Cell culture of sixteen-day-old rat embryo cerebra and associated changes in ganglioside pattern

Abstract— Cortical slices from rat brain were incubated in Krebs-Ringer phosphate medium. Activity of the pyruvate dehydrogenase complex (PDH) was measured in homogenates of the incubated tissue. Increasing the extracellular KCI concentration from 5 to 75 mM caused a dose-dependent increase in activity of this rate-limiting mitochondrial enzyme. The increase in PDH activity, produced by high concentration of KCI. was associated with a decrease in the tissue content of ATP. Omission of calcium, or replacement of sodium by choline, reduced, and addition of ouabain prevented, the activation of the enzyme in the depolarized tissue.
The mechanism by which extracellular potassium can affect PDH activity is unknown. However, it is most likely that the alterations in enzyme activity are related to changes in properties of cell membranes during depolarization leading to intracellular events directly affecting the enzyme complex. These could include alterations in the concentrations of adenine nucleotides or free calcium ions in the cell.     

The biological half life and distribution of 125Iodide and radioiodinated protein in the imported fire ant, Solenopsis invicta

The radioisotope ¹²⁵Iodide, a gamma emittor, was used in two different forms, as ¹²⁵I mixed with egg yolk and as ¹²⁵I covalently attached to egg albumin and mixed with egg yolk, to study food flow in the imported fire ant, Solenopsis invicta Buren. The biological half life of ¹²⁵I-albumin in egg yolk powder was determined to be 96 hr in isolated workers, 108 hr in individuals held with small groups of unlabelled workers, and 1,008 hr in workers held in colonies exposed to labelled food for 48 hr. In contrast, the biological half life of free ¹²⁵I mixed with egg yolk powder was 22 hr, 20 hr, and 40 hr, respectively.The internal distribution of radioactivity was checked after 24,48, and 380 hr. There was a significant difference in distribution of ¹²⁵I in ants fed either free ¹²⁵I or ¹²⁵I-albumin. Most of the free ¹²⁵I was rapidly excreted. A high percentage of ¹²⁵I-albumin was assimilated, apparently through protein digestion pathways with eventual storage in or below the cuticle. There was no evidence of gland involvement in food flow to either larvae or queens with the radio-iodinated protein.

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Résumé L'utilisation de l'iode radio-actif (¹²⁵I) a permis d'étudier le cheminement de la nourriture chez Solenopsis invicta Buren (Myrmicinée). Deux formes différentes de l'isotope ont été étudiées. L'iode 125 a été fixé d'une manière covalente à la tyrosine dans l'albumine des oeufs en utilisant la méthode chloramine T pour ioder les protéines. L'albumine marquée a été mangée ensuite à du jaune d'oeuf en poudre.La seconde forme contenait de l'iode 125 mélangé au jaune d'oeuf en poudre en absence de tout catalyseur, ce qui empêche la fixation chimique. La demi-vie biologique (T_biol) des deux formes a été déterminée chez des ouvrières isolées, chez des individus gardés avec de petits groupes d'ouvrières non-marquées, et chez des ouvrières gardées dans des colonies exposées à la nourriture radio-active pendant 48 h. La demi-vie biologique de l'albumine marquée était de 96 h, 108 h, et 1.008 h. En contraste, la demi-vie de l'iode 125 était de 22 h, 20 h, et 40 h. L'effet de groupe créé par des échanges répétés de nourriture entre les individus était négligeable avec la nourriture protéique. L'échange répété de nourriture entre les larves et les ouvrières a beaucoup augmenté la demi-vie de l'albumine marquée à I 125. Cet effet n'était pas aussi clair avec l'iode 125 par suite de son élimination rapide.La distribution de la radio-activité a été examinée chez des ouvrières au bout de 24, 48 et 380 h après les avoir nourries avec de l'albumine marquée à I 125 et de l'iode 125. Il y avait une différence considérable de distribution, avec un haut pourcentage d'albumine assimilé, sans doute par les voies de digestion de protéines. Le radio-isotope a été ensuite conservé sous forme d'iode 125 ou d'iodotyrosine, dans (ou sous) la cuticule de l'ensemble du corps. Les fourmis ont rapidement excrété l'iode 125 libre avec 5% de la radioactivité résiduelle après 380 h, peut-être fixée aux protéines cellulaires, et ensuite transportée vers la cuticule. Les différences considérables entre les demi-vies biologiques et la distribution interne de la radioactivité chez les fourmis nourries avec de l'iode 125 ou à l'albumine marquée à I 125, soulignent le danger de croire que le cheminement de la nourriture peut être définitivement étudié en utilisant des radio-isotopes qui ne sont pas fixés chimiquement à la substance étudiée.

     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

DIFFERENTIAL ENRICHMENT OF CELLS FROM EMBRYONIC RAT CEREBRA BY CENTRIFUGAL ELUTRIATION

—Centrifugal elutriation was used to obtain different populations of cells dissociated from 16-day-old rat embryo cerebra. The cell populations recovered were viable and could be maintained for several weeks in vitro. Sterile conditions were maintained throughout a preparation. Rat pups were removed by Caesarean section, the cerebra dissected and the cells dissociated by brief exposure to trypsin (0.125%, 6 min). An equivalent volume of elutriation medium (Dulbecco's medium containing 1% fetal calf serum, sodium bicarbonate, penicillin and streptomycin, EDTA, and deoxyribonuclease) was added to the trypsin-cell suspension, the dissociated cells pelleted, resuspended in elutriation medium and counted. Up to 4 x 10⁸ cells were injected into the previously sterilized elutriator. Seven fractions were usually recovered from a preparation. The first fraction contained primarily red blood cells and cell debris, which could not be maintained in vitro. Upon culture, fraction 2 consisted of predominantly non-neuronal cells, while fractions 3–6 contained neuronal and non-neuronal cells. The morphological characteristics of the neurons differed in these fractions. Fraction 7 contained cells that had reaggregated during the elutriation procedure and exhibited a variety of cell types in vitro.     

Seasonal patterns of CO2 and water vapor exchange of three salt marsh succulents

Summary Diurnal carbon dioxide exchange patterns of three salt marsh succulents, Borrichia frutescens, Batis maritima and Salicornia virginica, were determined on a seasonal basis in the marsh at Sapelo Island, Georgia. Year-round photosynthetic activity was observed in these species though winter rates of CO₂ exchange were reduced. Net primary productivity, estimated using gas exchange techniques, agreed with previously reported harvest data. The role of light and temperature in the control of seasonal photosynthetic changes was investigated. A similar variation in light utilization with season was found in all three species, while seasonal temperature acclimation was species dependent. Less than 20% of fixed CO₂ was lost through dark respiration in any of the species.Water use in the salt marsh succulents was found to be relatively inefficient. High rates of transpiration were observed both summer and winter in the succulents.The succulents were judged to be C₃ plants on the basis of several criteria.Contribution No. 391 from the University of Georgia Marine Institute     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

Lethal white foals in matings of overo spotted horses

Overo is a variable pattern of white coat color spotting which occurs in several breeds of horses in the United States. Occasionally, when overos are crossed inter se white foals are born which die soon after birth. Both intestinal tract abnormalities and isoerythrolysis have been reported in these foals. This report presents data which show that neonatal isoerythrolysis (NI) is not involved in the death of the white foals. Further research is needed to define the nature of the lethal anomaly, as well as the genetics, of overo and lethal white foals.     

An unusual allantoinase from Dolichos biflorus

An unusual allantoinase from Dolichos biflorus has been purified 62-fold. The purified enzyme has an unusual pH activity profile with a shoulder at pH 4 and a peak at pH 7.5. This is due to a single enzyme which does not need metal ions for activation. In the fully reduced state the enzyme exhibits a single sharp peak at 7.5; when it is not in the sulfhydryl form (in the fully oxidized SS form?) the enzyme shows a single pH optimum at pH 4. K_m values for (±)-allantoin were 5.5 mM at pH 4 and 1.43 mM at pH 7.5. Allantoinase activity has been demonstrated in the resting seed, and increased linearly with time during the first 5 days of seedling growth.     

Intracellular recording of cerebral cortical actions of prostaglandin F2α (PGF2α)

Our reported data on the cortical inhibitory actions of prostaglandin F_2α (PGF_2α) and the diversity of data in the literature on cerebral PG actions are examined here in the light of intracellular recording which provides the requisite membrane data for the first time. Thus, 1) intracellular recording from the cat cerebral cortex is obtained for the actions of PGF_2α and for norepinephrine (NE) and serotonin (5HT). 2) The parallel changes in firing and polarization and the simultaneous transmembrane conductance changes are qualitatively identical for PGF_2α, NE and 5HT. 3) The reduction in firing accompanied by hyperpolarization indicates that PGF_2α, NE and 5HT all inhibit these cells. 4) The ionic species responsible for this inhibition is such that it increased the transmembrane resistance, and this was true for all three. 5) The changes in membrane parameters, identical in direction for PGF_2α and NE, but stronger for the latter, constitute conditions that can lead to competitive inhibition and therefore conote, presumably, actions at the same or related receptors. Such competition with evoked cortical field potentials is shown in the preceding paper.