Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

Abstract: Plasma membranes were isolated from C6 glioblastoma cells by two methods. In the first method cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with a-methyl-D-mannoside. In the second method untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtaincd by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of mem- brane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a "lighter")membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a "heavier" membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of ¹²⁵I-labeled cell surface proteins. Their specific (Na⁺+ K⁺)-ATPase activity was seven- to eightfold the homogenate activity (recovery, 13.1%). Both fractions were, however, still contaminated by smooth endo- plasmic reticulum, as judged from the activity 0: NADPH-dependent cytochrome c reductase (recovery, 2.4%). It is suggested that plasma membrane fragments present in the two fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.     

IN-SITU MORPHOLOGY AND OCCURRENCE OF EUCARYOTIC PHOTOTROPHS OF BACTERIAL SIZE IN THE PICOPLANKTON OF ESTUARINE AND OCEANIC WATERS1

Concentrates of the picoplankton (0.2–2.0 μm) sized fraction from the euphotic zone of estuarine and oceanic waters were examined by transmission electron microscopy. In addition to the numerous phototrophic procaryotes (chroococcoid cyanobacteria) previously reported, small phototrophic eucaryotes were observed in 20 of 25 samples examined. Micromonas pusilla (Butcher) Manton and Parks, a 1 × 1.5 μm flagellate, was abundant in estuarine samples in summer. Similar sized cells of non-flagellated chlorophytes, either Nannochloris Naumann or Chlorella Beijerinck, were observed sporadically in many samples. The most ubiquitous microalga was a scaled, non-flagellated prasinophyte that occurred at 9 of 15 different locations on 15 of 20 sampling dates in water samples from Iceland to the Caribbean Sea, This tiny alga (0.5 to 1.0 μm in diam.) is probably the smallest known photo-trophic eucaryote and has not heretofore been described. Enrichment cultures using conventional techniques on several cruises yielded only the Chlorella-type of green alga, as well as numerous isolates of unicellular chroococcoid cyanobacteria.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

Time-correlated flights of juvenile and mature locusts: A comparison between free and tethered animals

The flights of free and tethered Locusta migratoria were followed from initiation with a high-speed film camera. A longer sequence of wing-beat cycles can thus be correlated unequivocally with the animals's movement in time and space. In both flight situations the locusts start with approximately the same instantaneous wing-beat frequency. During the early flight phase free-flying animals increase their wing-beat frequency, whereas for tethered locusts this parameter remains constant or even decreases. The general flight pattern is similar in juvenile and mature locusts; the juveniles however, fly with alower wing-beat frequency and flight speed. The differences in the wing-beat frequencies for both flight performances are discussed with respect to differences in the sensory inputs to the flight motor centre.     

Biochemistry and ultrastructure of iridescent virus type 29

Physical and chemical parameters of iridescent virus type 29, isolated from the mealworm, Tenebrio molitor, have been analyzed. The icosahedral capsid is 130–135 nm in diameter and is surrounded by a fringe of coarse filaments. The virus has a buoyant density in CsCl of 1.31 g cm^?3 and contains 20 to 25 structural proteins as analyzed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. The DNA has a buoyant density in CsCl of 1.6874 g cm^?3 indicating a G + C content of approximately 28%. The lipid components of this virus differ from those of the host cell; the virus contains about 80% cardiolipin and 20% phosphatidyl choline.     

Reduction in Brain Glucose Utilization Rate after Tryptophol (3-Indole Ethanol) Treatment

Abstract: 3-Indole ethanol has been recently identified as the hypnotic agent in trypanosomal sleeping sickness, and because it is formed in vivo after ethanol or disulfiram treatment, it is also associated with the study of alcoholism. When administered intraperitoneally to rats (250 mg/kg) tryptophol induced a sleep-like state that lasted less than an hour (no righting reflex was apparent 2 min after injection, but it returned at 11 min in bovine serum albumin solution, and 47 min in 40% ethanol solution). In ethanol solutions, tryptophol reduced brain cortical glucose utilization by 55% to the basal brain metabolic rate, and this effect lasted less than 1 h. Synergistic effects of tryptophol and ethanol were suggested by the observation that in albumin solution, tryptophol reduced brain glucose utilization by 35%, but a normal rate was not observed until 2 h postinjection.     

Effectors of lipoprotein lipase secretion from isolated cardiac muscle cells incubated in vitro

Cycloheximide, colchicine, tunicamycin, glucagon, dibutyryl-3′–5′-cyclic AMP, dexamethasone and hydrocortisone had no effect on the lipoprotein lipase activity associated with rat cardiac muscle cells incubated $\text{in vitro}$. However, the steroid hormones and inhibitors affected profoundly the appearance of extracellular enzyme during the incubations. The pattern of effects, was consistent with lipoprotein lipase being a normal secretory product of heart muscle cells.     

Spinal analgesia: Comparison of the mu agonist morphine and the kappa agonist ethylketazocine

The sites of analgesic action of the mu agonist morphine and the purported kappa agonist ethylketazocine (EKC) were compared. Using local drug injections and parenteral administration of drugs to spinalized rats, our data support a predominantly spinal site of action for EKC and a major supraspinal action for morphine in antinociceptive tests. This spinal analgesic action of EKC was dose dependent and naloxone reversible indicating opiate receptor involvement. The possibility that EKC activates a spinal kappa receptor population is under further study.     

Age-related motor and catecholomine alterations in mice on levodopa supplemented diet

Old mice reared on regular diet show reduced motor activity, decreased basal adenylate cyclase, and increased MAO activities compared to adults. Brain DDC and COMT activities, DA, NE levels and DA-stimulated adenylate cyclase remained unchanged. By contrast, mice fed levodopa for life did not develop decreased motor activity with aging, lived about 50% longer, had slightly elevated whole brain DA and NE levels and failed to develop the expected rise in MAO activity with aging. Levodopa did not alter the number of dopaminergic and muscarinic cholinergic receptors or the adenylate cyclase activity in the striatum during aging. On levodopa, hepatic and renal DA, dopa, and HVA increased but the latter two returned to basal levels by mid life. In liver, DDC was unchanged but MAO tended to be higher in levodopa-fed mice. Thus, motor impairment is an age-related phenomenon in mice associated with selective alterations in brain dopaminergic systems, which may be prevented by dietary levodopa. Extracerebral tissues, through possibly adaptive metabolic mechanisms, play a significant role in regulating brain catecholamines during chronic administration of large doses of levodopa.     

Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe

Summary A total of 59 new temperature sensitive cdc mutants are described which grow normally at 25°C but become blocked at DNA replication or mitosis when incubated at 36°C. Thirtynine of the mutants are altered in cdc genes which have been identified previously. The remaining 20 mutants define 10 new cdc genes. These have been characterised physiologically, and 6 of the genes (cdc 17, 20, 21, 22, 23, 24) were found to be required for DNA replication, 2 for mitosis (cdc 27, 28), and 2 (cdc 18, 19), could not be unambigously assigned to either DNA replication or mitosis but were definitely required for one or the other.Three genes, the previously identified cdc 10, and cdc 20, 22 are likely to be required for the initiation of DNA replication. Mutants in two genes, cdc 17, 24 undergo bulk DNA synthesis at 36°C, but this DNA is defective. In the case of cdc 17 the defect is in the ligation of Okazaki fragments. cdc 23 is required for bulk DNA synthesis, whilst cdc 21 may possibly be required for the initiation of a particular sub-set of replicons.A previously isolated mutant cdc 13.117 is also further described. This mutant becomes blocked in the middle of mitosis with apparently condensed chromosomes.