A Comparison of the Polypeptide Pattern Between a Brain Primary Culture and Fractions of Bulk-Isolated Glial Cells

Abstract: This paper reports on the electrophoretic protein/polypeptide pattern of a rat brain primary culture. For comparison, the polypeptide pattern of neuronal and glial enriched fractions from adult rat brain and cerebral hemispheres from newborn and adult rat have been analysed. Water-soluble and SDS-extractable polypeptide fractions appeared and/or increased in amount in the cultures until confluency. The polypeptide pattern of the cultures most resembled that of the glial cell fractions, showing some of this fraction's specificity. Removal of fetal calf serum and addition of 0.1 mM dibutyryl cyclic adenosine monophosphate (dB-cAMP) produced few changes in the electrophoretic pattern. The study thus provides evidence in favour of the astroglial nature of the brain primary culture. It also shows that the cells undergo some maturation in the culture.     

Abiotic formation of particles from extracellular organic carbon released by phytoplankton

¹⁴C-labeled extracellular organic carbon (EOC) released by the phytoplankton in a Danish Estuary was shown immediately to form particles (>0.2m) when the products were added to a natural water sample. About 14%–20% of the added activity could be recovered as particles. Any bacterial assimilation of the extracellular products was thus masked. The abiotic origin of the particulate EOC was verified, and it was shown that the particle formation was due to some factors present in the estuarine water with a nominal diameter >0.2m. Precaution must be taken to avoid misinterpretations in studies concerning carbon flow from algae to bacteria.     

Role of H-2 antigens in the host response to methylcholanthrene-induced tumors

H-2 loss variant sublines of a sarcoma (M-AS), induced by methylcholanthrene in an (A × A.SW)F₁ mouse, were used to study the role of the MHC products in the recognition of MC-TSTA. The two reciprocal variant sublines (M-A and M-S) were found to express the TSTA of the original tumor as shown by cross-reactions in graft rejection experiments performed in (A × A.SW)F₁ mice. In the A/Sn and A.SW mice the presence of the reciprocal parental H-2 antigens on the immunizing cells decreased the response against the tumor antigens. An admixture of lymphocytes derived from hyperimmune mice inhibited the outgrowth of the tumor cells. The growth inhibition was mediated by T cells and was H-2 restricted. Cells derived from hyperimmune syngeneic mice inhibited the outgrowth of the variant subline used for immunization but had no effect on the reciprocal variant subline.     

Control of activity states of heart mitochondrial ATPase. Role of the proton-motive force and Ca2+

The ATPase complex of submitochondrial particles exhibits activity transitions that are controlled by the natural ATPase inhibitor (Gómez-Puyou, A., Tuena de Gómez-Puyou, M. and Ernster, L. (1979) Biochim. Biophys. Acta 547, 252–257). The ATPase of intact heart mitochondria also shows reversible activity transitions; the activation reaction is induced by the establishment of electrochemical gradients, whilst the inactivation reaction is driven by collapse of the gradient. In addition it has been observed that the influx of Ca²⁺ into the mitochondria induces a rapid inactivation of the ATPase; this could be due to the transient collapse of the membrane potential in addition to a favorable effect of Ca²⁺-ATP on the association of the ATPase inhibitor peptide to F₁-ATPase. This action of Ca²⁺ may explain why mitochondria utilize respiratory energy for the transport of Ca²⁺ in preference to phosphorylation. It is concluded that the mitochondrial ATPase inhibitor protein may exert a fundamental regulatory function in the utilization of electrochemical gradients.     

Role of Heme in Synthesis and Membrane Binding of Succinic Dehydrogenase in Bacillus subtilis

A 5-aminolevulinic acid-requiring mutant of Bacillus subtilis was isolated. When the mutant is shifted from medium containing 5-aminolevulinic acid to medium lacking this growth factor, the bacteria continued to grow at undiminished rate for about three generations. The membranes from these bacteria contained severely reduced amounts of cytochrome. The mutant was used to study the role of heme synthesis on synthesis and membrane binding of succinic dehydrogenase (SDH). The amount of SDH in whole-cell lysates in the soluble cytoplasmic fraction and in membranes was determined by one-dimensional (rocket) immunoelectrophoresis with an SDH-specific antiserum. After heme synthesis was blocked, the relative amount of SDH in the membrane decreased, whereas increasing amounts of SDH antigen were found in the cytoplasm. When heme synthesis was resumed on readdition of 5-aminolevulinic acid, the amount of membrane-bound SDH antigen increased at a much faster rate than net synthesis. During a 3-h growth period without 5-aminolevulinic acid, there was little change in the pattern of membrane proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioactively labeled membranes, as compared to membranes from control cultures. However, both the 65,000-dalton and the 28,000-dalton polypeptides of the SDH complex (L. Hederstedt, E. Holmgren, and L. Rutberg, J. Bacteriol. 138:370-376, 1979) were present in decreasing amounts in membranes from 5-aminolevulinic acid-starved bacteria. From these results we suggest that SDH in B. subtilis is synthesized as a soluble protein and becomes membrane bound only when it attaches to a site in the membrane, (part of) which is a cytochrome of b type.     

Fluorescence-microscopical demonstration of a population of gastro-intestinal nerve fibres with a selective affinity for Quinacrine

Summary A population of nerve fibres in the gastro-intestinal tract of mice showing a high affinity for quinacrine was revealed by fluorescence microscopy. Similar results were obtained in rats and guinea pigs. Whole-mounts of sheets of the smooth muscle layer following incubation in 10^-6-10^-7 M quinacrine for 15–60 min revealed fine fluorescent varicose nerve fibers in the myenteric plexus of Auerbach both around nerve cell bodies and in the interconnecting strands. Many fibers were also present between the strands of the plexus, especially running parallel to the circular muscle layer. Such fibers were not seen in similarly quinacrine-incubated irides. A proportion of the cell bodies in Auerbach's plexus also showed quinacrine accumulation. These cells were apparently smaller neurons, sometimes with fluorescent processes. Intraperitoneal injections of quinacrine failed to demonstrate nerve fibers, but some cell bodies in Auerbach's plexus were positive. Subsequent paraformaldehyde treatment for monoamine visualization showed persistent adrenergic nerve terminals in the intestine and iris. These nerves seemed to be fewer and had a more yellow fluorescence than normally. The identity of the quinacrine-positive fibers is discussed with respect to recent suggestions that purinergic, substance P, enkephalin, and somatosin-containing nerves, in addition to adrenergic and cholinergic nerves, are present in the gut wall.Supported by the Swedish Medical Research Council (04X-03185). Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. For generous gifts of Mepacrine we thank Winthrop, Skärholmen, Stockholm, Sweden. The skilful technical assistance of Miss Gerd Boetius and Miss Maud Eriksson is gratefully acknowledged     

Formation of a functional adrenergic input to intraocular cerebellar grafts: Ingrowth of inhibitory sympathetic fibers

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10⁻⁶ or 10⁻⁵ M α-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic β-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the β-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.