Microtubular response to colchicine in adult and fetal rat hepatocytes

1. Colchicine and related anti-microtubular drugs impair plasma protein secretion from adult rat liver explants 2-3-fold more than from fetal tissue. 2. Indirect immunofluorescence microscopy of cultured adult and fetal hepatocytes demonstrated that hepatocytes of both ages contain large numbers of densely packed microtubules which are equally disassembled by 10 microM colchicine. 3. Colchicine (10 microM) reduced secretion of [14C]leucine-labelled proteins from cultured adult hepatocytes by about 50% but did not significantly impede fetal secretion. 4. These results confirmed that plasma protein secretion can proceed without an intact microtubular system in fetal hepatocytes.     

Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications

The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

Cytogenetic Analysis of Chromosome 3 in DROSOPHILA MELANOGASTER: The Homoeotic Gene Complex in Polytene Chromosome Interval 84a-B

Cytogenetic evidence is presented demonstrating that the 84A-B interval in the proximal portion of the right arm of chromosome 3 is the residence of a homoeotic gene complex similar to the bithorax locus. This complex, originally defined by the Antennapedia (Antp) mutation, controls segmentation in the anterior portion of the organism. Different lesions within this complex homoeotically transform portions of the prothorax, proboscis, antenna and eye and present clear analogies to similar lesions within the bithorax locus.     

Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.)

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. `Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10⁻⁵ and 10⁻⁶ molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10⁻⁵ and 10⁻⁶ molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin α (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.     

Effect of NOS inhibition on central response to atrial distension during pregnancy

Atrial distension increases c-fos expression in the paraventricular nucleus of virgin, but not pregnant, rats. We proposed that nitric oxide (NO), biosynthesis of which increases during pregnancy, blunts this reflex and that blocking NO biosynthesis would restore the response. Female rats were implanted with indwelling intracardiac balloons. On day 14 of pregnancy, osmotic minipumps containing either D- or N(G)-nitro-L-arginine methyl ester (L-NAME) (120 mg/2 ml at 10 microg/min) were implanted. On day 20, the rats were infused with saline (3 ml/h) with or without atrial balloon inflation (1 h). The brains were then processed for quantitation of c-fos expression. In the virgin rats, and in the pregnant rats treated with L-NAME, atrial distension significantly increased hypothalamic c-fos expression. In the pregnant animals treated with D-NAME, the response was greatly attenuated. NO had no effect on the increase in atrial receptor afferent discharge (single-fiber recordings) elicited by atrial distension. We conclude that, during pregnancy, NO attenuates central processing of the reflex response to atrial distension but does not alter the transducer properties of the volume receptors.     

Shutoff on Neuroblastoma Cell Protein Synthesis by Semliki Forest Virus: Loss of Ability of Crude Initiation Factors to Recognize Early Semliki Forest Virus and Host mRNA's

A crude ribosomal wash containing the initiation factors of protein synthesis was isolated from mouse neuroblastoma cells 8 h after infection with Semliki Forest virus (SFV). The activity of this wash was compared with that of a wash from control cells in a cell-free protein-synthesizing “pH5” system, with early SFV mRNA (42S), late SFV mRNA (26S), encephalomyocarditis virus (EMC) mRNA, or neuroblastoma polyadenylated mRNA templates. A pronounced loss of activity (±80%) of the crude ribosomal wash from infected cells was observed with host mRNA (neuroblastoma polyadenylated mRNA) and early SFV mRNA, messengers which contain a cap structure at the 5′ terminus. However, these washes were only slightly less active in systems programmed with (noncapped) EMC mRNA and late SFV mRNA. Although late SFV mRNA (26S) is capped, the synthesis of late (= structural) proteins in infected lysates was insensitive to inhibition by cap analogs. Purified initiation factors eIF-4B (M_r, 80,000) and cap-binding protein (M_r, 24,000) from reticulocytes (but none of the others) were able to restore the activity of infected factors to about 90% of control levels in systems programmed with early SFV mRNA and host mRNA. These observations indicate that infection-exposed crude initiation factors have a decreased level of eIF-4B and cap-binding protein activity. However, after partial purification of these and other initiation factors from infected and control cells, we found no significant difference in activity when model assay systems were used. Furthermore, both eIF-4B and cap-binding protein from infected cells were able to restore the activity of these infection-exposed factors to the same level obtained when these factors isolated from control cells or reticulocytes were added. A possible mechanism for the shutoff of host cell protein synthesis is discussed.     

RNAi analysis of Deformed, proboscipedia and Sex combs reduced in the milkweed bug Oncopeltus fasciatus: novel roles for Hox genes in the hemipteran head

Insects have evolved a large variety of specialized feeding strategies, with a corresponding variability in mouthpart morphology. We have, however, little understanding of the developmental mechanisms that underlie this diversity. Until recently it was difficult to perform any analysis of gene function outside of the genetic model insects Drosophila melanogaster and Tribolium castaneum. In this paper, we report the use of dsRNA-mediated interference (RNAi) to dissect gene function in the development of the milkweed bug Oncopeltus fasciatus, which has specialized suctorial mouthparts. The Hox genes Deformed (Dfd), proboscipedia (pb) and Sex combs reduced (Scr) have previously been shown to be expressed in the gnathal appendages of this species. Strikingly, the milkweed bug was found to have an unusual expression pattern of pb. Here, by analyzing single and combination RNAi depletions, we find that Dfd, pb and Scr are used in the milkweed bug to specify the identity of the mouthparts. The exact roles of the genes, however, are different from what is known in the two genetic model insects. The maxillary appendages in the bug are determined by the activities of the genes Dfd and Scr, rather than Dfd and pb as in the fly and beetle. The mandibular appendages are specified by Dfd, but their unique morphology in Oncopeltus suggests that Dfd's target genes are different. As in flies and beetles, the labium is specified by the combined activities of pb and Scr, but again, the function of pb appears to be different. Additionally, the regulatory control of pb by the other two genes seems to be different in the bug than in either of the other species. These novelties in Hox function, expression pattern and regulatory relationships may have been important for the evolution of the unique Hemipteran head.     

Regulation of basolateral membrane potential after stimulation of Na+ transport in proximal tubules

Summary We have previously shown that stimulation of apical Na-coupled glucose and alanine transport produces a transient depolarization of basolateral membrane potential (V _bl) in rabbit proximal convoluted tubule (PCT. Sl segment). The present study is aimed at understanding the origin of the membrane repolarization following the intial effect of addition of luminal cotransported solutes. Luminal addition of 10–15mMl-alanine produced a rapid and highly significant depolarization ofV _bl (20.3±1.1 mV,n=15) which was transient and associated with an increase in the fractional K⁺ conductance of the basolateral membrane (t _K) from 8 to 29% (P<0.01,n=6). Despite the significant increase int _K, the repolarization was only slightly reduced by the presence of basolateral Ba²⁺ (2mM,n=6) or quinine (0.5 mM,n=5). The repolarization was greatly reduced in the presence of 0.1 mM 4-acetamino-4isothiocyamostilbene-2,2-disulfonic acid (SITS) and blunted by bicarbonate-free solutions. Intracellular pH (pH _i ) determined with the fluorescent dye 2, 7-bis-2-carboxyethyl-5(and-6)-carboxyfluorescein (BCECF), averaged 7.39±0.02 in control solution (n=9) and increased to 7.50±0.03 in the first 15 sec after the luminal application of alanine. This was followed by a significant acidification averaging 0.16±0.01 pH unit in the next 3 min. In conclusion, we believe that, contrary to other leaky epithelia, rabbit PCT can regulate its basolateral membrane potential not only through an increase in K⁺ conductance but also through a cellular acidification reducing the basolateral HCO ₃ ^– exit through the electrogenic Na-3(HCO₃) cotransport mechanism.