Quantitation of endogenous liver apolipoprotein B mRNA editing

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.     

Salivary deposits and plant damage associated with specific probing behaviors of the potato leafhopper, Empoasca fabae, on alfalfa stems

The feeding behaviors and salivary deposits of the potato leafhopper, Empoasca fabae (Harris), on stems of a susceptible cultivar of alfalfa (Medicago sativa, L.) were studied in relation to the cellular damage and initiation of hopperburn they cause. An AC electronic monitoring system (EMS) was used to quantify the number and duration of probes made on alfalfa stems. Light and electron microscopy were then used to examine the effects of these probes. Most probes were short in duration as determined by EMS and comprised primarily of multiple-cell laceration behavior (represented by the I_a waveform). Probes containing still-stylet ingestion behavior (represented by the I_c waveform) were longer in duration and occurred less frequently. There was a close correspondence between the number of probes containing the I_c waveform and number of salivary deposits. Histological examinations of the stems revealed six types of salivary deposits but only one of these resembled the solid, distinct (or true) salivary sheaths characteristic of sheath-feeders. Less than 25% of E. fabae probes left any salivary deposit, and only 1% left true sheaths. Therefore, it seems unlikely that these deposits could physically block the phloem to cause hopperburn. Cellular disruptions, such as abnormally divided and enlarged cells, were found in the vascular cambium and phloem, as well as other tissues. Enlarged vascular cambial cells appeared to crush the phloem cells, and thus probably caused hopperburn. This microscopic damage was initiated by vigorous, active probing, along with the copious watery saliva that is secreted during the behavior represented by I_a. These observations strongly support Medler's (1941) hypothesis that hopperburn is caused by a substance in the insect's saliva that induces plant cells to enlarge and block the phloem.

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Résumé Le comportement alimentaire et les dépôts salivaires d'E. fabae Haris ont été examinés en relation avec les dégâts cellulaires sur tiges de cultivars sensibles de Medicago sativa. Un système de contrôle électronique AC (EMS) a été utilisé pour quantifier le nombre et la durée des sondages dans les tiges de luzerne. Les microscopies optique et électronique ont permis d'examiner les effets de ces condages. EMS a montré que la majorité des sondages étaient brefs (1 à 2 min) et commençait par la lacération multiple de cellules (révélé par un onde I_a). Les sondages comprenant un comportement d'ingestion avec immobilisation des stylets (révélé par une onde I_c) sont plus longs et moins fréquents. Il y a une bonne correspondance entre le nombre de sondages contenant un onde I_c et le nombre de dépôts salivaires. L'association des sondages et des dépôts salivaires montre que la durée du sondage était plus liée à la solidité des dépôts salivaires que la durée des ondes individuelles. L'examen histologique des tiges a révélé 6 types de dépôts salivaires, mais seul l'un d'entre eux ressemblait à celui donnant la gaine solide, individualisée (ou véritable) gaine salivaire caractéristique des consommateurs avec gaines salivaires. Moins de 25% des sondages d'E. fabae laissent un dépôt et seulement 1% laisse une véritable gaine. Ainsi, il semble improbable que ces dépôts puissent bloquer le phloème et provoquer la brûlure des ciccadèles. Des dégâts cellulaires, tels que divisions anormales et cellules hypertrophiées, ont été observés dans la cambium vasculaire et dans le phloème, ainsi que dans d'autres tissus. Dans des tissus fortement abîmés, le phloème est complètement désorganisé et contient des cellules ratatinées et affaissées. Des cellules vasculaires cambiales hypertrophiées paraissaient aplatir les cellules du phloème, et ainsi interférer avec la translocation des photosynthétats. Les dégâts microscopiques sont produits par les sondages actifs et vigoureux, associés à la copieuse détrempe salivaire secrétée pendant le comportement représenter par I_a. Ces observations appuient fortement l'hypothèse de Medler (1941) suivant laquelle la brûlure des ciccadèles est provoquée par une substance de la salive de l'insecte qui induit l'hypertrophie cellulaire et bloque le phloème.

     

NT-3, like NGF, Is Required for Survival of Sympathetic Neurons, but Not Their Precursors

Superior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491-500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115-3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor.     

Intracellular chloride and calcium transients evoked by γ‐aminobutyric acid and glycine in neurons of the rat inferior colliculus

Microfluorometric recordings showed that the inhibitory neurotransmitters γ‐aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl⁻ concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin‐perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO and characterized by a continuous shift of their reversal potential (E_GABA/gly) in the positive direction. In HCO‐buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca²⁺ concentration. Ca²⁺ transients occurred only with large depolarizations and were blocked by Cd²⁺, suggesting an activation of voltage‐gated Ca²⁺ channels. However, in the absence of HCO, only a small rise, if any, in the intracellular Ca²⁺ concentration could be evoked by GABA or glycine. We suggest that the activation of GABA_A or glycine receptors results in an acute accumulation of Cl⁻ that is enhanced by the depolarization owing to HCO efflux, thus shifting E_GABA/gly to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca²⁺ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 386–396, 1999     

Changes in conduction within the diffuse sinoatrial node of the chicken following premature atrial stimulation

Premature atrial stimulation was used to estimate sinoatrial conduction within the diffuse sinoatrial node of the bird (chicken), and compare its conduction with that reported for mammals. While sinoatrial conduction could not be determined in the chicken because reset did not occur, the premature wavefront did have an effect on the sinoatrial node because the recovery interval following the premature stimulus became less than compensatory with shortening of the premature stimulus interval. This less than compensatory non-reset recovery interval is interpreted as a conduction dependent response in which the intrinsic wavefront leading to the first recovery atrial activation conducts out of the node faster than normal. This conduction dependent recovery interval is seen infrequently in mammals (rabbit, dog and man). The absence of reset and the presence of a less than compensatory non-reset response in the chicken suggests that while the general organization of the sinoatrial node of the chicken is similar to that in mammals, a larger transitional cell network in the chicken prevents a premature wavefront from reaching the pacemaker cells and resetting them.     

Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro

Mitochondria are key regulators of cellular energy and are the focus of a large number of studies examining the regulation of mitochondrial dynamics and biogenesis in healthy and diseased conditions. One approach to monitoring mitochondrial biogenesis is to measure the rate of mitochondrial DNA (mtDNA) replication. We developed a sensitive technique to visualize newly synthesized mtDNA in individual cells to study mtDNA replication within subcellular compartments of neurons. The technique combines the incorporation of 5-bromo-2-deoxyuridine (BrdU) and/or 5-ethynyl-2′-deoxyuridine (EdU) into mtDNA, together with a tyramide signal amplification protocol. Employing this technique, we visualized and measured mtDNA biogenesis in individual cells. The labeling procedure for EdU allows for more comprehensive results by allowing the comparison of its incorporation with other intracellular markers, because it does not require the harsh acid or enzyme digests necessary to recover the BrdU epitope. In addition, the utilization of both BrdU and EdU permits sequential pulse–chase experiments to follow the intracellular localization of mtDNA replication. The ability to quantify mitochondrial biogenesis provides an essential tool for investigating the alterations in mitochondrial dynamics involved in the pathogenesis of multiple cellular disorders, including neuropathies and neurodegenerative diseases. (J Histochem Cytochem 58:207–218, 2010)     

Influence of plant ontogeny and abiotic factors on resistance of glandular-haired alfalfa to potato leafhopper (Hemiptera: Cicadellidae)

Laboratory experiments were conducted to characterize the trichome-based defense of glandular-haired alfalfa, Medicago sativa L., against the potato leafhopper, Empoasca fabae (Harris). Within-plant variability in leafhopper resistance was examined by caging adult leafhoppers to either basal or apical stem internodes of the leafhopper-resistant, glandular-haired M. sativa genotype G98A or the susceptible, nonglandular-haired M. sativa 'Ranger'. Young, actively secreting glandular trichomes are located on apical internodes of G98A, whereas senesced gland heads are found on older, basal internodes of G98A. After 96 h, the highest cumulative leafhopper mortality and lowest number of excretory droplets were associated with apical internodes of G98A. No difference was detected in mortality and feeding levels among insects caged to basal internodes of G98A and basal and apical internodes of Ranger. The influence of abiotic factors on leafhopper resistance was evaluated by caging adult leafhoppers to either G98A or Ranger under four combinations of low and high light (250 and 1,000 micromol s(-1) m(-2)) and temperature regimes (17 and 30 degrees C). After 96 h, the highest cumulative mortality was associated with leafhoppers confined to G98A under high light and high temperature conditions. Temperature level and plant type also had an effect on the production of excretory droplets, resulting in the highest number of excretory droplets being associated with Ranger under the high temperature regime. These results indicate that certain regions of M. sativa G98A are better protected against the potato leafhopper than others and that temperature influences resistance levels of glandular-haired alfalfa.     

Correlation of stylet activities by the glassy-winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms

Glassy-winged sharpshooter, Homalodisca coagulata (Say), is an efficient vector of Xylella fastidiosa (Xf), the causal bacterium of Pierce's disease, and leaf scorch in almond and oleander. Acquisition and inoculation of Xf occur sometime during the process of stylet penetration into the plant. That process is most rigorously studied via electrical penetration graph (EPG) monitoring of insect feeding. This study provides part of the crucial biological meanings that define the waveforms of each new insect species recorded by EPG. By synchronizing AC EPG waveforms with high-magnification video of H. coagulata stylet penetration in artifical diet, we correlated stylet activities with three previously described EPG pathway waveforms, A1, B1 and B2, as well as one ingestion waveform, C. Waveform A1 occured at the beginning of stylet penetration. This waveform was correlated with salivary sheath trunk formation, repetitive stylet movements involving retraction of both maxillary stylets and one mandibular stylet, extension of the stylet fascicle, and the fluttering-like movements of the maxillary stylet tips. Waveform B1 was ubitquious, interspersed throughout the other waveforms. B1 sub-type B1w was correlated with salivation followed by maxillary tip fluttering. This tip fluttering also occurred before and during B1 sub-type B1s, but was not directly correlated with either the occurrence or frequency of this waveform. Waveform B2 was correlated with sawing-like maxillary stylet movements, which usually occurred during salivary sheath branching. Waveform C was correlated with ingestion. Fluid outflow was also observed as a mechanism to clear the maxillary tips from debris during waveform C. This detailed understanding of stylet penetration behaviors of H. coagulata is an important step toward identifying the instant of bacterial inoculation which, in turn, will be applied to studies of disease epidemiology and development of host plant resistance.     

Direct utilization of mannose for mammalian glycoprotein biosynthesis

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2- 3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic- derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.