Stimulatory Effect of Histamine on Cyclic AMP Formation in Chick Pineal Gland

Abstract: Histamine (HA) potently stimulated cyclic AMP accumulation in intact pineal glands taken from light-exposed chicks. The action of HA was stronger in the presence of forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The effect of HA was mimicked by HA H₁- and H₂-receptor-selective agonists in the following order of potency: HA > 4-methylhistamine (H₂) > 2-methylhistamine (H₁) > 2-thiazolylethylamine (H₁) ≫ dimaprit (H₂). The HA H₃-receptor-selective agonist (R)α-methylhistamine was poorly active. The effect of HA was antagonized by selective H₂-receptor blockers (tiotidine > oxmetidine > cimetidine = ranitidine) and was not significantly affected by the selective H₁- and H₃-receptor blockers mepyramine and thioperamide. A detailed analysis of an antagonistic action of ranitidine (versus HA) revealed a noncompetitive mode of action of the H₂ blocker. The stimulatory action of the H₁ agonist 2-thiazolylethylamine (both under basal conditions and in the presence of forskolin or IBMX) was not significantly influenced by three H₁-receptor-selective blockers (mepyramine, triprolidine, and diphenhydramine), but it was totally counteracted by ranitidine. Using accepted selective agonists and antagonists of the HA H₁, H₂, and H₃ receptor we were unable to identify clearly the receptor subtype mediating the HA action on the cyclic AMP-generating system of the chick pineal. It is suggested that the receptor under consideration may represent either an H₂-like (in terms of mammalian criteria) or avian-specific HA receptor. The data suggest that HA may be considered a modulator of the pineal activity in chicks.     

Thyrotropin-Releasing Hormone Release Is Enhanced in Hippocampal Slices After Electroconvulsive Shock

Abstract: Hippocampal thyrotropin-releasing hormone (TRH) release was examined after seizures were induced by electroconvulsive shock (ECS). Rat hippocampal slices taken 12, 24, or 48 h after 3 days of alternate-day ECS treatment or sham-ECS treatment were stimulated with potassium with or without calcium in a superfusion system containing in-line charcoal adsorbent to concentrate TRH. Released TRH and tissue TRH were measured by radioimmunoassay. The TRH content of hippocampal slices was increased fivefold over sham-ECS levels 12, 24, and 48 h after ECS, but this was not associated with an increase in basal TRH release. Potassium-stimulated TRH release was significantly elevated over basal release 12, 24, and 48 h after ECS. Potassium-stimulated calcium-dependent TRH release increased linearly after ECS, reaching its highest level 48 h after seizure. Thus, although enhanced calcium-dependent TRH release was associated with elevated tissue levels, this relationship was not proportional in that tissue TRH was elevated to the same extent at all times after ECS, whereas potassium-evoked calcium-dependent TRH release increased gradually over time after seizure. These results suggest that postictal elevations in TRH are associated with an enhanced capacity for release that develops as a result of a time-dependent shift of TRH from a storage compartment to a readily releasable pool. The observed elevation in stimulated TRH release may be relevant to seizure-induced modulation of TRH receptors in vivo.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.     

Genetic structure and mating system in the palila, an endangered Hawaiian honeycreeper, as assessed by DNA fingerprinting

We conducted DNA fingerprinting analyses to ascertain the mating system and population genetic structure of the palila, an endangered Hawaiian honeycreeper, which occupies a fragmented range on the Mauna Kea volcano of the island of Hawai'i. DNA fingerprinting of twelve complete families from the Pu'u La'au population revealed no evidence of extrapair fertilization or intraspecific brood parasitism. Band-sharing coefficients from fingerprints produced with two probes revealed that the large Pu'u La'au population on the southwest slope of Mauna Kea, and a smaller, geographically separate population on the east slope (at Kanakaleonui) had relatively high and virtually identical levels of minisatellite variability (mean S of 0.27 for each population based on combined data of M13 and Jeffreys 33.15 probes). The two populations also had nearly identical allele frequencies based on their mean corrected similarity, S_ij, of 0.98. These data suggest that the two populations have not been fragmented long and/or have sufficient current gene flow to ameliorate any affects of genetic drift. We conclude that present levels of inbreeding are low within both populations, and that proposed translocations of individuals from Pu'u La'au to Kanakaleonui appear appropriate from a genetic standpoint.     

Sound localisation in crickets

Intracellular recordings were made in the brain of the cricket Gryllus bimaculatus from an ascending auditory interneuron (AN1). Acoustic stimuli with calling song temporal pattern were delivered via earphones in a preparation with the acoustic trachea cut (attenuation of crossing sound > 30 dB). The input-output function of this cell was then determined by recording its responses to stimulation of the ipsilateral ear alone, of the contralateral ear alone and to stimulation of both ears simultaneously with the same or different carrier frequencies and intensities.This interneuron was excited by the ear ipsilateral to its axon and dendritic field and unresponsive to stimuli presented to the axon-contralateral ear alone. However, in binaural stimulation experiments, the response to a constant ipsilateral stimulus was progressively reduced as the intensity of a simultaneous contralateral stimulus was increased, above a threshold intensity.Tuning curves for threshold of this inhibition, determined in binaural stimulation experiments, indicated significant inhibition in the range 3–20 kHz with lowest threshold at 4–5 kHz. The inhibition was unaffected by sectioning of the contralateral circumoesophageal or neck connective, indicating that the inhibitory influence crosses the midline at the level of the prothoracic ganglion. Intracellular recordings from AN1 in the prothoracic ganglion confirmed that it was indeed neurally inhibited by inputs from the contralateral ear.Tuning curves for excitation of an omega neuron (ON1) by the ear ipsilateral to its soma and also the tuning of inhibition of ON1 by its contralateral ON1 partner, closely match the tuning of inhibition of AN1 and to a lesser extent, of AN2. This was taken as evidence that each AN1 is inhibited by the contralateral ON1. The significance of this interaction for directional hearing and phonotaxis is discussed.Abbreviations AP/CHP action potentials per chirp - AN1, AN2 ascending auditory interneurons 1, 2 - ON1 omega neuron 1 - ipsi ipsilateral contra contralateral - PTG prothoracic ganglion loc lateral ocellar nerve - On optic nerve an antennal nerve - coc circum-oesophageal connective so sound off     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

Self-fertility and associated flower head traits in the Iberian taxa ofLactuca and related genera (Asteraceae: Lactuceae)

Sexual reproduction is the main reproduction mechanism among the 14 wild Iberian taxa ofLactuca, Prenanthes, Cicerbita, andMycelis. High levels of self-fertilization occur inLactuca, as well as facultative and obligate xenogamy. Xenogamy is strongly correlated with large capitula having blue or bright yellow colouration, high P/O ratios, and long anthesis, whereas self-fertilization is correlated with smaller capitula having pale yellow colouration, small P/O ratios, and short anthesis. Large variations occur in P/O index among taxa showing similar fertilization mechanism, probably in relation to the number of florets included in flower heads. Interesting differences in reproductive systems have been detected between subspecies ofLactuca viminea.     

Maximization of recombinant protein yield in the insect cell/baculovirus system by one-time addition of nutrients to high-density batch cultures

Suspension cultures of Sf-9 cells at different stages of growth were infected with a recombinant baculovirus expressing -galactosidase, using a range of multiplicities of infection (MOI) of 0.05 to 50. Following infection, the cells were resuspended either in the medium in which they had been grown or in fresh medium. Specific -galactosidase yields were not markedly affected by either MOI or medium change in cultures infected in early exponential phase (3×10⁶ cells mL^–1). In cultures infected at later growth stages, -galactosidase yields could only be maintained by medium replacement. The possibility that this requirement for medium replacement is due either to the accumulation of an inhibitory byproduct or nutrient limitation was examined. Alanine, a major byproduct of cultured insect cell metabolism, did not significantly reduce recombinant protein yield when added to infected cultures in concentrations of up to 40 mM. Following a factorial design, various nutrient concentrates were added alone or in combination to cultures infected in late exponential phase. Additions that included both yeastolate ultrafiltrate and an amino acid mixture restored specific -galactosidase yields to levels observed at earlier growth stages or in late stages with medium replacement; the addition of these concentrates, by permitting production at higher cell density, led to increases in the volumetric yield of recombinant protein. Together or separately, the concentrates when added to uninfected late exponential phase cultures, lead to a doubling of the maximum total cell protein level normally supported by unamended medium.     

Identification and characterization of a gene cluster involved in nitrate transport in the cyanobacterium Synechococcus sp. PCC7942

Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O₂ evolution (photosynthetic O₂ evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O₂ evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system.