Establishment and characterization of photosynthetic hairy root cultures of Datura stramonium

Twelve different lines of Datura stramonium (normal and hairy) root cultures were subjected to conditions which induce photoautotrophy. Two of the hairy root lines responded to induction, showing clearly a diminished growth rate when compared to heterotrophic cultures, an increase in chlorophyll, a net O₂ evolution, CO₂ fixation, and de novo synthesis of the ribulose 1,5 biphosphate carboxylase enzyme. A time course of growth and tropane alkaloid levels in the tissue and medium, revealed a correlation between the development of the photosynthetic apparatus and the increase in scopolamine. Although normal cultures did not grow photosynthetically, they showed some greening response under the first step of the induction. The correlation between development of photosynthesis and increase in scopolamine synthesis were corroborated with normal root cultures. This experimental model is used for the basic study of the regulatory enzymes involved in the biosynthesis of tropane alkaloids, as well as for the study of their mechanisms of transport.     

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression

Abstract: Two forms of pituitary adenylate cyclase-activating polypeptide (PACAP), the 38- and 27-amino-acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100- to 1,000-fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP-selective type I receptor. PACAP38 elicited a sustained seven- to ninefold increase in the rate of NPY secretion and three-fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro-NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro-NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.     

Spinal Cord Dynorphin Precursor Intermediates Decline During Late Gestation

Abstract: This laboratory has previously reported that the maternal opioid analgesia associated with pregnancy and parturition is mediated, at least in part, by a maternal spinal cord dynorphin/κ opioid system. This analgesia is accompanied by an increase in dynorphin peptides (1–17 and 1–8) in the lumbar spinal cord. Levels of trypsin-generated arginine⁶-leucine-enkephalin (Leu-Enk-Arg)-immunoreactive determinants were also determined and used to reflect the content of dynorphin precursor intermediates. In spinal tissue, the amount of dynorphin A (1–17) contained in the form of precursor is, at a minimum, 10-fold higher than the content of mature dynorphin A (1–17) or dynorphin (1–8). During gestational day 22, the content of dynorphin precursor is reduced significantly (∼50%). The decline in the magnitude of dynorphin precursor intermediates in the spinal cord of pregnant rats vastly exceeds the magnitude of increase in the content of dynorphin peptides (1–17 and 1–8). This difference can best be explained by postulating a corresponding increase in the rate of release of spinal cord dynorphin (1–17). It is suggested that enhanced processing of dynorphin precursor intermediates represents the initial biochemical level of adaptation of spinal dynorphin neurons to increased demands of pregnancy.     

Synthesis of nitrodiazirinyl derivatives of neurotoxin II fromNaja naja oxiana and their interaction with theTorpedo californica nicotinic acetylcholine receptor

Five singly modified nitrodiazirine derivatives of neurotoxin II (NT-II) fromNaja naja oxiana were obtained after NT-II reaction with N-hydroxysuccinimide ester of {2-nitro-4 [3-(trifluoromethyl)-3H-diazirin-3yl]phenoxy}acetic acid followed by Chromatographic separation of the products. To localize the label positions, each derivative was first UV-irradiated and then subjected to reduction, carboxymethylation, and trypsinolysis. Tryptic digests were separated by reversed phase-HPLC, the labeled peptides being identified by mass spectrometry. The derivatives containing the photolabel at the position Lys 25, Lys 26, Lys 44, or Lys 46 were [¹²⁵I]iodinated by the chloramine T procedure. Each iodinated derivative was found to form photoinduced cross-links with the membrane bound nicotinic acetylcholine receptor (AChR) fromTorpedo californica. The pattern of labeling the receptor's, , , or subunits was dependent on the photolabel position in the NT-II molecule and differed from that obtained earlier with an analogous series ofp-azidobenzoyl derivatives of NT-II. The results obtained indicate that (i) different sides of the neurotoxin molecule are involved in the AChR binding, and (ii) fragments of the different AChR subunits are located close together at the neurotoxin-binding sites.Abbreviations AChR Acetylcholine receptor - NDPA [2-nitro-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]]phenoxy]acetyl - NT-II neurotoxin II     

Arthropod biodiversity loss and the transformation of a tropical agro-ecosystem

The coffee (Coffea arabica) agro-ecosystem in the Central Valley of Costa Rica was formerly characterized by a high vegetational diversity. This complex system has been undergoing a major transformation to capital-intensive monocultural plantations where all shade trees are eliminated. In this study we examined the pattern of arthropod biodiversity loss associated with this transformation. Canopy arthropods were sampled in three coffee farms: a traditional plantation with many species of shade trees, a moderately shaded plantation with only Erythrina poeppigeana and coffee, and a coffee monoculture. An insecticidal fogging technique was used to sample both canopy and coffee arthropods. Data are presented on three major taxonomic groups: Coleoptera, non-formicid Hymenoptera, and Formicidae. Data demonstrate that the transformation of the coffee agro-ecosystem results in a significant loss of biological diversity of both canopy arthropods as well as arthropods living in coffee bushes. Percentage of species overlap was very small for all comparisons. Furthermore, species' richness on a per tree basis was found to be within the same order of magnitude as that reported for trees in tropical forests. If results presented here are generalizable, this means that conservation efforts to preserve biological diversity should also include traditional agro-ecosystems as conservation units.     

Some environmental factors affecting the reproduction of Aphanius dispar (Rüppell, 1828)

In order to determine the suitability of thecyprinodont fish Aphanius dispar as a biologicalcontrol agent for mosquito larvae, the effects oftemperature, salinity, feeding rate and photoperiod onits reproductive condition were studied. The factorsexamined were the gonado-somatic index (GSI) and fourdistinct stages of cell maturation in the ovaries(oogonia, primary oocytes, secondary oocytes andmature ova). The fish were found to be tolerant to awide range of the examined factors. GSI's were notaffected by ambient temperatures over the range of18 °C to 37 °C. However, oocyte maturationstages increased as temperatures rose from 18 °Cto 27 °C, with mature ova first being found at thelatter temperature. No significant differences amongmaturation stages were found between27 °C–37 °C. At salinities ranging from of0 ppt to 56 ppt, no significant differences were found betweenmean GSI's, except for the two extremes of the testrange. The ovaries of fish kept in distilled watercontained primordial germ cells only. In feedingexperiments, significant differences were found onlybetween the ovaries (GSI's and oocyte maturationstages) of fish deprived of food and those fed at1% body weight per day. Regarding photoperiod,a decrease in day length from 14L:10D (the longest dayof the year) to 10L:14D (the shortest) caused adecrease in oocyte maturation stages. Fish at theintermediate level (12L:12D) still had ovariessuitable for spawning, whereas those at 10L:14D didnot. The results of this study show that limitinglevels for adequate reproductive condition were foundfor some of the factors investigated, however, rangesof the factors above these levels were relativelylarge. That Aphanius dispar's reproductivecondition may be controlled in an artificialenvironment is essential for it to be mass produced,and a prerequisite for its use in the biologicalcontrol of mosquito larvae.     

Nonlinear autoregressive analysis of the 3/s ictal electroencephalogram: implications for underlying dynamics

 In a previous study, nonlinear autoregressive (NLAR) models applied to ictal electroencephalogram (EEG) recordings in six patients revealed nonlinear signal interactions that correlated with seizure type and clinical diagnosis. Here we interpret these models from a theoretical viewpoint. Extended models with multiple nonlinear terms are employed to demonstrate the independence of nonlinear dynamical interactions identified in the ‘NLAR fingerprint’ of patients with 3/s seizure discharges. Analysis of the role of periodicity in the EEG signal reveals that the fingerprints reflect the dynamics not only of the periodic discharge itself, but also of the fluctuations of each cycle about an average waveform. A stability analysis is used to make qualitative inferences concerning the network properties of the ictal generators. Finally, the NLAR fingerprint is analyzed in the context of Volterra-Weiner theory. Received: 6 April 1994/Accepted in revised form: 18 November 1994     

Therapy of bovine ocular squamous-cell carcinoma with local doses of interleukin-2: 67% complete regressions after 20 months of follow-up

We have tested the therapeutic potency of peritumorally injected low doses of interleukin-2(IL-2). Seventy tumours of the bovine ocular squamous-cell carcinoma (BOSCC), 1–3 cm in diameter, were treated with 5000, 20 000 or 200 000 U IL-2 from Eurocetus (Chiron) to find the optimal dose for treatment. Injections were given peritumorally on Monday to Friday on 2 consecutive weeks. The size of the tumours was measured before treatment and 1, 3, 4, 9 and 20 months after treatment. After 9 months complete regression was observed in 89% of the tumours treated with 5000 U IL-2, 80% treated with 20 000 U and 67% treated with 200 000 U. After 20 months, there was complete regression of 35%, 31% and 67% of the tumours respectively. The 9-and 20-month results of the 200 000-U treatment are significantly better than those of the 5000-U and 20 000-U treatments taken together. This protocol may be useful to treat advanced inoperable tumours (e.g. of the nasopharynx or skin) of human patients.     

Facile removal of the N-indole-mesitylenesulfonyl protecting group using HF cleavage conditions

Summary Nitrogen indole protection of the -methyltryptophan side-chain residue is important for avoiding undesired side reactions during peptide synthesis. Of great importance is the choice of a side-chain protecting group for orthogonal peptide synthesis and its stability under a variety of chemical conditions required for synthesis of the four isomers of this unusual amino acid. We report here the successful use of the mesitylenesulfonyl (Mts) protecting group for -methyltryptophan in the synthesis of melanotropin and CCK peptide analogues and the ready cleavage of this protecting group under HF conditions.