Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Electrophysiological Evidence for Intrinsic Pacemaker Currents in Crayfish Parasol Cells

I used sharp intracellular electrodes to record from parasol cells in the semi-isolated crayfish brain to investigate pacemaker currents. Evidence for the presence of the hyperpolarization-activated inward rectifier potassium current was obtained in about half of the parasol cells examined, where strong, prolonged hyperpolarizing currents generated a slowly-rising voltage sag, and a post-hyperpolarization rebound. The amplitudes of both the sag voltage and the depolarizing rebound were dependent upon the strength of the hyperpolarizing current. The voltage sag showed a definite threshold and was non-inactivating. The voltage sag and rebound depolarization evoked by hyperpolarization were blocked by the presence of 5–10 mM Cs²⁺ ions, 10 mM tetraethyl ammonium chloride, and 10 mM cobalt chloride in the bathing medium, but not by the drug ZD 7288. Cs⁺ ions in normal saline in some cells caused a slight increase in mean resting potential and a reduction in spontaneous burst frequency. Many of the neurons expressing the hyperpolarization-activated inward potassium current also provided evidence for the presence of the transient potassium current I_A, which was inferred from experimental observations of an increased latency of post-hyperpolarization response to a depolarizing step, compared to the response latency to the depolarization alone. The latency increase was reduced in the presence of 4-aminopyridine (4-AP), a specific blocker of I_A. The presence of 4-AP in normal saline also induced spontaneous bursting in parasol cells. It is conjectured that, under normal physiological conditions, these two potassium currents help to regulate burst generation in parasol cells, respectively, by helping to maintain the resting membrane potential near a threshold level for burst generation, and by regulating the rate of rise of membrane depolarizing events leading to burst generation. The presence of post-burst hyperpolarization may depend upon I_A channels in parasol cells.     

Evidence for Distinct Aggregation Factors and Receptors in Cells

SINCE the original observations by Wilson¹ that dissociated sponge cells could reassociate in vitro, cell aggregation (or reaggregation) has been widely used as an operational criterion for the study of intercellular adhesion². The introduction of rotation-mediated methods to promote cell aggregation^3,4 led to the possibility of obtaining reproducible quantitative data. In these methods, suspensions of dissociated single cells are shaken under defined conditions of speed and temperature and cell aggregation is measured by either the size of aggregates or the number of single cells. The aggregation of dissociated cells from sponges⁵, chick and mouse embryos⁴ and tissue culture cells⁶ has been investigated with this method. Cells maintained in vitro seemed particularly suitable for studying mechanisms of cell aggregation as they represent a histotypically homogeneous population.     

Peculiarities of Cardiac Activity in the Crayfish Astacus astacus in Different Functional States

Analyzed are changes of temporal and amplitude characteristics of the cycles in animals in different functional states, using a non-invasive method of recording of cardiac activity in the adult noble crayfish Astacus astacus L. under laboratory conditions. It has been shown that the state of excitation in the crayfish after action of an intense stimulus lasts for a long time (more than 1 h) and can have no manifestations in some observed behavioral responses. Based only on the cardiac response (the heart rate), the real physiological state of the animal could be evaluated. Characteristic of the excited animal is a short-term response to the stimulus. In a high excitement of the animal (a high heart rate), significant transformations of the heart activity in response to other current stimuli may be barely noticeable. For the quiet animal, the response to a stimulus is longer than for the excited animal at the same stimulus intensity; for a comparative search for significant readjustments of the animal state, based on the heart activity parameters, the cardiograms typical of the quiet animal should be used. In the quiet animals, the cardiogram parameters change to a greater extent than in the excited ones. The quiet animals have longer diastole duration (DD) than the systole duration (SD). The diastole amplitude (DA) is higher than the systole amplitude (SA), and DA changes more markedly than SA. The healthy animals are characterized by equal values of DD and SD, sometimes by longer SD than DD, but, on the background of a continuing action, their values and the values of DA and SA become equal. There were noted a negative correlation between the systolic and diastolic phases by temporal parameters for healthy animals and an increase of the correlation significance in transition of the animal into the excited state. Based on the cardiovascular system response, the chief features of a shift of the functional crayfish state to pathological direction seem to include a stable heart rate reduction, a decrease of the heart contraction amplitude, a more frequent predominance of SD over DD, an increase of the means and the standard deviations of the parameters, especially of the cycle amplitudes. Changes of the sign and significance of correlation relations between inotropic and chronotropic parameters of cardiac activity in sick animals appear to indicate a disturbed mechanism of central coordination of the cardiovascular system work with its autonomic mechanisms. The results are discussed on the basis of known mechanisms of the heart activity control in Decapoda. The records of cardiac activity, such as laser light cardiograms (LLC), in testaceous invertebrates may be useful and interesting in studies on mechanisms and processes of internal control and management of the cardiovascular system activity of the animals.     

Evidence for the Existence of Granzyme-Like Serine Proteases in Teleost Cytotoxic Cells

Granzymes are granule-associated serine proteases, which are important effector molecules in NK cell and CTL functions. The granzyme family poses a perplexing problem in phylogenetics due to the lack of nonmammalian sequence information. We now report the identification of a cDNA that codes for a granzyme homologue, channel catfish granzyme-1 (CFGR-1), from nonspecific cytotoxic cells (NCC) of a teleost. NCC are the first identified and extensively studied cytotoxic cell population in teleosts. Ictalurus punctatus (channel catfish) granzyme cDNA encodes a protein with ~50% similarity to granzymes A and K. Highly conserved catalytic triad residues of serine proteases and other motifs common to granzymes were also identified. Conserved amino acid sequences, structure–function data available for the serine protease family, and the crystal structure of human granzyme K supported a model of CFGR-1. It suggested an Arg/Lys primary substrate specificity that is shared with granzymes A and K. Furthermore, CFGR-1 has the four conserved disulfide bonds of granzymes A, K, and M. Phylogenetic analysis suggested that this molecule is a member of the granzyme family. Expression of CFGR-1 in NCC was confirmed by RT-PCR analysis. Presence of a granzyme-like molecule that might play an important role in the effector functions of NCC indicates that cell-mediated immunity with granule exocytosis and Fas pathways have been conserved for more than 300 million years.     

Evidence for Two Distinct Forms of Fatty Acid Cyclooxygenase in Brain

Abstract: The enzymatic metabolism of [¹⁴C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E₂ (PGE₂) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F_2a (PGF_2a) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE₂-9-ketoreductase activity. Inhibition of the PGH₂→ PGE₂ isomerase by arachidonic acid or its metabolites could not explain the abrupt halt in PGE₂ biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE₂ biosynthesis while PGF2o production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE₂ production at concentrations that did not affect PGF_2a biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH₂→ PGE₂ isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH₂→ PGF_2a reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.     

About the Existence of Circadian Activity in Cave Crayfish

Evidence of a circadian clock mechanism was found in the cave crayfish Procambarus cavernicola. Analysis of motor activity recorded in this species during 12 consecutive days in either free running (constant darkness, DD or constant light, LL) or entrainment conditions (12 h of light alternated with 12 h of darkness, 12 : 12 LD) showed a well recognized circadian rhythm. In this rhythm however, the absence of synchronization by periodical external signals was notorious. The comparison between the motor circadian rhythm in cave crayfish and epigeous crayfish Procambarus clarkii (these last studied during juvenile and adult stages), evidenced strong similitude between the motor circadian rhythm of cave crayfish and juvenile epigeous crayfish.     

Fatty Acid- and Retinoid-binding Proteins Have Distinct Binding Pockets for the Two Types of Cargo

Parasitic nematodes cause serious diseases in humans, animals, and plants. They have limited lipid metabolism and are reliant on lipid-binding proteins to acquire these metabolites from their hosts. Several structurally novel families of lipid-binding proteins in nematodes have been described, including the fatty acid- and retinoid-binding protein family (FAR). In Caenorhabditis elegans, used as a model for studying parasitic nematodes, eight C. elegans FAR proteins have been described. The crystal structure of C. elegans FAR-7 is the first structure of a FAR protein, and it exhibits a novel fold. It differs radically from the mammalian fatty acid-binding proteins and has two ligand binding pockets joined by a surface groove. The first can accommodate the aliphatic chain of fatty acids, whereas the second can accommodate the bulkier retinoids. In addition to demonstrating lipid binding by fluorescence spectroscopy, we present evidence that retinol binding is positively regulated by casein kinase II phosphorylation at a conserved site near the bottom of the second pocket. far-7::GFP (green fluorescent protein) expression shows that it is localized in the head hypodermal syncytia and the excretory cell but that this localization changes under starvation conditions. In conclusion, our study provides the basic structural and functional information for investigation of inhibitors of lipid binding by FAR proteins.     

Evidence for the Existence of Two Arginyl-Transfer Ribonucleic Acid Synthetase Activities in Escherichia coli

Two arginyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.13, arginine: ribonucleic acid ligase adenosine monophosphate) activities were found in extracts of Escherichia coli strains AB1132 and NP2. The two arginyl-tRNA synthetase activities in extracts of strain AB1132 were found to be separable by diethylaminoethyl-cellulose column chromatography, Sephadex column fractionation, and by sucrose density gradient centrifugation. In addition, in the standard assay using extracts of strain AB1132 there were two pH optima for arginyl-tRNA synthetase activity. Furthermore, when arginyl-tRNA synthetase of strain NP2 was fractionated by hydroxylapatite column chromatography, two activities were observed which were similar to those of strain AB1132.     

Crayfish freshwater adaptation starts in eggs: ontogeny of osmoregulation in embryos of Astacus leptodactylus

Osmoregulation was studied throughout the embryonic development of Astacus leptodactylus. Egg-carrying females were held in freshwater (FW) and in three dilute seawater media (200, 400, 600 mosm kg(-1), 6.8, 13.6, 20.4 per thousand salinity). In FW, changes in peri-embryonic fluid (PEF) and (when available) embryonic hemolymph osmolality were followed from newly-laid eggs to hatching (for an embryonic eye index, EI, of 430-450 microm) and in first-stage juveniles. The PEF and/or hemolymph osmolality remained stable at about 360-380 mosm kg(-1) from early to late (EI 410 microm) embryos; it decreased prior to hatching (EI 420 microm) and in newly-hatched juveniles, down to 290 mosm kg(-1). Artificial opening and removal of the egg membranes, followed by direct exposure to FW, demonstrated that the ability to hyper-osmoregulate, and consequently to survive, in FW appears in embryos with EI > or = 410 microm, i.e., only a few hours or days before hatching. Following a transfer to the dilute seawater media, the PEF/hemolymph osmolality increased slowly over 18-20 days and became isosmotic with the external media at 13.6 and 20.4 per thousand. The embryos died at EI 380-395 microm in these media, and only at 6.8 per thousand was the development completed until successful hatch. These results demonstrate that (1) the embryos become able to osmoregulate in FW shortly before hatching, (2) the embryos are osmo-protected in the eggs during their development, (3) embryonic development and hatching are possible up to a salinity of 7 per thousand. These results are discussed in relation to freshwater adaptation of crayfish.     

Glial Cells in Abdominal Ganglia of Crayfish

Glial cells of abdominal ganglia of crayfish have been studied by transmission electron microscopy. Four cell types can be defined: (1) perivascular glial cells, close to the vascular spaces; (2) perineuronal glial cells, the processes of which ensheathe neuron perikarya; (3) adaxonal glial cells ensheathing axons; (4) neuropilar glial cells, associated with synapsing terminals in the neuropile. Neuropilar glia, adaxonal glia and the system formed by perineuronal and perivascular glia separate different functional zones of the neurons from the hemolymph or the electron dense extracellular matrix. These glial arrangements could play a similar role in hemato-neuronal transport. Gap-like junctions between glia and neuron cell bodies are frequent and could be involved in direct triggering of glial activities related to neurons.     

Two Types of Pharmacologically Distinct Ca2+ Currents with Voltage-dependent Similarities in Zona Fasciculata Cells Isolated from Bovine Adrenal Gland

Voltage-activated Ca²⁺ currents, in zona fasciculata cells isolated from calf adrenal gland, were characterized using perforated patch-clamp recording. In control solution (Ca²⁺: 2.5 mm) a transient inward current was followed, in 40% of the cells, by a sustained one. In 20 mm Ba²⁺, 61% of the cells displayed an inward current, which consisted of transient and sustained components. The other cells produced either a sustained or a transient inward current. These different patterns were dependent upon time in culture. Current-voltage relationships show that both the transient and sustained components activated, peaked and reversed at similar potentials: −40, 0 and +60 mV, respectively. The two components, fully inactivated at −10 mV, were separated by double-pulse protocols from different holding potentials where the transient component could be inactivated or reactivated. The decaying phase of the sustained component was fitted by a double exponential (time constants: 1.9 and 20 sec at +10 mV); that of the transient component was fitted by a single exponential (time constant: 19 msec at +10 mV). Steady-state activation and inactivation curves of the two components were superimposed. Their half activation and inactivation potentials were similar, about −15 and −34 mV, respectively. The sustained component was larger in Ba²⁺ than in Sr²⁺ and Ca²⁺. Ni²⁺ (20 μm) selectively blocked the transient component while Cd²⁺ (10 μm) selectively blocked the sustained one. (±)Bay K 8644 (0.5 μm) increased the sustained component and nitrendipine (0.5–1 μm) blocked it selectively. The sustained component was inhibited by calciseptine (1 μm). Both components were unaffected by ω-conotoxin GVIA and MVIIC (0.5 μm). These results show that two distinct populations of Ca²⁺ channels coexist in this cell type. Although the voltage dependence of their activation and inactivation are comparable, these two components of the inward current are similar to T- and L-type currents described in other cells. Received: 12 July 1999/Revised: 5 October 1999     

Existence of Two Types of Electrostatic Interaction in Reactions Catalyzed by Ferredoxin-NADP$ Oxidoreductase

Effects of salt and pH on diaphorase and NADP^$ photoreductionactivities were studied with broken spinach chloroplasts andpurified ferredoxin-NADP^$ oxidoreductase. Two types of electrostatic interactions, which regulate thereaction rate, were observed. One is the long-range electrostaticinteraction which determines the local concentrations of reactantsin the surface-mediated processes due to the change in the surfacepotential. In addition to the salt-induced change in the reactionrate, the pH optimum shift by salt was also remarkable: theoptimum pH in the diaphorase activity of chloroplasts shiftedto the more acidic pH region with an increase of salt concentration,while that of the membrane-free enzyme was not affected by salts. A more specific, short-range electrostatic interaction in reactionsbetween NADP(H) and ferredoxin-NADP^$ oxidoreductase was observed.This interaction became clearer when fixed charges on the membranesurface were masked by an addition of salts. Complete dissociationof the 2'-phosphate group of NADP(H) was necessary for its associationwith the enzyme. The eletrostatic attraction between the negativelychargedpart of NADPH and the positively-charged part of the enzyme(probably lysyl and arginyl residues) may play a role in theshort-range interaction. ¹Present address: Department of Agronomy, University of Kentucky,Lexington, Kentucky 40546, U.S.A.²Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received February 21, 1983; Accepted March 17, 1984)     

Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey

Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara''s owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.     

Evidence for Coexistence of Two Distinct Functional Groups of Sulfate-Reducing Bacteria in Salt Marsh Sediment

Oxidation of acetate in salt marsh sediment was inhibited by the addition of fluoroacetate, and also by the addition of molybdate, an inhibitor of sulfate-reducing bacteria. Molybdate had no effect upon the metabolism of acetate in a freshwater sediment in the absence of sulfate. The inhibitory effect of molybdate on acetate turnover in the marine sediment seemed to be because of its inhibiting sulfate-reducing bacteria which oxidized acetate to carbon dioxide. Sulfide was not recovered from sediment in the presence of molybdate added as an inhibitor of sulfate-reducing bacteria, but sulfide was recovered quantitatively even in the presence of molybdate by the addition of the strong reducing agent titanium chloride before acidification of the sediment. Reduction of sulfate to sulfide by the sulfate-reducing bacteria in the sediment was only partially inhibited by fluoroacetate, but completely inhibited by molybdate addition. This was interpreted as showing the presence of two functional groups of sulfate-reducing bacteria—one group oxidizing acetate, and another group probably oxidizing hydrogen.     

Evidence for Two Main Domestication Trajectories in Saccharomyces cerevisiae Linked to Distinct Bread-Making Processes

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