Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal,normal and neoplastic gastrointestinal tract

Summary Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for 26 sialylated type 2 chain) and IB9 (for the 26 sialylated type 2 chain and glycoproteins having NeuAc26GalNAc), and 188C1 (for short- and long-chain Le^x antigens) and FH2 (for the long-chain Le^x antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The 26 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Le^x antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its 26 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Le^x antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc26GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.Abbreviations Fuc fucose - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - MAb monoclonal antibody - NeuAc N-acetylneuraminic acid - PBS phosphate-buffered saline     

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.     

Progeny analysis of the interspecific somatic hybrids: Nicotiana tabacum (CMS) + Nicotiana sylvestris with respect to nuclear and chloroplast markers

Summary The progeny of a fusion experiment involving N. sylvestris protoplasts and X-irradiated protoplasts of the cytoplasmic male sterile Line 92 (N. tabacum nucleus and alien, male-sterility inducing, cytoplasm) were analyzed. Three groups of somatic hybrid plants resulted: Type A, Type B-1 and Type B-2. These as well as their androgenic progenies and the progenies resulting from their pollination with N. tabacum or N. sylvestris were followed with respect to several nuclear and cytoplasmic traits. Those controlled by the nuclear genome were plant and flower morphologies; those controlled by genetic information in the cytoplasm were tentoxin sensitivity (affecting the coupling factor of chloroplast ATPase), the large subunit of ribulose bisphosphate carboxylase and the restriction endonuclease pattern of plastid DNA. A further cytoplasmic trait investigated (exact site of genetic control not known) was male sterility. The examinations of the somatic-hybrid groups and their respective progenies indicated that: Type A plants have N. sylvestris nuclei and Line 92 plastids; Type B-1 plants also have Line 92 plastids but their genome is composed of N. sylvestris and N. tabacum nuclei; Type B-2 plants with impaired male fertility had N. sylvestris plastids and N. sylvestris nuclei.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Variation in preleptotene chromosome contraction among three cultivars of Lilium longiflorum

Preleptotene chromosome contraction was observed in several clones of Lilium longiflorum cultivars Ace, Nellie White and Croft. The degree of chromosome contraction was variable, among microsporocytes of single anthers, buds of individual plants, and clones of single cultivars. It is suggested that one of the sequence of genetically controlled events that determine the orderly development of meiosis provides for the temporary inhibition of mitotic coiling, thus allowing the development of leptotene; and if this gene action is delayed or out of sequence, a period of mitotic-like chromosome contraction precedes leptotene. These cultivars appear to share an inherited predisposition for such an irregularity in sequence of gene action. Differences between clones may be attributed to differences in other genes affecting these earliest stages of meiosis. There is some evidence of external environmental influence upon this genetic predisposition for preleptotene chromosome contraction, that is, there appears to be an inverse correlation between degree of preleptotene contraction and temperature. The degree of chromosome contraction may also be influenced by different metabolic conditions affecting microsporocytes developing sequentially in the anther. Experiments are presently underway to determine whether the degree of preleptotene chromosome contraction is related to chiasma frequency.     

Hybrid genes in the analysis of transformation conditions

Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR₁ tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR₁ Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl₂ and PEG. The technique yielded up to 1400 transformants per 3×10⁵ treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the competence for transformation has a species-specific component.Abbreviations PEG polyethyleneglycol - RTF relative transformation frequency - ATF absolute transformation frequency     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

Hyoscyamus muticus + Nicotiana tabacum fusion hybrids selected via auxotroph complementation

Protoplasts of the nicotinamide-deficient Hyoscyamus muticus cell line nic^– IVH2 and of the nitrate reductase-deficient Nicotiana tabacum cell line NR^– cnx 68 were induced to fuse. Selection for putative interspecific hybrid clones was via auxotroph complementation. Controls included tests for cross-feeding, cross inhibition, PEG-induced variation, culture-induced variation, reversion, viability, delayed selection. Protrotrophic cell lines were recovered exclusively from PEG-treated mixtures of both protoplast types. The putative hybrid clones grew independent of exogenously supplied auxins and cytokinins, and at a faster rate than either parent. The morphogenic potential of different clones varied from non-morphogenic to regeneration of fertile plants. Indirect evidence for the hybrid nature of the clones is provided from a) tight selection, b) hormone-independent growth, c) hybrid vigour, d) extreme morphological variation, e) isoenzyme bands from both parents, f) morphogenic potential. Definite proof for the hybrid nature was, however, provided from species-specific DNA hybridization. Chimerism could be excluded since only the large subunit of Hyoscyamus muticus ribulose bisphosphate carboxylase was found and since species-specific DNA hybridization identified clones which gave no Nicotiana tabacum signal.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA Naphthaleneacetic acid - BAP 6-Benzylaminopurine - PEG Polyethyleneglykol - MES (2[N-morpholino]ethane sulfonic acid - Tris-HCl Tris(hydroxymethyl)-aminoethane-HCl % is given throughout in w/v - FMI Friedrich Miescher-Institut     

Localization of melanotropin-like peptides in the central nervous system of two insect species,the migratory locust,Locusta migratoria,and the fleshfly,Sarcophaga bullata

Summary By use of well characterized antisera in the peroxidase-antiperoxidase method, we were able to demonstrateMSH andMSH immunoreactive cells and nerve fibres within the nervous system of adults and larvae ofLocusta migratoria and 3-, 5- and 8-day-old adultSarcophaga bullata. In neither of these insect species, any immunoreaction was obtained with a ₃MSH-antiserum. Double immuno-histochemical stainings revealed thatMSH-like andMSH-like substances are located in different cells. These cells show no immunoreactivity to a number of antisera against other POMC-derivatives (anti-lipotropin, anti-endorphin, anti-ACTH_1–24); thus they appear to containMSH- orMSH-like material in a specific way. The function of the immunologically detected peptides remains to be demonstrated. The distribution of the immunoreactive material suggests that, like in amphibians and other lower vertebrates, the synthesis or release of melanotropins might be under the influence of external stimuli. The present observations support the recently developed concept that even some of the smallest neuropeptides, the melanotropins, have been highly conserved during a long period of evolution.     

Effects of competition and N and P supply on carbon isotope discrimination and <Superscript>15</Superscript>N-natural abundance in four grassland species

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (¹³C) and enriched ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species ¹³C while caused enrichment of ¹⁵N of P. pratensis and F. valida but strong depletion of ¹⁵N of T. officinale. Phosphorous had no significant effect on ¹³C but depleted ¹⁵N of all species.     

Selective feeding by larval dytiscids (Coleoptera:Dytiscidae) and effects of fish predation on upper littoral zone macroinvertebrate communities of arctic lakes

Carbon and nitrogen stable isotopic data from the primary producers in mangrove ecosystems are needed to investigate trophic links and biogeochemical cycling. Compared with other mangrove species (e.g. Rhizophora mangle) very few measurements have been conducted on the white mangrove, Laguncularia racemosa. The carbon and nitrogen stable isotopic and elemental compositions of L. racemosa were analyzed and compared from Florida and Belize. ¹³C values of L. racemosa from Florida (mean = –26.4) were slightly higher than those from Twin Cays, Belize (mean = -27.4), which may be due to higher salinity in some parts of the Florida site. There was no difference between the ¹⁵N values from L. racemosa from these two sites (Florida mean = 0.6; Belize mean = 0.3), which are indicative of nitrogen derived from nitrogen fixation in a planktonic marine system. However, higher ¹⁵N values from L. racemosa at Man of War Cay in Belize (11.4 and 12.3), which is fertilized by roosting marine birds (14.0), illustrate that L. racemosa can sensitively reflect alternative nitrogen sources. Although the isotopic data could not distinguish between Avicennia germinans, R. mangle and L. racemosa in Belize the L. racemosa had considerably higher C/N ratios (46.5 – 116.1) compared with the Florida samples (42.2 – 76.0) or the other mangrove species. Unlike some previous findings from R. mangle, substrate characteristics (e.g. salinity, NH₄ ⁺, and H₂S) were not related to the isotopic or elemental composition of L. racemosa. ¹³C, ¹⁵N and C/N were analyzed for ecosystem components from L. racemosa habitats at Twin Cays, including other plants (e.g. R. mangle, A. germinans and seagrass), detritus, microbial mats and sediments. Results from mass-balance calculations show that mangrove detritus composes very little of the sediment, which is principally composed of microbial biomass (80 – 90%). Detritus at some sites is also influenced by sources other than that from L. racemosa, including seagrass leaves.     

The Hawk—Dove game revisited: Effects of continuous variation in resource-holding potential on the frequency of escalation

Summary The classic Hawk—Dove game is extended to deal with continuous variation in resource-holding potential or RHP, when RHP is observable (via any sensory modality) but RHP difference is less than perfectly reliable as a predictor of the outcome of an escalated contest. The relationship between sensory and physical magnitudes of RHP is assumed to be governed by Fechner's psychophysical law, whose effect is that contestants interact as if they had perfect information about their relative RHP (as opposed to RHP difference). Thus, an animal is aggressive if its RHP exceeds a certain fraction, called its threshold, of its opponent's RHP and otherwise is non-aggressive; and the classic Hawk and Dove strategies correspond to zero and infinite thresholds, respectively. For RHPs drawn at random from an arbitrary Gamma distribution there is a unique evolutionarily stable strategy or ESS, which depends on a parameter measuring the reliability of RHP as a predictor of the outcome of a fight, on the ratio of the valueV of winning to the costC of losing (both measured in units of reproductive fitness) and on the mean µ and variance ² of the RHP distribution. In a population at this ESS, ifV/C < 1 then the threshold is 1 and there is no fighting. AsV/C increases beyond 1 to a second critical value , however, the threshold decreases steadily from 1 to 0 and remains 0 forV/C > ; is an increasing function of , but a decreasing function of ². That a lower variance of RHP can imply a lower escalation frequencyp is a novel insight of the analysis. The prediction is at first counterintuitive, because if the aggression threshold were fixed then larger variance would imply lowerp (dispersion effect of variance). When natural selection acts on the threshold, however, increasing the variance not only reduces the probability that an animal with larger RHP will be attacked by an animal with lower RHP at the existing threshold, but also reduces the expected costs of adopting that particular threshold, so that a mutant with a somewhat lower threshold can invade the population (selection effect of variance). Forp, the selection effect dominates toward the upper end of the interval 1 V/C .     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Human λ light chain locus: Organization and DNA sequences of three genomicJ regions

Evidence for the genomic organization of human lambda light chain joining (J) region gene segments is presented. A mouse J probe was used in Southern hybridizations to localize joining region sequences in a cosmid clone containing the genomic cluster of six human lambda constant (C) region gene segments. The results of these hybridizations suggest the presence of at least one J gene segment upstream from each constant region gene segment. The DNA sequences indicate that the human JI, J2, and J3 gene segments have consensus nonamer and heptamer sequences, proposed to be involved in V-J joining, are capable of encoding the known amino acid sequences for the respective J peptides, and have a sequence which could give functional RNA splice site at the end of their coding regions. Our data show that a single functional J is located 1.3 or 1.6 kb upstream of each of the C gene segments known to encode the Mcg, Kern^– Oz^–, and Kern^–Oz⁺ isotypes. Therefore, the gene organization of this region of the human lambda locus is J1 CI -J2C2-J3C3. The DNA sequences ofJ 1,J 2, andJ 3 presented in this paper establish that a singleJ gene segment precedes each expressed C gene segment, and support a model for the evolution of the human JC clusters where JICI andJ2C2-J3C3. arose from different ancestral JC units.     

Studies on the E. coli groNB (nusB) gene which affects bacteriophage lambda N gene function

Summary Escherichia coli mutants, called groNB, which block the growth of bacteriophage at the level of action of the gene N product, have been isolated as survivors at 42°C of bacteria carrying a) the defective prophage bio1 1 i cI857 H1 or b) the pcR1 plasmid containing the EcoRI immunity fragment of phage cI857. In addition, groNB bacterial mutants have been isolated at 37° C, as large colony formers in the presence of i cI h ⁴³⁴, i cI h , and i cI h ⁸⁰ phage. The groNB locus is located at 9 minute of the E. coli genetic map with the order of the neighboring loci being proC tsx groNB purE. Most groNB mutations isolated at 42° C were found to interfere in addition with bacterial growth at low temperatures, since (a) the GroNB phenotypes of growth inhibition and bacterial cold sensitivity cannot be separated by P1 transduction, and (b) some cold resistant revertants simultaneously become Gro⁺ for growth. Lambda transducing phages carrying the groNB ⁺ bacterial gene have been isolated. GroNB mutant bacterial lysogenized by the transducing phage acquire the Gro⁺ phenotype and simultaneously the cold resistant phenotype, suggesting that the groNB mutations are recessive to the wild-type gene.     

Expression of a Synthetic Porcine α-Lactalbumin Gene in the Kernels of Transgenic Maize

The main nutritional limitation of maize used for feed is the content of protein that is digestible, bioavailable and contains an amino acid balance that matches the requirements of animals. In contrast, milk protein has good digestibility, bioavailability and amino acid balance. As an initial effort to create maize optimized as a source of swine nutrition, a codon-adjusted version of a gene encoding the milk protein porcine -lactalbumin was synthesized. Maize expression vectors containing this gene under the control of the Ubi-1 promoter and nos 3 terminator were constructed. These vectors were used to transform maize callus lines that were regenerated into fertile plants. The -lactalbumin transgenes were transmitted through meiosis to the sexual progeny of the regenerated plants. Porcine -lactalbumin was detected in callus and kernels from transgenic maize lines that were transformed by two constructs containing the 27-kDa maize gamma-zein signal sequence at the 5 end of the synthetic porcine -lactalbumin coding sequence. One of these constructs contained an ER retention signal and the other did not. Expression was not observed in kernels or callus from transgenic maize lines that were transformed by a construct that does not contain an exogenous protein-targeting signal. This suggests that the signal peptide might play an important role in porcine -lactalbumin accumulation in transgenic maize kernels.     

Development of medium components for the production of glucosyl-transferring enzyme by Aureobasidium

Aureobasidium sp. ATCC 20524 produced a glucosyl-transferring enzyme which produced panose (O--D-glucopyranosyl-(1»6)-O--D-glucopyranosyl-(1»4)-d-glucose) from maltose. Optimum production for the enzyme was with maltose at 2% (w/v) and yeast extract at 1.5% (w/v). Enzymatic activity reached 0.7×10³ U/g dry cells after 48 h.     

Expression of S-locus glycoprotein genes from Brassica oleracea and B. campestris in transgenic plants of self-compatible B. napus cv Westar

Summary Self-compatible Brassica napus var Westar was transformed with SLG, the S-locus-derived gene that encodes S-locus-specific glycoproteins (SLSG). Four allelic variants of SLG isolated from self-incompatible B. oleracea and B. campestris strains homozygous for different S alleles were used. We show that the transgenic plants synthesized SLSG with the same apparent charge, molecular weight, and antigenic properties as that produced by the corresponding self-incompatible strains from which the cloned SLG genes were isolated. In addition, transgene-encoded SLSG was detected specifically in the papillar cells of the stigma, and was correctly targeted to the papillar cell wall. However, SLSG was produced at reduced levels in transgenic plants relative to self-incompatible strains. The introduction of the SLG genes did not confer a self-incompatibility phenotype on the Westar cultivar.