU were significantly different from controls. These results suggest that even as early as 2 weeks of age protein and lipid metabolism are adversely affected in mice exposed to the higher dose of the alkylating agent at a critical time in retinal development, but general protein and lipid synthesis is not affected in animals exposed to 1 mg kg-1 MNU at least up to 12 weeks of age. These studies suggest further investigation of more subtle derangement in the retinal function in animals exposed to low levels of MNU.

Animals↗

PubMed1991

Isolation of brain parenchymal lymphocytes for flow cytometric analysis. Application to acute viral encephalitis.

A strategy for the isolation of mononuclear cells from the brain parenchyma of mice with ongoing central nervous system (CNS) inflammation has been developed in order to permit flow cytometric (FCM) analysis of these cell populations. Sindbis virus (SV) encephalitis in mice is characterized morphologically by an infiltration of mononuclear cells into both brain parenchyma and cerebrospinal fluid (CSF). Perfused brain tissue from infected animals is collected, homogenized, and subjected to a mild enzymatic digestion. A sedimentation at unit gravity is performed to remove any large particulate debris, and the remaining tissue is then centrifuged over a modified density gradient which separates intact cells from smaller tissue fragments. Cells collected directly from these gradients can be stained with monoclonal antibodies and analyzed by FCM without further manipulation. Data generated by this method correlates with previous studies of SV encephalitis using immunohistochemical analysis of brain tissue sections to quantify mononuclear cell types. This suggests that representative samples of the cellular infiltrate are obtained using this technique. The approach however, offers the possibility of more sophisticated and quantitative analyses of CNS inflammatory cells which is unobtainable by tissue section staining.

Animals↗

PubMed1987

Initial adherence of human capillary endothelial cells to Dacron.

Successful endothelialization of vascular grafts by seeding with endothelial cells (EC) at implantation is related to the number of EC which initially adhere to the graft. Using an in vitro system we examined the initial adherence of EC from human perinephric fat capillaries to woven Dacron that was either unmodified or precoated with several substrates. We studied capillary EC because they have not been investigated as a source of EC for graft seeding, although transinterstitial capillary ingrowth is one possible mechanism for spontaneous graft endothelialization. EC were isolated using collagenase and characterized morphologically and functionally including positive factor VIII-related antigen staining. EC were studied at three phases in culture: (A) primary EC with no subcultivations (EC-0); (B) EC after two subcultivations with trypsin (EC-2); and (C) EC after 10 subcultivations with trypsin (EC-10). EC were seeded onto graft material at a density of 10(5) cells/cm2 (100% confluence) and examined for cell counts and morphology after one day in culture by light and electron microscopy. Results are as follows: (table; see text) The conclusions are: (1) All capillary EC demonstrated adherence to Dacron, but this initial adherence was strongly influenced by graft pretreatment with collagen or plasma. (2) Serially subcultivated EC (EC-2 and EC-10) had significantly higher initial adherence to pretreated Dacron compared to the primary cells (EC-0) (P less than .05). This suggests that briefly cultured and subcultivated EC have superior initial adherence characteristics to treated dacron compared to primary EC with no subcultivations. (3) Fat capillary EC are easily procured and cultured and provide a rich source of human EC for endothelializing vascular prostheses.

Blood Vessel Prosthesis↗

PubMed1977

Satellite cells and muscle regeneration in diseased human skeletal muscles.

By virtue of the lanthanum nitrate staining technique applied to biopsied muscle we are able to demonstrate interaction between satellite cells and parent myofibers, as well as development of premyocytes from activated satellite cells. The process of regeneration in diseased muscle appears to differ from that described in experimental myogenesis. Transformation of activated satellite cells to two types of premyocytes in the process of muscle regeneration seems to rely primarily on the state of innervation and recovery rate of the parent cell after injury. Activated satellite cells are characterized morphologically by proliferation of caveolae, first on the parent fiber side, and early T-tubule and myofilament formation and central displacement. In diseased human muscle the satellite cells appear to play significant roles in muscle regeneration both as a source of reinforcement for failing metabolism in the parent cell and as potential replacements for the necrotic segment of the parent cell. This study also demonstrates that the satellite cells are capable of developing into independent myocytes which may fuse with or replace the parent cell, dependent upon the type and extent of the injury sustained. Abnormal fusion among premyocytes or with their parent fiber, resulting in formation of split-or ring-fibers, becomes conceivable when both innervation and recovery from the injury of the parent cell are delayed. Thus, myotube formation, characteristic of usual myogenesis, seldom takes place in the regenerative process instituted by satellite cells in diseased human skeletal muscles.

Humans↗