Frog stomach enteric plexuses in culture: isolation, morphological characterization and bioelectrical recordings

We have succeeded in the isolation, culture and morphological characterization of Rana ridibunda stomach enteric plexuses. We have furthermore obtained intra and extracellular bioelectric recordings from the explants in culture. The culture medium used (Eagle MEM), the collagenase digestion and the general culture conditions followed are similar to those applied to mammal enteric plexus explant cultures. The most striking difference is that the solutions were diluted to 70% in order to maintain the osmolar conditions required by the amphibian cells. Acetylcholinesterase, osmium tetroxide-zinc iodide- and para-formaldehyde-induced fluorescence methods reveal similar morphological images from the perivascular fibre plexuses. The different cell types observed by phase contrast light microscopy from the myenteric explants in culture have been identified by comparison with those revealed by the acetylcholinesterase method. The prevailing neurons show piramidal somas; other neurons are bipolar with oval somas and a third type shows oval somas tightly aligned, following sinusoidal courses. The intra and extracellular bioelectric recordings from the explants in culture show that the culture conditions we have applied preserve the electrophysiological properties of the neuronal membranes. These preliminary recordings will allow us to undertake the synaptic characterization of the gastrointestinal neurotransmitters in frogs.     

The effect of kinetin and naphthalenacetic acid upon localized accumulation as related to senescence in detached leaves

Summary Naphthaleneacetic acid, when applied together with kinetin to defined areas of detached leaves of Xanthium pensylvanicum Wallr. or broccoli (Brassica oleracea L., var. italica), reduced the senescence-retarding effect of kinetin but synergistically increased the kinetin-induced accumulation of ¹⁴C from labeled glycine. Naphthaleneacetic acid by itself did not accelerate senescence. The ability of kinetin to delay senescence in these detached leaves therefore can not be explained as a result of the gross accumulation (mobilization) of protein precursor.     

Cytological and molecular observations on Solanum phureja-induced dihaploid potatoes

Summary Seventeen potato dihaploids, produced by pollinating the tetraploid (2n = 48) cv Pentland Crown with pollen from Solanum phureja (2n = 24) dihaploid inducer clones, were studied. Since dihaploids are thought to develop parthenogenetically from unfertilized ovules they were expected to be euploid (2n = 24), but somatic chromosome counts showed that 15 of the 17 dihaploids were aneusomatic. Ten of the clones were predominantly diploid (2n = 24) with a proportion of hyperploid cells that contained 25 or 26 chromosomes. Five of the dihaploids contained variable numbers of triploid cells (2n = 36). RFLP analysis was used to determine whether the additional chromosomes were from S. phureja or S. tuberosum. Unique hybridizing fragments present in S. phureja but not in Pentland Crown were identified. These S. phureja-specific restriction fragments were present in some of the dihaploid offspring of Pentland Crown. Of the 5 clones that contained triploid cells 4 had S. phureja type banding. Four of the 10 aneusomatic clones that contained hyperploid cells had the unique S. phureja hybridizing fragments. We propose that ovules of Pentland Crown were fertilized by pollen from S. phureja and that the aneusomatic clones were derived from triploid zygotes from which some of the S. phureja chromosomes were eliminated. We consider that this is an additional mechanism of dihaploid formation in potato.     

Glutamate Receptor Requirement for Neuronal Death from Anoxia–Reoxygenation: An in Vitro Model for Assessment of the Neuroprotective Effects of Estrogens

1.Previous studies demonstrated that estrogens, specifically 17-estradiol, the potent, naturally occurring estrogen, are neuroprotective in a variety of models including glutamate toxicity. The aim of the present study is twofold: (1) to assess the requirement for glutamate receptors in neuronal cell death associated with anoxia–reoxygenation in three cell types, SK-N-SH and HT-22 neuronal cell lines and primary rat cortical neuronal cultures, and (2) to evaluate the neuroprotective activity of both 17-estradiol and its weaker isomer, 17-estradiol, in both anoxia-reoxygenation and glutamate toxicity.2.SK-N-SH and HT-22 cell lines, both of which lack NMDA receptors as assessed by MK-801 binding assays, were resistant to both anoxia–reoxygenation and glutamate-induced cell death. In contrast, primary rat cortical neurons, which exhibit both NMDA and AMPA receptors, were sensitive to brief periods of exposure to anoxia–reoxygenation or glutamate. As such, there appears to be an obligatory requirement for NMDA and/or AMPA receptors in neuronal cell death resulting from brief periods of anoxia followed by reoxygenation.3.Using primary rat cortical neuronal cultures, we evaluated the neuroprotective activity of 17-estradiol (1.3 or 133 nM) and 17-estradiol (133 nM) in both anoxia–reoxygenation and excitotoxicity models of cell death. We found that the 133 nM but not the 1.3 nM dose of the potent estrogen, 17-estradiol, protected 58.0, 57.5, and 85.3% of the primary rat cortical neurons from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively, and the 133 nM dose of the weak estrogen, 17-estradiol, protected 74.6, 81.7, and 85.8% of cells from anoxia–reoxygenation, glutamate, or AMPA toxicity, respectively. These data demonstrate that pretreatment with estrogens can attenuate glutamate excitotoxicity and that this protection is independent of the ability of the steroid to bind the estrogen receptor.     

In vitro regeneration from hypocotyl and seedling cotyledons of tronchuda (Brassica oleracea var. tronchuda Bailey)

Four tronchuda (Brassica oleracea var. tronchuda Bailey) cultivars were tested for their ability to regenerate in vitro on Murashige & Skoog (MS) medium supplemented with 3 different combinations of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Explants were either axillary bud-free whole cotyledons or hypocotyls from 7-day-old darkgrown seedlings. The ability to regenerate varied by cultivars, explants and the concentration of growth regulators. Hypocotyl explants of all 4 cultivars, and cotyledon explants of 2 cultivars, developed plantlets within 4 weeks. Hypocotyl explants produced more shoots than cotyledons. Cotyledon explants produced more roots than hypocotyls. Best shoot regeneration was on MS medium supplemented with 2 mgl^-1 BAP and 0.1 mgl^-1 NAA. Portuguesa produced the most shoots. Some regenerants varied in leaf shape and phyllotaxy.Abbreviations BAP benzylaminopurine - NAA napththaleneacetic acid - IBA indolebutyric acid     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Regeneration of Cucumis sativus var. sativus and C. sativus var. hardwickii,C. melo,and C. metuliferus from explants through somatic embryogenesis and organogenesis

Regeneration in six inbred lines or F₁ hybrids of Cucumis sativus was achieved on Murashige & Skoog's medium containing various concentrations of 2,4-D/BA, NAA/BA, NAA/Z or NAA/K. The range of regeneration frequency for cotyledon, leaf and petiole explants was 0–38, 0–75 and 14–96%, respectively, after 6–8 weeks in culture. Only one subculture of calli to growth regulator-free medium was required for regeneration. Preincubation of explants in the dark for 2–3 weeks was essential to achieve optimal regeneration. Highest frequency of plantlet formation occurred with petiole explants incubated on NAA/BA (5.0/2.5 M), NAA/Z (5.0/5.0 M) or 2,4-D/BA (5.0/5.0 M). Approximately 80% of these plantlets survived after transplanting to greenhouse soil, and they flowered and set fruit. The F₁ hybrid, Endeavor, gave the highest regeneration frequency of 91% on 2,4-D/BA at 5.0/5.0 M. Formation of somatic embryos was observed on 2,4-D/BA, while organogenesis and embryogenesis both were evident on NAA/BA and NAA/Z. Cotyledonary explants yielded the lowest frequency (ca. 7%) of plantlet formation in this study. Plantlets of C. sativus var. hardwickii and an F₁ hybrid of C. sativus x C. s. var hardwickii were regenerated on NAA/Z and NAA/K at frequencies of 15–65%, predominantly by the formation of somatic embryos. Shoots were obtained from cotyledon and leaf explants of C. metuliferus on IAA/BA (7.5/5.0 M) and from leaf and petiole explants of C. melo on NAA/BA (5.0/2.5 M), but plantlets were recovered only in C. melo.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indoleacetic acid - K kinetin - MS Murashige & Skoog's medium - NAA naphthaleneacetic acid - Z zeatindihydroside     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Concluding remarks

On the basis of symposium contributions onChlorella, Hibbertia, Eucalyptus, Ambrosia and on numerical approaches some fundamental problems of (bio)systematics, evolution, and taxonomic categories are discussed: Methods available for analysing affinities; conflicting evidence from phenetic, biochemical, cytogenetic and other analyses; further classification problems in cases of intermediacy, etc. While sibs of various levels and their natural hierarchy often can be objectively defined, this appears impossible for particular taxonomic levels itself (e. g. species). A single objective taxonomic system of organisms is unrealistic. Certain guiding lines for relative and practicable concepts of species and genus are proposed.Presented at the symposium Speciation and the Species Concept during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Assessment of the Respiratory Costs of Adaptation in Plant Species That Differ in Their Responses to Insufficient and Excessive Mineral Nutrition

The effects of insufficient and excessive mineral nutrition on the relative growth rate (RGR); the rate of total dark respiration (R); gross photosynthesis (Pg); and the contents of endogenous IAA, ABA, and cytokinins (CK) were studied in 20–60-day-old seedlings of plant species that differed in their responses to these factors: beet root (Amaranthus retroflexusL.), orchard-grass (Dactylis glomerataL.), and common wheat (Triticum aestivumL.). The changes in the level of mineral nutrition lowered the RGR and Pgvalues and the IAA/ABA and CK/ABA ratios and increased the Rand R/Pgvalues and ABA content, especially in A. retroflexusand T. aestivum. The authors propose a new way to assess the respiratory cost of adaptation to stress based on the relation between the RGR and the R/Pgvalues. The adaptation component of Ris shown to relate to the ABA content.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Effect of mutant host RNA polymerase on the bifunctional activities of P22 gene c1.

Summary A phage has been isolated which specifically transduces the Escherichia coli pheS and pheT genes coding for the and subunits of the phenylalanyl-tRNA synthetase (PRS). This phage transduces with high frequency (i) several temperaturesensitive PRS mutants to thermoresistance and (ii) a p-fluorophenylalanine resistant PRS mutant to sensitivity against this amino-acid analog. The in vitro PRS activities of such lysogens suggest that the and subunits coded by the transducing phage complement the mutant host PRS-subunits in vivo by means of formation of hybrid enzymes.The transducing phages were also used to infect UV light irradiated cells. The SDS-gel electrophoretic analysis of the proteins synthesized in such cells revealed that the phage codes at least for four different E. coli proteins. Two proteins with molecular weights of 94,000 and 38,000 daltons cross-reacted with an anti PRS serum and were thus identified as the and subunits of PRS, respectively. A third protein with w molecular weight of 22,000 daltons is identical with the ribosomal initiation factor IF3 (Springer et al., 1977b). The other protein (M_r 78,000) is still unidentified.     

Discrimination of sunflower volatiles by the red sunflower seed weevil

Five principle monoterpenoid and other constituent volatile chemicals of sunflower heads were combined to resemble two lines of sunflower (Helianthus annuus L.): one U.S.D.A. standard line and one French line which was poorly visited by insects (Etievant et al., 1984). Field trials of attraction to red sunflower seed weevils (Smicronyx fulvus Le Conte, Coleoptera: Curculionidae) showed that one was clearly preferred over the other. The more attractive mixture contained -pinene, -pinene, limonene, camphene and bornyl acetate in a ratio resembling that of Flath et al. (1985) rather than that described by Etievant et al. (1984). One or two volatiles were deleted from the optimal blend but only mixtures of five volatiles showed the highest attraction. Substitution of sabinene, another volatile prominent in sunflower, for one of the five in the optimal blend also decreased attraction of seed weevils. When the monoterpenoid components and green leaf volatiles in the traps resembled the ratios of most of the prominent volatiles of sunflower, attraction was significantly greater than controls.     

Characterization of sodium and pyruvate interactions of the two carrier systems specific of mono- and di- or tricarboxylic acids by renal brush-border membrane vesicles

Summary The experiments reported in this paper aim at characterizing the carboxylic acid transport, the interactions of pyruvate and citrate with their transport sites and specificity. The study of these carriers was performed using isotopic solutes for the influx measurements in brush-border membrane vesicles under zerotrans conditions where the membrane potential was abolished with KCl preloading with valinomycin or equilibrium exchange conditions and =0.Under zerotrans condition and =0, the influence of pyruvate concentrations on its initial rates of transport revealed the existence of two families of pyruvate transport sites, one with a high affinity for pyruvate (K _t =88 m) and a low affinity for sodium (K _t =57.7mm) (site I), the second one with a low affinity for pyruvate (K _t =6.1mm) and a high affinity for sodium (K _t =23.9mm) (site II). The coupling factor [Na]/[pyruvate] stoichiometry were determined at 0.25mm and 8mm pyruvate and estimated at 1.8 for site I, and 3 when the first and the second sites transport simultaneously.Under chemical equilibrium (0) single isotopic labeling, transport kinetics of pyruvate carrier systems have shown a double interaction of pyruvate with the transporter; the sodium/pyruvate stoichiometry also expressed according to a Hill plot representation wasn=1.7. The direct method of measuring Na⁺/pyruvate stoichiometry from double labeling kinetics and isotopic exchange, for a time course, gives an=1.67.Studies of transport specificity, indicate that the absence of inhibition of lactate transport by citrate and the existence of competitive inhibition of lactate and citrate transports by pyruvate leads to the conclusion that the low pyruvate affinity site can be attributed to the citrate carrier (tricarboxylate) and the high pyruvate affinity site to the lactate carrier (monocarboxylate).     

Release of endogenous excitatory amino acids in the neostriatum of the rat under physiological and pharmacologically-induced conditions

Summary There is immunohistochemical evidence suggesting that glutamate (Glu) is released from nerve terminals and acts, via several receptor subtypes, as a major excitatory neurotransmitter in the cortico-striatal pathway of the rat. Aspartate (Asp) is also present in cortico-striatal neurons, but its role as a neurotransmitter has been questioned, since, in contrast to Glu, it has not been demonstrated in presynaptic vesicles. Glu and Asp can be found at subM concentrations in the extracellular compartment of most areas of the basal ganglia. Their concentrations are largely regulated by transport mechanisms, but also by a synaptotagmin-dependent exocytotic release, and are sufficiently high to occupy junctional and extrajunctional receptors.We have investigated whether Glu and Asp release in the neostriatum can be selectively modulated by different neuronal systems. Dopamine (DA) and cholecystokinin (CCK) selectively stimulate Asp release, via D₁ and CCK_B receptor subtypes, respectively. Also opioid -agonists increase Asp release. We propose that the selective modulation of Asp release by D_1–, CCK_B- and agonists involves striatal neurons containing Asp, but not Glu. In contrast, local perfusion with the ,-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH₂ (CTOP) increases both Glu and Asp release. This effect is probably exerted on cortico-striatal terminals, via presynaptic inhibitory -receptors. Thus, these results demonstrate that extracellular levels of Glu and Asp are modulated differentially by different neuronal systems, and suggest that in the neostriatum of the rat there are neuronal populations using Glu and/or Asp as messenger(s).     

Phenylalanyl-tRNA synthetase fromE. coli MRE-600: Localization of the phenylalanine binding sites on the subunits by affinity reagents

The phenylalanine and the phenylalanyl-tRNA^Phe binding sites on the subunits of phenylalanyl-tRNA synthetase fromE.coli MRE-600 were localized using p-azidoanilidate of [¹⁴C]phenylalanine and N-bromoacetyl[¹⁴C]phenylalanyl-tRNA^Phe. The phenylalanine recognizing site was shown to be situated on the subunit of the enzyme in close proximity to the contact region of the and subunits and the phenylalanyl-tRNA^Phe recognizing site on the subunit. Transfer of the aminoacyl moiety from the subunit to the subunit of the enzyme was assumed to take place in the process of catalysis of the aminoacylation reaction.     

Effects of exercise training and diabetes on cardiac myosin heavy chain composition

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. -MHC and -MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of -MHC. Sedentary diabetics exhibited a shift to -MHC. Exercise trained diabetic rats showed a predominance of -MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the -MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.     

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter--glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. Pharo. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 g l–1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants     

Structure function analysis of theH-2 Ab p gene

The gene encoding the H-2A ^p class II chain was isolated from a B10.P genomic library and sequenced. This gene was also used to construct transfectants of the CH12 lymphoma clone CH12.LX, which express the Ab^p gene product in association with the endogenous A ^k chain. We present here the first report of the complete nucleotide coding sequence of Abp. The predicted amino acid sequence of Abp reveals only five residues different from Ab q, four of which are present in the mature peptide. These four amino acid changes could account for the differential susceptibility of H-2 q vs H-2 p mice to the development of collagen-induced arthritis (CIA). Antibodies specific for the transfected Abp protein induce CH 12. LX cells to secrete immunoglobulin in the presence of antigen. Comparison of the amino acid sequence with other A chains that have been tested in signal transduction experiments suggests that amino acid 9 may be important to the signaling ability of class II A molecules.     

Structure of cells and nerve endings in abdominal vagal paraganglia of the rat

Summary The abdominal vagal paraganglia of the rat consist of small groups of cells, interspersed by blood vessels and nerve bundles and lying close to, or within, the vagus nerve or its branches. Each cell group consists of 2–10 Type I cells incompletely invested by 1–3 satellite cells. Type I cells are characterised by the presence of numerous dense-cored vesicles in their cytoplasm and may exhibit synaptic-like contact with each other.Small efferent nerve endings make synaptic contacts with Type I cells. Larger cup-shaped afferent nerve endings also make synaptic contacts of two kinds with Type I cells. Nerve-nerve synapses are often seen within or close to paraganglia.Attention is drawn to the close similarity of fine structure of abdominal vagal paraganglia, carotid body and small intensely fluorescent cells of the superior cervical ganglion in rats. Possible functional implications of this morphological similarity are discussed.