Effect of Quinine on Autoreceptor-Regulated Dopamine Release in the Rat Striatum

In vivo brain microdialysis was used to examine the role of potassium channel activation in dopamine (DA) autoreceptor function in the striatum of freely moving rats. Local application of the D2 receptor agonists quinpirole or N-0437 through the dialysis probe significantly reduced extracellular concentrations of DA. Local application of the D2 antagonist (-)-sulpiride produced significant increases in DA. Local perfusion with quinine, a K+ channel blocker, completely blocked the (-)-sulpiride-induced increases in DA but did not affect the DA agonist-induced decreases. (-)-Sulpiride completely blocked the effect of quinpirole on DA both in control and in quinine-treated animals. At the highest dose used, quinine caused a large transient increase in extracellular DA. Local application of tetrodotoxin or infusion of Mg2+ in the absence of Ca2+ did not prevent this quinine-induced transient increase in extracellular DA. These results demonstrate that DA autoreceptors in the striatum regulate DA release in awake, behaving animals. Local application of (-)-sulpiride increases DA levels by blocking the tonic activation of autoreceptors by endogenous DA. Quinine blocks the neuroleptic-induced increase in DA, perhaps by preventing the K+ channel opening that would normally accompany endogenous autoreceptor activation. The fact that exogenously applied DA receptor agonists can decrease extracellular DA levels in the presence of quinine suggests that they may be acting at extrasynaptic autoreceptors that are not tonically active in vivo. The effect of DA agonists on this site is via a DA receptor because it is blocked by (-)-sulpiride. However, this receptor does not appear to be coupled to a quinine-sensitive potassium channel.     

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

In vitro propagation and cytology of wild yams,Dioscorea abyssinica Hoch. and D. mangenotiana Miège

Two wild yams of West Africa, Dioscorea abyssinica Hoch, and D. mangenotiana Miège were micropropagated from nodal cultures. Both species produced 4–5 nodes per each node cultured. The size of nodal cuttings was critical, segments shorter than 0.5 cm being less suitable for micropropagation. The number of nodes produced was constant even after 5 cycles of subculture; however, D. abyssinica continuous subculture decreased propagation efficiency, resulting in a reduced number of reculturable nodes at each cycle. In D. mangenotiana, the decrease in multiplication efficiency affected both the number of total and reculturable nodes. Large-sized microtubers were induced on nodal segments maintained under 8-h daylength in both species. In D. abyssinica, however, microtubers were induced on media containing 20, 40, 60 and 80 g l^-1 sucrose, whereas in D. mangenotiana only 40 and 60 g l^-1 sucrose favoured tuberization. Cytological studies confirmed that the chromosome number of D. abyssinica was 2n=40, although a high incidence of cytochimerism and cells with 2n=38 were observed in root meristems. In D. mangenotiana clones, the chromosome number was 2n=40, as against 2n=72 and 2n=80 reported in literature. This species also displayed karyological stability.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - PPF photosynthetic photon flux     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

A linkage map of distal mouse chromosome 12

To refine the linkage map of distal mouse Chromosome 12, we have identified DNA restriction fragment variants associated with a creatine kinase gene (Ck-3), the Akt proto-oncogene, an Abelson proviral integration site (D12N1), and the immunoglobulin heavy chain V_H3609 variable region family (Igh-V36). The patterns of inheritance of these markers in backcross progeny and recombinant inbred mouse strains allowed their localization with respect to previously mapped genes to yield the linkage map: Aat-15.8 cM-Ck-3-0.9 cM-(Crip, Akt, Igh-C)-0.3 cM-(D12N1, Igh-V). This map confirms genetically the localization of the Igh-V gene complex distal to Igh-C on the chromosome. It differs from previous maps in placing D12N1 distal to Igh-C, and in suggesting that the Igh-V gene complex spans less than one centiMorgan (cM).Other DNA sequence variants detected with the creatine kinase probe allowed definition of four additional genetic loci: Ck-1 near Lmyc-1 on Chromosome 4; Ck-2 between Upg-1 and Hprt-ps1 (D17Rp10) on distal Chromosome 17; Ck-4 near Mpmv-17 and Mls-3 on Chromosome 16; and Ck-5 near Hba on Chromosome 11.     

Biocontrol of the apple sawfly,Hoplocampa testudinea,with entomogenous nematodes

Four types of experiments were conducted to assess the pathogenicity of entomogenous nematodes against the apple sawfly,Hoplocampa testudinea Klug (Hymenoptera: Tenthredinidae). In Petri dish,Steinernema carpocapsae (Weiser) DD 136 strain,S. carpocapsae All strain,S. feltiae (Filipjev) andHeterorhabditis bacteriophora Poinar caused 100% larval mortality 72 h after the treatments. In semi-field conditions under dwarf apple trees, a single treatment with either 40 or 80S. carpocapsae All strain/cm² caused significant (>80%) larval mortality. No significant increase in mortality was obtained with a second application. In 1990, late treatment with 80S. carpocapsae All strain/cm² caused significant (39.1%) pupal mortality. In 50×50 cm quadrats under dwarf apple trees, treatment with 40 and 80S. carpocapsae All strain/cm² were done in spring 1989. In spring 1990, respectively 22 and 25 apple sawfly adults emerged from quadrats treated with 40 and 80S. carpocapsae All strain/cm², whereas 78 adults emerged from untreated quadrats. In a foliar application experiment, a single treatment withS. carpocapsae All strain significantly reduced the percent of fruit showing secondary damage. The possibility of using entomogenous nematodes as part of an integrated pest management in apple orchards is discussed, with particular reference to the integration of programs aimed at managing plum curculio,Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae) populations.      

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.     

Nucleotide sequence and evolution of a mammalian α-Tubulin messenger RNA

Bacterial clones containing complementary DNA sequences specific for rat brain α-tubulin messenger RNA were constructed. One plasmid, pILαTl, contains >95% of the sequences found in the mRNA: the entire coding sequence as well as extensive 5′ and 3′ untranslated sequences. Comparison of the rat amino acid sequence with the known chicken α-tubulin sequence (Valenzuela et al., 1981) reveals the extraordinary evolutionary stability of α-tubulin protein. The presence of only two interspecies amino acid differences within analogous 411 amino acid sequences predicts that amino acid substitutions in this protein are fixed with a unit evolutionary period (Wilson et al., 1977) of 550 million years (i.e. the time required for a 1% difference to arise within a specific protein in two diverging evolutionary lineages). An analysis of the silent nucleotide differences, permissible because of the degeneracy of the genetic code, demonstrates that these might not occur in a random fashion. The high guanine-cytosine bias in silent codon positions within the chicken α-tubulin sequence, previously noted by Valenzuela et al. (1981), is not conserved within the rat sequence. This decrease in guanine-cytosine bias is accompanied by a selective loss of CpG dinucleotides in the rat sequence.