Correlation of Rates of Calcium Entry and Release of Endogenous Norepinephrine in Rat Brain Region Synaptosomes

Voltage-dependent 45Ca2+ uptake and endogenous norepinephrine (NE) release were measured simultaneously in synaptosomes isolated from rat hypothalamus, brainstem, and cerebellum at 1, 3, 5, 15, and 30 s. In synaptosomes depolarized by 125 mM KCl, 45Ca2+ uptake and NE release exhibited fast and slow components. Rates of NE release and 45Ca2+ uptake were fastest from 0 to 1 s. NE release and 45Ca2+ uptake rates from 1 to 5 s were less than 15% of 0-1 s rates. Both resting (5 mM KCl) and depolarization-induced (125 mM KCl) NE release paralleled 45Ca2+ uptake from 1 to 30 s. Voltage-dependent NE release was approximately 1% and 2% of total synaptosomal NE content at 1- and 30-s measurement intervals, respectively, and did not differ between the three brain regions studied. Calcium and potassium dependence studies showed that NE release was stimulated by increased potassium and that depolarization-induced NE release was dependent on the presence of external calcium. These results show that calcium-dependent NE release from synaptosomes is correlated with calcium entry. Both processes exhibit fast and slow temporal components.     

Structure and possible ureide degrading function of the ubiquitous urease of soybean

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.

The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection (“Western Blot”). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.

The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

     

Transport of AMP by Rickettsia prowazekii.

Rickettsia prowazekii possesses an exchange transport system for AMP. Chromatographic analysis of the rickettsiae demonstrated that transported AMP appeared intracellularly as AMP, ADP, and ATP, and no hydrolytic products appeared in either the intracellular or extracellular compartments. The phosphorylation of AMP to ADP and ATP was prevented by pretreatment of the cells with 1 mM N-ethylmaleimide without inhibiting the transport of AMP. Although no efflux was demonstrable in the absence of nucleotide in the medium, the intracellular adenine nucleotide pool could be exchanged with external unlabeled adenine nucleotides. Both ADP and ATP were as effective as AMP at inhibiting the uptake of [3H]AMP. Although this transport system was inhibited by low temperature (0 degrees C) and partially inhibited by the protonophore carbonyl cyanide-m-chlorophenyl hydrazone (1 mM), it was relatively insensitive to KCN (1 mM). The uptake of AMP at 34 degrees C had an apparent Kt for influx of 0.4 mM and a Vmax of 354 pmol min-1 per mg. At 0 degrees C there was a very rapid and unsaturable association of AMP with these organisms. Correction of the uptake data at 34 degrees C for the 0 degrees C component lowered the apparent Kt to 0.15 mM. Both magnesium and phosphate ions are required for optimal transport activity. Chemical measurements of the total intracellular nucleotide pools demonstrated that this system was not a net adenine nucleotide transport system, but that uptake of AMP was the result of an exchange with internal adenine nucleotides.     

Regulation of product synthesis in cell cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle Catharanthus roseus (L) G. Don were maintained on Gamborg's B5 medium and their growth monitored by measuring cellular fresh and dry weight, cell number and mitotic activity. Samples of cells of different ages and physiological states were subcultured onto an alkaloid production medium and their rates of growth and alkaloid accumulation measured over a period of 30–45 days. In two experiments the rate of biomass accumulation was directly related to the rate of cellular serpentine accumulation. Possible mechanisms underlying this phenomenon are discussed in relation to the properties of cells comprising the inocula.     

Captan alters transcription in Escherichia coli permeabilized by toluene

RNA synthesis was measured in toluenized E. coli by the incorporation of radiolabeled precursor into either acid precipitable or phenol extracted RNA. Exposure to captan (100 microM) caused a 2.6 fold increase in the apparent rate of RNA synthesis. When captan was tested for its effect on the initiation of RNA synthesis, using either rifampicin-treated cells or by measuring the incorporation of gamma [32P]ATP or gamma [32P]GTP, no change was observed in the number of RNA chains being initiated. Thus, captan does not exert its influence at the level of initiation of nascent chains. However, captan did have an effect on chain growth. From calculations of the incorporation of precursors molecules, RNAs isolated from treated cells were measured to be an average of 2.7 times longer than those from untreated cells. RNA chain lengths were also analyzed by polyacrylamide gel electrophoresis. By this latter technique it was also shown that cells exposed to captan synthesized RNAs that were longer than those of untreated cells. Alterations in the degradation of RNA molecules do not account for the captan mediated response in RNA synthesis.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

Isolation and Immunological Characterization of a Glycoprotein from Adrenal Chromaffin Granules

A glycoprotein (s-GP III) was isolated from the soluble lysate of chromaffin granules by chromatography with immunoaffinity and lectin columns. An identical protein (m-GP III) was shown to be present in the granule membranes. The apparent molecular weight of these glycoproteins as determined by the electrophoresis system of Laemmli (1970) was 43,000 under reducing conditions. In the absence of mercaptoethanol they aggregated to dimers. Antisera were raised against both the soluble and the membrane-bound forms of this glycoprotein. With these antisera GP III was further characterized: Immunoreplicas were obtained after two-dimensional electrophoresis of soluble and membrane-bound proteins of chromaffin granules. GP III was identified as a protein with a rather broad pI (4.6-5.3), indicating microheterogeneity. As shown by subcellular fractionation, m-GP III is specifically confined to chromaffin granules. GP III can therefore be used as a marker for the membranes of these organelles. The soluble form is secreted from adrenal medulla during stimulation with carbamylcholine chloride. An immunologically identical antigen was detected in adeno- and neurohypophysis. The physiological function of GP III is still unknown. It does not demonstrate any of the enzymatic activities so far known to occur in chromaffin granules.     

How do food passage rate and assimilation differ between herbivorous lizards and nonruminant mammals?

Summary What digestive adaptations permit herbivorous nonruminant mammals to sustain much higher metabolic rates than herbivorous lizards, despite gross similarity in digestive anatomy and physiology? We approached this question by comparing four herbivorous species eating the same diet of alfalfa pellets: two lizards (chuckwalla and desert iugana) and two mammals (desert woodrat and laboratory mouse). The mammals had longer small and large intestines, greater intestinal surface area, much higher (by an order of magnitude) food intake normalized to metabolic live mass, and much faster food passage times (a few hours instead of a few days). Among both reptiles and mammals, passage times increase with body size and are longer for herbivores than for carnivores. The herbivorous lizards, despite these much slower passage times, had slightly lower apparent digestive efficiencies than the mammals. At least for chuckwallas, this difference from mammals was not due to differences in body temperature regime. Comparisons of chuckwallas and woodrats in their assimilation of various dietary components showed that the woodrat's main advantage lay in greater assimilation of the dietary fiber fraction. Woodrats achieved greater fiber digestion despite shorter residence time, but possibly because of a larger fermentation chamber, coprophagy, and/or different conditions for microbial fermentation. We conclude with a comparative overview of digestive function in herbivorous lizards and mammals, and with a list of four major unsolved questions.     

Transmembrane electrical potential in Rickettsia prowazekii and its relationship to lysine transport.

The transmembrane electrical potential (delta psi) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a delta psi as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R.prowazekii concentrated lysine to a gradient indicating a delta psi of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a delta psi of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. delta psi as measured with tetraphenyl-phosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of O2 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.     

Developmental regulation of 16S acetylcholinesterase and acetylcholine receptors in a mouse muscle cell line

We have studied the appearance, distribution and regulation of acetylcholinesterase (AChE) and acetylcholine receptors (AChRs) in a mouse skeletal muscle cell line (C2), that was originally isolated and described by Yaffe & Saxel [54]. In culture, cells from this line form spontaneously contracting myotubes, with overshooting action potentials that are TTX-sensitive. After fusion of myoblasts into myotubes, there was a dramatic increase in the amount of both AChE and AChR. Three forms of AChE, distinguished by their sedimentation on sucrose gradients, were synthesized: 4-6S, 10S, and 16S. The 4-6S and 10S forms appeared 1 day after the cells began to fuse, whereas the 16S form appeared only 2 days after fusion began. Maximal levels of the 16S AChE form (25-30% of the total) were obtained by reducing the concentration of horse serum in the fusion medium. Prevention of myoblast fusion by reducing the calcium levels in the medium decreased the total AChE by 70%, and only the 4-6S form was synthesized. Blocking spontaneous contractile activity of the myotubes by tetrodotoxin (TTX) led to a 50% reduction in all three esterase forms. Thus, the 16S, or endplate form of AChE is not specifically regulated by electrical or contractile activity in the C2 cell line. After fusion the number of AChRs increased rapidly for 3-4 days and then stabilized. Receptor clusters, ranging from 10-30 micron in length, appeared 1 day after myoblast fusion began. When cells were grown in medium containing reduced Ca2+, the total number of AChRs was decreased by 20-50%. Reduction of Ca2+ after myotubes and AChR clusters had formed resulted in dispersal of AChR clusters. Inhibition of muscle contractions with TTX did not affect the number of AChRs or their distribution.