Carbohydrate Structural Units in Glycosphingolipids as Receptors for Gal and GalNAc Reactive Lectins

Glycosphingolipids (GSLs) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. Many of them are in the nervous system and function as important receptors in various life processes. During the past two decades, 11 mammalian structural units have been used to express the binding domain of applied lectins. They are: F, GalNAc1 3GalNAc; A, GalNAc1 3Gal; T, Gal1 3GalNAc; I, Gal1 3GlcNAc; II, Gal1 4GlcNAc; B, Gal1 3Gal; E, Gal1 4Gal; L, Gal1 4Glc; P, GalNAc1 3Gal; S, GalNAc1 4Gal, and Tn, GalNAc1 Ser(Thr). Although 10 of them occur in GSLs, only 3 (L , S , and T ) are found in human brain, and 2 (L and II ) are present in the inner structures of human blood group active GSLs. In the families of gangliosides, L and II represent 55% of the total structural units, while the other three units (T , P , and S ) constitute the rest. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding specificities of Gal/GalNAc reactive lectins have been classified according to their highest affinity for the structural units and their binding properties expressed by decreasing order of reactivity. Hence, the binding relation between GSLs and Gal/GalNAc specific lectins can be established.     

Compartmentation of α- and β-Carbonic Anhydrases in Cells of Halo- and Alkalophilic Cyanobacteria Rhabdoderma lineare

The organization of carbonic anhydrase (CA) system in halo- and alkalophilic cyanobacteria Rhabdoderma lineare was studied by Western blot analysis and immunocytochemical electron microscopy. The presence of extracellular -CA (60 kD) in the glycocalyx, forming a tight sheath around the cell, and of two intracellular -CA is reported. One -CA (60 kD) is associated with polypeptides of photosystem II (PSII) and is a constitutive enzyme. Another -carbonic anhydrase (25 kD) was induced by low content of bicarbonate in the culture medium; this inducible CA was found in the fraction of total soluble proteins. The expressed synthesis of inducible -CA was accompanied by the increase in the intracellular pool of inorganic carbon, which suggests an important role of this enzyme in the functioning of CO₂-concentrating mechanism.     

UDP-galactose:lactosylceramide α-galactosyltransferase activity in human placenta

The activity of UDP-Gal: LacCer galactosyltransferase in human placenta was studied by using crude homogenate and Triton CF-54 extract as the source of enzyme. Transfer of galactose to lactosylceramide was optimal in the presence of 0.1% Triton CF-54 and Mn²⁺ at pH 6.3, and the reaction product was susceptible to -galactosidase.Abbreviations LacCer lactosylceramide (Gal1-4Glc1-1Cer) - Gb₃ globotriaosylceramide (Gal1-4Gal1-4Glc1-1Cer) - Gb₄ globoside (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer) - TLC thin-layer chromatography - GC/MS gas chromatography/mass spectrometry - NMR nuclear magnetic resonance - EDTA ethylenediamine tetraacetic acid - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate     

Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

Summary Thermostable, extracellular -amylase and -glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an -amylase and an -glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The -amylase and -glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The -glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl--d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the -amylase and -glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.     

Mutants of Arabidopsis with alterations in seed lipid fatty acid composition

Summary A diverse collection of mutants of Arabidopsis with altered seed lipid compositions was isolated by determining the fatty acid composition of samples of seed from 3,000 mutagenized lines. A series of mutations was identified that caused deficiencies in the elongation of 181 to 201, desaturation of 181 to 182, and desaturation of 182 to 183. In each of these cases the wild type exhibited incomplete dominance over the mutant allele. These results, along with results from earlier studies, point to a major influence of gene dosage in determining the fatty acid composition of seed lipids. A mutation was also isolated that resulted in increased accumulation of 183. On the basis of the effects on fatty acid composition, the nature of the biochemical lesion in three of the mutants could be tentatively attributed to deficiencies in activities of specific enzymes. The other mutant classes had relatively less pronounced changes in fatty acid composition. These mutants may represent alterations in genes that regulate lipid metabolism or seed development. The availability of the mutants should provide new opportunities to investigate the mechanisms that control seed lipid fatty acid composition.Abbreviations FAMES (fatty acid methyl esters) - PC (phosphatidylcholine) - 181 (oleic acid) - 182 (linoleic acid) - 183 (linolenic acid) - 201 (eicosenoic acid) Supported in part by grants from the USDA (No. 89-37262-4388), USDA/NSF/DOE Plant Science Center Program, the U.S. Department of Energy (No. AC02-76ER01338), Karlshamns Research Foundation, and the WSU Research and Arts Committee     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Analysis of kafirin promoter activity in transgenic tobacco seeds

Sequences corresponding to 855 bp of 5 promoter region and the transit peptide from GK.1, a genomic clone encoding a 22 kDa -kafirin seed protein from sorghum, were translationally fused to a cloned -glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10–15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.     

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.     

Genetic analysis of two wheat cultivars, ‘Sonalika’ and ‘WL 711’ for reaction to leaf rust (Puccinia recondita)

Summary Genetic analysis for leaf rust reaction of two widely adapted cultivars, Sonalika and WL 711, has been done using 21 near isogenic Lr lines and rust culture IL004 — avirulent on the two cultivars and all the Lr lines used. The segregation pattern in the F₂ generation indicated the presence of a recessive gene in Sonalika and of a dominant gene in WL 711. These genes in cultivars Sonalika and WL 711 have been identified as Lr 11 and Lr 13, respectively. Gene Lr 13 is no longer effective in WL 711 but it continues to give field resistance in the backgrounds of Chris, Prelude and Thatcher. There has been no significant change in the virulence spectrum of the leaf rust pathogen in India with the release of WL 711. High susceptibility of WL 711 seems to be due to the evolution of more aggressive forms of the pathogen to this cultivar. The gene Lr 11, which behaves as a recessive in Sonalika, was effective against leaf rust when this cultivar was released. The high susceptibility of Sonalika is probably due to an increase in the frequency of race 77 virulent on Lr 11. Lr 11 has shown a dominance reversal in the background of Sonalika. Present results suggest that interaction of resistance genes with the background genotype must be studied for their effective use in breeding programme.     

Tetramethyl Rhodamine Methyl Ester (TMRM) is Suitable for Cytofluorometric Measurements of Mitochondrial Membrane Potential in Cells Treated with Digitonin

A new method for cytofluorometric analysis of mitochondrial membrane potential has been developed by using TMRM as a cationic, mitochondrial selective probe. The method is based on limited treatment of cultured cells with digitonin which permeabilises the plasma membrane and leaves mitochondria intact. The resulting signal of TMRM-stained cells thus represents only the probe accumulated in mitochondria. Fibroblasts and cybrids were used as a model cell systems and optimal conditions for digitonin treatment and staining by TMRM were described. The TMRM signal collapsed by valinomycin, KCN and antimycin A and FCCP titration was used to gradually lower and characterise the stability of . The method is suitable for sensitive measurement of in different types of cultured cells.     

Concerted evolution in a segment of the first domain exon of polymorphic MHC class II β loci

Genetic exchange of sequence information between members of a gene family, generally denoted gene conversion, causes a phenomenon called concerted evolution meaning that non-allelic genes do not evolve independently. The possible significance of this phenomenon in the evolution of major histocompatibility complex (MHC) class II genes has been investigated in the present study. The results of a phylogenetic analysis of human, mouse, bovine, and chicken class II sequences were consistent with the occurrence of gene conversion between polymorphic class II genes (i. e. DPB, DQB, and DRB) but not between these genes and the monomorphic DOB gene or between class II genes. Gene conversion between polymorphic genes appears to be restricted to a gene segment between approximately nucleotide positions 94–286 in the first domain exon. Due to this genetic exchange, there is a greater interlocus similarity both at the DNA and protein level in this region than in the rest of the sequence. The region encodes a functionally important part of the class II molecule including more than half of the -chain residues of the antigen binding site and the residues in the helix assumed to form contact with the T-cell receptor. The observed similarity in the -helical region of class II molecules may be functionally significant for the utilization of the T-cell repertoire for antigen recognition in the immune system.     

The inheritance of host plant resistance and its effect on the relative infection efficiency of Magnaporthe grisea in rice cultivars

The inheritance of host plant resistance and its effect on the relative infection efficiency for leaf blast was studied in the crosses IR36/CO39 (partially resistant × highly susceptible) and IR36/IR64 (both partially resistant). On the natural scale, gene action appeared multiplicative. After log transformation, additive effects described most of the genetic variation in the cross IR36/CO39, while additive and dominance effects were about equal in magnitude in the cross IR36/IR64. Dominance was towards increased resistance. No transgressive segregation occurred in the cross IR36/CO39. The number of genes that reduce lesion number was estimated to be zero in CO39 and five or more in IR36. The cross IR36/IR64 showed transgressive segregation in both directions, and IR36 and IR64 each contain at least one gene that is not present in the other cultivar. The heritabilities (narrow sense) in the F₂ were low (range 0.06–0.16), while narrow sense heritabilities based on F₃ lines were much higher (range 0.41–0.68). Lesion numbers in F₃ lines were reasonably correlated with those in F₅ progenies derived from the same F₂ plant (r was±0.6 in both crosses). Partial resistance can be effectively improved by selecting the most resistant plants from the most resistant F₃ lines.     

The effect of some macronutrients on adventitious root development on scion apple cultivars in vitro

The influence of some macronutrients, especially NH₄NO₃ and KNO₃, on root development of microcuttings from 3 apple scion cultivars is discussed. A reduction of the level of NH₄NO₃ in the medium from full strength to 1/4 strength significantly increased the percentage rooting of Gala and Royal Gala, but not Jonagold. Further reduction of NH₄NO₃ level from 1/4 strength to zero significantly reduced the percentage of rooting in Gala but not Royal Gala. Jonagold rooted best at zero concentration NH₄NO₃. Without NH₄NO₃, rooting percentages were as high as 100% for all 3 cultivars when KNO₃ was provided at full strength. The results show that adventitious roots can be induced on apple scion cultivars by media manipulation.     

The status of general reproductive-strategy theories,illustrated in winkles

Summary Populations of the rough winkle, Littorina rudis from two spatially-adjacent but distinct habitats have been compared in terms of reproductive effort, size at maturity and embryo size and number. In each case, the basis for the comparison has been examined critically. The suites of characteristics exhibited by the two populations do not fit the conventional r-K scheme which has come to dominate life-history theory.Specifically, individuals in a boulders population make a smaller reproductive effort, delay maturity longer and produce more numerous, smaller young than individuals in a crevice population.The effects in the two populations of various environmental factors on shell thickness, animal size and these three life-history traits are examined and discussed. These factors adequately explain the characteristics exhibited by the populations. It is argued that the conventional scheme is inadequate in that it fails to take such specific effects into account.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

The final thermal preferendum of fishes: Shuttling behavior and acclimation overshoot

The concept of final preferendum is reexamined in light of data concerning acclimation in a cyclic thermal regime. Because of Shuttling behavior to temperatures above and below the mean preferendum, and because of faster acclimation upward than downward, which result in acclimation to a temperature exceeding the mean of the cycle, a fish may finally gravitate to a preferendum which does not equal acclimation temperature. It is suggested that a distinction be made between the crossover-point preferendum, where preference and acclimation are equal, and the ultimate preferendum to which a fish will ultimately gravitate.     

Efficient determination of angles subtended by Cα-Hα and N-HN vectors in proteins via dipole-dipole cross-correlation§

The angle CH,NHN subtended by the internuclear vectors 13C-H and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13C and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle CH,NHN. Assuming planarity of the peptide bond, the dihedral angle (between C and C) can be determined from the knowledge of CH,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

Thidiazuron substitution for chilling requirement in three apple cultivars

Thidiazuron [(TDZ)N-phenyl-N-1,2,3-thidiazol-5-ylurea] at 750 M was applied to buds of apple trees to determine if it could substitute for the chilling requirement to induce bud break. Shoots of cv. Anna (low chill), Delicious cv. Redchief (medium chill), and Northern Spy (high chill) were untreated, treated with TDZ prior to chilling (before-chill), and treated with TDZ at various intervals after the accumulation of specific amounts of chilling (after-chill). Shoots were stored in a cold room at 4°C. TDZ applied prior to chilling reduced the chill unit (CU) requirement (1 CU = 1 h at 4°C) for the promotion of bud break on 1-year-old shoots of Anna and Northern Spy and 1- and 2-year-old wood of Delicious. TDZ applied after-chill promoted bud break only for Anna and buds on 2-year-old wood of Delicious. While accumulating CUs, untreated buds or buds treated with TDZ on 1-year Delicious and Northern Spy did not respond to the cold treatment even after 1848 h of CU accumulation. For all three cultivars, TDZ treatment was more effective in promoting bud break when applied before the initiation of chilling.The use of a company or product name does not constitute an endorsement by USDA or the University of Maryland nor imply approval to the exclusion of other suitable products.