Thermoregulatory Activity of Dermorphin Fragments

We studied the effect of C-terminal truncation of the dermorphin (DM) molecule and analogs of its N-terminal tetrapeptide, [DOrn²]-DM_1–4, [DArg²]-DM_1–4, [DAla⁴]-DM_1–4, [DArg², DAla⁴]-DM_1–4, Arg-DM_1–4, Arg-[DArg²]-DM_1–4, Arg-[DAla⁴]-DM_1–4, and Arg-[DArg², DAla⁴]-DM_1–4, on the functional status of the thermoregulation system in rats at different ambient temperatures. For the first time, we demonstrate that the N-terminal tetrapeptide is the minimal fragment with the hypothermic effect. Only the N-terminal octapeptide exerted the vasomotor effect. Amino acid substitutions in the tetrapeptide affected its hypothermic effect. [DArg²]-DM_1–4 and [DArg², DAla⁴]-DM_1–4 had the greatest effect. Addition of Arg to the N-terminus of DM_1–4 analogs changed their thermoregulatory activity. The greatest thermoregulatory effect was observed for Arg-[DArg²]-DM_1–4 and Arg-[DArg², DAla⁴]-DM_1–4.     

Effect of [DAla4]-Dermorphin and Its Analogs on the Functional Status of the Thermoregulation System in Rats at Different Environmental Temperature

We studied the influence of [DAla⁴]-dermorphin ([DAla⁴]-DM), its fragments ([DAla⁴]-DM_2–7, [DAla⁴]-DM_3–7, and [DAla⁴]-DM_4–7) and analogs ([Trp¹, DAla⁴]-DM, [DArg², DAla⁴]-DM, and [DOrn², DAla⁴]-DM) on the functional status of the thermoregulation system in rats at different thermal environments: cold (4–6°C), thermoneutral (27–28°C) and hot (31–32°C). [DAla⁴]-DM administration proved to induce temperature- and dose-dependent hypothermia and vasomotor responses. No activity of the peptide was observed in the hot environment. N-terminal shortening of the peptide inhibits its thermoregulatory activity. Tyr¹ to Trp¹ substitution nearly completely suppressed the thermoregulatory effects. DAla² to DArg² substitution decreased the hypothermic effect and only the vasodilatory response was observed in the comfortable environment. DAla² to DOrn² substitution completely suppressed the hypothermic effect in the cold environment and considerably attenuated the vasodilatory response in the thermoneutral environment.     

Comparative analysis of analgesic activities of dermorphin, [DPro<Superscript>6</Superscript>]-dermorphin,and their C-terminal tripeptides

Analgesic activities of dermorphin (DM), [DPro⁶]-DM, and their C-terminal tripeptides were comparatively studied. Analgesic activity was evaluated in tail flick, hot plate, tail pinch, formalin, and acetic acid writhing tests describing different levels of organization of pain sensitivity. Intraperitoneal administration of the peptides decreased the pain threshold in all these tests. The C-terminal tripeptides DM_5–7 and [DPro⁶]-DM_5–7 demonstrated analgesics activity comparable or sometimes higher than that of the full-length molecules. The effect of DM, [DPro⁶]-DM, and C-terminals fragments DM_5–7 and [DPro⁶]-DM_5–7 decreased after co-administration with naloxone, which points to the opioid nature of analgesic activity of the peptides.     

Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature

Summary The histochemical activities of succinic dehydrogenase (SDH) and Ca⁺⁺-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. Red and white muscle fibres, as distinguished by SDH, showed high and low Ca⁺⁺-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K₂-EDTA solution. The ultrastructural investigation of CA⁺⁺-ATPase reaction at pH 7.4 by the Ca⁺⁺-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and Z bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb⁺⁺ precipitation technique demonstrated Mg⁺⁺-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail red muscle fibres are possible slow, and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, red muscle fibres of the anuran tail musculature are not equivalent to Type I fibres of higher chordates.     

Effect of some metals and related metal-orgaic compounds on ALA-dehydratase activity of corn

Summary The activity of ALA-dehydratase from corn seedlings is affected by Mn⁺⁺, Fe⁺⁺, Pb⁺⁺, Cu⁺⁺, Zn⁺⁺ and Sn⁺⁴ ions, in vivo Mn⁺⁺ and Fe⁺⁺ are ativators while Pb⁺⁺ and Sn⁺⁴ are inhibitors; in vitro Cu⁺⁺ and Zn⁺⁺ are inhibitors. The kinetic parameters (V_max and K_M) support the hypothesis that Mn, Fe, Sn and Pb ions act on the biosynthesis of the enzyme and Zn and Cu ions on the enzyme-substrate affinity. Some related metal-organic compounds interrere in vivo on the ALA-dehydratase activity modifying the kinetic parameters, therefore the enzyme biogenesis and/or enzyme-sustrat affinity are affected.     

High-level Expression of Maize <Emphasis Type="Italic">γ</Emphasis>-zein Protein in Transgenic Soybean (<Emphasis Type="Italic">Glycine max</Emphasis>)

Primers were developed for 118 microsatellites isolated from grape (Vitis vinifera) genomic libraries enriched for (AC)n repeats. Only one microsatellite sequence matched other grape SSR-sequences in the GeneBank database. Genotyping was carried out in the parental lines and four offspring of two pseudo-test-cross populations, Cabernet Sauvignon x Seyval and Chardonnay x Bianca, and a further six other grape genotypes (V. vinifera Sultanina, Merlot, Syrah, Müller-Thurgau, Vitis Regent and V. riparia Gloire de Montpellier). A total of 108 microsatellites showed easily scorable alleles and 100 of them segregated according to a configuration suitable for mapping in either cross. A further 8 SSRs, although unsuitable for mapping in those crosses, showed polymorphism in the other genotypes tested. This set of markers was used, along with 75 microsatellites of other repeat-types, to fingerprint 46 offspring of the cross Chardonnay x Bianca. For each full-sib, individual heterozygosity and distance in repeat units between pairs of alleles at each locus (mean d²) were calculated as a tool for predicting highly outbred recombinant individuals. Six microsatellites with segregation ratios significantly distorted towards the lack of homozygous sibs were identified and mapped to linkage groups LG 3 and LG 5. Estimation of heterozygosity at genome-wide level and genotyping at loci for which homozygous sibs are discriminated against are discussed for marker-assisted background selection in outcrossing grapevines.     

Codon recognition mechanisms in plant chloroplasts

In chloroplasts, all 61 sense codons are found in chloroplast (cp) DNA sequences coding for proteins. However among the sequenced cp tRNAs or tRNA genes, tRNAs with anticodons complementary to codons CUU/C (Leu), CCU/C (Pro), GCU/C (Ala) and CGC/A/G (Arg) [or CGC/A (Arg) in Marchantia] have not been found. In this paper we show that cp tRNA^Ala(U^*GC), cp tRNA^Pro(U^*GG) and cp tRNA^Arg(ICG) are able to decode the corresponding four-codon family. In the case of leucine codons CUU/C, we show that U:U and U:C wobble mechanisms can operate to allow the reading of these codons by cp tRNA^Leu (UAm⁷G).     

Life tables of Moina macrocopa (Straus) in successive generations under food and temperature adaptation

Life tables of Moina macrocopa (Straus) cultured at seven foodconcentrations (FC) (Scenedesmus sp., 1.49–1490 mg wet weightl^-1, 10⁴–10⁶ cellml^-1) were investigated for animals of the first generation(nonadapted animals) and for animals of the third generation (adaptedanimals) cultivated at these FC. Adapted animals showed a trophicpreferendum, i.e. a narrow FC-range at which maximal R_ovalues were observed in comparison with nonadapted animals. In adaptedanimals, the maximal R_o was 115.3 individual, observed at 20°C and a FC of 74.5 mg wet weight l^-1.     

Effects of soil pH and associated cations on growth of apple trees planted in old orchard soil

Summary Trunk circumferences were measured on apple trees which had been grown for 5 to 10 years on land previously in apple orchard. Soil beneath each tree was analysed for soil acidity characteristics and basic cations. Tree growth (trunk circumference), was generally retarded but highly variable, but correlated well with the soil measurements in four of the six orchards investigated. Size of Delicious, Tydeman and Rome Beauty cultivars correlated positively with soil pH and negatively with 0.02M CaCl₂-soluble Al and Mn, while that of McIntosh grown adjacent to the Delicious and Tydeman trees in two of those orchards correlated little or not at all with these soil acidity characteristics. However, in the three orchards where McIntosh trees were grown, their size correlated (r=0.72^**, 0.71^**, 0.54^**) well with exchangeable soil Mg. The effects of soil acidity and associated nutrient deficiencies on growth of young apple trees is discussed.Contribution No. 602.     

Kinetic studies of peptide bond formation. Effect of chloramphenicol

There might be an undetermined order in the interaction of the substrates with the ribosomes in the reaction of CACCA-Leu-Ac with puromycin to form Ac-Leu-puromycin and CACCA (fragment reaction). Km 0°=6×10^-4 M for the puromycin · ribosome interaction. Chloramphenicol totally blocks the fragment reaction as a consequence of a single interaction with the ribosome of Kd 0°=2.2×10^-6 M. The inhibition by chloramphenicol of the fragment reaction is mixed competitive for puromycin.     

The effect of leaf source and developmental stage on shoot organogenic potential of sweetgum (Liquidambar styraciflua L.) leaf explants

Leaf explants harvested from shoot proliferating cultures and intact plants of Liquidambar styraciflua Variegata were placed on solidified Woody Plant medium supplemented with 0.1 mgl^-1 (0.5 M) naphthaleneacetic acid and 2.5 mgl^-1 (11.1 M) benzyladenine to initiate shoot meristems directly. Leaves from intact plants produced over 4 times more adventitious shoots than leaves from in vitro shoots and had a greater tendency to form shoots on the lamina. The relative developmental age of leaf tissue used dramatically influenced the shoot organogenic response observed for leaf explants from intact plants of L. styraciflua Variegata and Moraine.-Leaves that were either 20% or 50% of full size and still actively expanding were superior to other developmental stages for shoot organogenesis. As developmental leaf age increased throughout the period of leaf expansion, the number of shoots forming on the petiole stub remained constant, whereas shoot formation on the lamina increased 8 fold. Shoots derived from Variegata leaves rooted well and grew normally as plants. Differences in rooting ability and plant size could be detected between groups that had been separated according to explant source (in vitro vs. intact plant) and the location of shoot formation (petiole vs. lamina).     

Further studies on the biosynthesis of the avermectins

Summary The biosynthesis of avermectins was studied further inStreptomyces avermitilis MA5502 by feeding experiments with labeled precursors.¹³C-NMR analysis of the compounds biosynthesized from [2-¹³C]acetate, [1,2-¹³C₂]acetate, [3-¹³C]propionate and [2,3-¹³C₂]propionate confirmed that the aglycone of avermectins is made from seven intact acetate and five propionate units. Feeding experiments with [1-¹³C]2-methylbutyrate and [1-¹³C]isobutyrate have shown that 2-methylbutyrate and isobutyrate are immediate precursors of the starter units of the polyketide chains of avermectin a and b components, respectively. The³H/¹⁴C doublelabeling experiments suggest that the two oleandrose moieties are derived from glucose.     

Sublittoral macrozoobenthic assemblages in the enclosed sediment-polluted Rhine-Meuse Delta; their relationship to environmental conditions

Composition, densities and biomass of sublittoral macrozoobenthos were investigated at 14 sites in the enclosed Rhine-Meuse Delta. The aim was to identify the main macrozoobenthic assemblages and to establish their relationship with environmental conditions, including sediment pollution. Most sites were dominated by the tubificidsLimnodrilus hoffmeisteri andL. claparedeanus. Four zoobenthic assemblages could be identified. They are related to different geomorphological phases of a basin filling up with fluvial sediment causing a longitudinal sediment gradient. The Rhine river assemblage was found in the Nieuwe Merwede, filled up in the 1970's, the soft mud assemblage in the Hollands Diep at sites with high sedimentation rates; the basin silt assemblage in the Amer, Hollands Diep and Haringvliet, at sites with lower sedimentation rates, and the basin shoal assemblage at former flats with low sedimentation rates. CANOCO was used to relate the TWINSPAN indicator species to a selection of environmental parameters. Sediment pH, water content, sand fraction, total polycyclic aromatic hydrocarbons (PAH) and current velocity explained 44% of the variance of the average abundance of the indicator taxa per site. Sediment firmness, indicated by sediment water content, and high ammonium concentrations probably were together responsible for the very low densities in the soft mud assemblage. Densities and biomasses in the other assemblages were low as well, except for theDreissena polymorpha habitat, where very high total zoobenthic biomass values (max. 55.8 g m^–2) were observed. It is argued that sediment contamination has a considerable impact on macrozoobenthic densities and species composition in all assemblages, the basin shoal assemblage excepted.     

First copulation increases longevity and fecundity of <Emphasis Type="Italic">Histiostoma feroniarum</Emphasis> (Acari: Astigmata: Acaridida) females

I investigated the influence of insemination at different life stages on female fitness in the mite Histiostoma feroniarum. In this species, males guard immature females at the tritonymph stage to inseminate them immediately after the last moulting. Four groups of females were studied (1) females inseminated naturally, i.e. mating occurred immediately after guarding, and then the male was removed (IF/0M), (2) naturally inseminated females, where after insemination the male was replaced by two additional males (IF/2M), (3) virgin females reared without males (VF/0M) and (4) mature, virgin females to which two virgin males were added 3 days after last moulting (VF/2DM). The results show that females inseminated naturally (IF/0M) have higher longevity and fecundity than either virgin (VF/0M) or late-inseminated females (VF/2DM). Furthermore, longevity and fecundity of the former (IF/0M) was also greater than that of females naturally inseminated and subsequently exposed to males (IF/2M). One may suggest that seminal fluids have a positive effect on female fitness. When delayed insemination occurs, such positive effect may not be observed due to a change in features of the sperm access system. Harassment may explain decreased longevity and fecundity of females inseminated naturally compared to females that received additional males.This revised version was published online in May 2005 with a corrected cover date.     

Effects of enkephalin in analogues on prolactin release in the rat.

The effects of nineteen enkephalin analogues on the circulating levels of prolactin in the male rat following intraventricular injection of the peptides were determined and compared with that of Met- and Leu-enkephalin. Eleven of the 19 analogues stimulated prolactin secretion. It was found, in general, that the structure activity relationship for enkephalin stimulation of prolactin secretion was similar to that for opiate receptor activity. Analogues which contained a [DAla²] substitution were generally effective in stimulating prolonged prolactin release. Some, but not all analogues containing [DTrp²] or [DLeu⁵] were active. Analogues containing the [DTrp¹], [DPhe⁴] or [DMet⁵] substitutions were ineffective. The prolactin releasing effect of intravenous Tyr-DAla-Gly-Phe-DLeu was reversed by naloxone. Naloxone had no effect on the haloperidol- and alpha-methylparatyrosine induced increases in plasma prolactin levels. The results of these studies are discussed in the light of the suggestion that the enkephalins may function as neuroendocrine modulators.     

Ion Regulation of the Kinetics of Potential-Dependent Potassium Channels

We apply a theoretical approach developed earlier. The interaction ofions that permeate a channel with slowly relaxing charged channel-forminggroups (ion-conformational interaction – ICI) is addressed by thisapproach. One can describe the ion concentration influence (ion regulation)on channel functioning in this manner. A patch-clamp method in awhole-cell configuration is used to study the ICI. For this purpose theinfluence of an external concentration of potassium ions on thepotential-dependent potassium current (I_A) in the externalmembrane of GH₃ cells was studied. The increase of[K⁺ _out] from 5 mM to 100 mM causes anon-monotonous shift of current-voltage dependencies. The dependence of bothan activation time constant tgr_n and a steady-state activation(n) on [K⁺]_out have a minimum andmaximum respectively. The analysis of the results suggests that the observedeffects are caused by ICI. A physical model is developed to describe thedependence of the potassium channel kinetics on the external concentrationof the ions and the membrane potential. The deformation of the closedstate of the gate and the corresponding energy shifts cause the observednon-monotonous dependencies due to ICI. Thus, the general theoreticalapproach has an experimental confirmation and is applied to concreteexamples. Formulas for concentrational dependencies of the channel kineticsare given for practical uses.     

Visualization of chitin-wall formation in hyphal tips and anastomoses of Diplodia natalensis by fluorescein-conjugated wheat germ agglutinin and [3H] N-acetyl-D-glucosamine

Colonies of the fungus Diplodia natalensis produce ample anastomoses which are visible 0.5–1.0 mm inwards of the colony's periphery. Anastomose formation as well as other morphogenetic features, were followed by autoradiography, lectin binding and application of the chitin synthase inhibitor polyoxin D.Hyphal tips and septae were strongly labelled by short pulses of [³H] N-acetyl-D-glucosamine ([³H]GlcNAc) and were showing marked fluorescence after exposure to fluorescein isothiocyanate (FITC) conjugated wheat germ agglutinin (WGA).The dynamics of wall formation was followed by pulse and chase as well as by pulse and wash treatments in which the colony was shortly exposed to [³H]GlcNAc and then freed from the radioactive chitin precursor.Application of the chitin synthase inhibitor polyoxin D caused hyphal tip swellings as well as inflations and balloons along the hyphae at sites of initial new outgrowths and anastomoses. These structures were strongly fluorescenting after FITC-WGA application, indicating imbalance of wall formation and wall lysis.FITC-WGA binding, [³H]GlcNAc labelling and/or exposure to polyoxin D, indicated a process of anastomose formation which starts with short outgrowths of two juxtapositioned hyphae and ends with a complete bridge formation.Abbreviations FITC fluorescein isothiocyanate - WGA wheat germ agglutinin - GlcNAc N-acetyl-D-glucosamine     

Evaluation of an exotic maize population adapted to a locality

Summary An exotic Zea mays L. population (Tuxpeno) was adapted to North Carolina conditions by first introducing genes for adaptability from two North Carolina varieties ([(Jarvis X Indian Chief)Tuxpeno]Tuxpeno) including four generations of intermating, and then selecting for adaptability using maturity as the primary measure. The study evaluated selection for adaptability and the diversity available between adapted Tuxpeno and the local varieties, Jarvis and Indian Chief. Analytical procedures were developed to quantify the diversity between populations and the complementation of local varieties by introduced germ plasms. The analyses utilized the specific effects available from the diallel mating design.Three replicate selections responded similarly under simple recurrent mass selection (1/10) for the earliest disease-free plants initially and additionally for plant types (primarily height) in the final generation. The 1/4 local germ plasm permitted rapid adaptation of Tuxpeno gene pool to local conditions. The adapted Tuxpeno populations yielded similarly to the local populations with an average heterosis for grain yield of 28% when crossed to the local populations used as source of genes for adaptability. The diversity found between adapted Tuxpeno lines and these local varieties based on genes affecting grain yield was 1.5 to 2.5 times that measured between the local varieties (Jarvis and Indian Chief). Diversity lost through intergradation with local material was a reasonable investment. Yield genes introduced from Tuxpeno complemented local gene pools through nonadditive, primarily dominance-associated, gene effects. Reassortment of major gene blocks apparently occurred leading to significant divergence among replicate selections involving both additive-associated and dominance-associated gene effects.Paper No. 6355 of the North Carolina Agri. Res. Ser., Raleigh, NC. Research supported in collaboration with the Rockefeller Foundation and CIMMYT, D.F. (Mexico)     

Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

Influence of the nitrogen level on root growth and morphology of two potato varieties differing in nitrogen acquisition

Two potato (Solanum tuberosum L.) cultivars (Astrid and Bodenkraft) differing in their nitrogen acquisition from the soil (Hunnius, 1981) were used in nutrient solutions to study the effect of increasing concentrations of nitrate (0.05; 0.5; 5.0 mol m^-3) particularly on root growth and morphology. In each variety increasing nitrogen concentrations stimulated shoot growth more than root growth. At all nitrate concentrations, the variety with higher nitrogen acquisition (Astrid) had a significantly larger root system. The larger root system of Astrid compared to Bodenkraft was particularly evident when surface area and total length of the roots, instead of root dry weight were used as parameters. The results stress the importance of root length and surface area for nitrogen acquisition from soils.