Investigation of the acetolysis products of the sulphated polysaccharide of Aeodes ulvoidea

Investigation of the acetolysis products of the sulphated polysaccharide of the seaweed Aeodes ulvoidea led to the isolation and characterization of the following oligosaccharides: 3-O-α- -galactopyranosyl- -galactose (1), 3-O-(2-O-methyl-α- -galactopyranosyl)- -galactose (2), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (3), 4-O-β- -galactopyranosyl-2-O-methyl- -galactose (4), O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose (5), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)- -galactose (6), O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (7), O-(2-O-methyl-α- -galactopyranosyl)-(1→3)-O-β- -galactopyranosyl-(1→4)-2-O-methyl- -galactose (10), and O-α- -galactopyranosyl-(1→3)-O-β- -galactopyranosyl-(1→4)-O-α- -galactopyranosyl-(1→3)- -galactose. In addition, the isolation of a tetrasaccharide possessing alternating - and -galactose residues demonstrates the hitherto unexpected presence of -galactose in the polysaccharide. The structure of the polysaccharide is discussed.     

High-Resolution Linkage Map in the Vicinity of the Host Resistance Locus Bcg

The mouse chromosome 1 locus Bcg determines natural resistance/susceptibility of inbred mouse strains to infection with antigenically unrelated intracellular parasites, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani. In our effort to clone Bcg, we have constructed a high-resolution genetic linkage map in the vicinity of the gene. We have developed eight new highly polymorphic markers (simple sequence repeats) corresponding to cloned genes (Vil, Inha, Des), microdissected chromosome 1 anonymous probes (λMm1C136, λMm1C163, λMm1C165), or novel DNA markers from the region obtained by chromosome walking (D1Mcg101 and D1Mcg105). We have followed the cosegregation of these markers with respect to Bcg in a novel panel of 1000 (C57L/J × C57BL/6J) × C57BL/6J segregating backcross mice. Additional segregation analyses were carried out in preexisting panels of intra- and interspecific backcross mice and recombinant inbred strains. Three of these markers were found to be very tightly linked to Bcg: λMm1C165 did not show recombination with Bcg in 1424 meioses analyzed, while D1Mcg105 and λMm1C136 were located 0.1 cM proximal and 0.2 cM distal to Bcg, respectively. This analysis enabled us to define further the proximal and distal boundaries of the Bcg interval: the proximal limit was defined by a single crossover occurring between D1Mcg105 and Bcg/λMm1C165/Vil, and the distal limit by 1 crossover between Bcg/λMm1C165/Vil and λMm1C136 in 1683 and 575 informative meioses, respectively, for a maximal interval of 0.3 cM. Combined pedigree analyses for the 30-cM segment overlaping Bcg on mouse chromosome 1 indicated the locus order and the interlocus distances (in cM): centromere-Col3a1-(8.8)-Cryg-(2.6)- λMm1C163 -(1.6)-Fn-1-(2.0)-Tp-1- (1.O)-D1Mcg105-(O.1)-λMm1C165/Vil/Bcg-(O.2)-λMm1C136-(0.3)-Des/D1Mit7-(0.1)-Inha-(2.8)-λMm1C153-(2.4)-λMm1C156-(1.2)-Pax-3-(5.6)-Akp-3-(O.8)-Acrg-(2.0)-Sag-(O.5)-Col6a3.     

Role of the immune-response region of the B complex in the control of the graft-vs-host reaction in chickens

The relative importance of the B and IR regions of the chicken B complex were compared as to their role in the graft-versus-host (GVH) reaction. Spleen enlargement (splenomegaly) on 19-day-old embryos, inoculated 5 days earlier with immune competent leukocytes, served as the test for the GVH reaction. The B blood group locus was the marker of the B region, and the Ir-GAT gene was the marker of the immune response (Ir) region of the B complex, the major histocompatibility system (MHS) of chickens. The test stocks consisted of B¹B¹ GAT-Low (1-Low) and B¹⁹B¹⁹ Gat-High (19-High) birds of our S1 Leghorn line plus the recombinant genotypes B¹B¹ Gat-High (1-High) and B¹⁹B¹⁹ GAT-low (19-Low). A dosage of 0.1 ml of donor white blood cells was injected into each of 191 recipient embryos on day 14, and the spleens were removed and weighed on day 19. Of 16 combinations of (donor blood)-(host embryos), arranged with respect to the four genotypes listed above, four were compatible, e. g., (1-Low)-(1-Low). There were four incompatible combinations at the B locus, four at the GAT locus, and four at both the B and GAT loci. All 16 combinations were replicated. Results were expressed as a splenomegaly index (SI), that is, the ratio of incompatible to compatible spleen weights corrected for differences in embryo weight. If (SI-1) is greater than 0, the GVH reaction is considered positive within sampling errors. The mean (SI-1) indexes obtained were: incompatible at GAT-0.5±0.07; incompatible at B-1.34±0.10. Thus, both GAT and B contributed to the GVH reaction, but the B region was much stronger than the IR region. The results were strongly asymmetrical: maximal stimulation occurred when the host embryo was B¹⁹B¹⁹ GAT-high and donor leukocytes were B¹B¹ GAT-Low. The parental donor-host paired combinations gave stronger GVH reactions than did the recombinant pairs. Effects of incompatibilities at the two regions proved additive when compared with two-locus differences of parental genotypes. In general, the results proved that the IR region, as specifically defined by recombinants obtained in our S1 line of Leghorns, plays a significant, but minor, role in the GVH reaction compared with the region of the B complex identified with the B blood-group locus.     

Cosegregation of the polymorphic C4 with the MHC in the frog,Xenopus laevis

Employing isoeletric focusing combined with enzyme-linked immunoelectrotransfer blot analysis, the fourth component of complement (C4) was analyzed in the two highly histocompatible, major histocompatibility complex homozygous groups (J and K) of Xenopus laevis. Each group had a characteristic C4 isoelectric focusing pattern, i. e., an isoelectric point range of 8.0–8.5 for J (C4 ^j C4 ^j ) and 7.6–8.1 for K (C4 ^k C4 ^k ). In (J x K)F₁ frogs, C4 proteins were expressed in a codominant fashion (C4 ^j C4 ^k ). In the backcrossed progeny B₁ [J × (J × K)F₁], those with C4 ^j C4 ^j rejected (J × K)F₁ skins hyperacutely (< 17 days), were high responders against (J × K)F₁ cells, and nonstimulators to J cells in mixed lymphocyte reaction (MLR), but they did not suffer from the graft-versus-host reaction (GVHR), even after the injection of (J x K)F₁ cell-stimulated J splenocytes. On the other hand, the B₁ frogs with C4 ^j C4 ^k acutely or chronically (> 17 days) rejected (J × K)F₁ skins, were low or nonresponders against (J × K)F₁ cells and high stimulators to J cells in MLR, and they suffered from GVHR after the injection of prestimulated J splenocytes. These results argue for the notion that the genes equivalent to mammalian class III map to the MHC at the phylogenetic level of the anuran amphibian.     

Haplotype frequencies of the collagen type-I genes in the Italian population

Summary We have determined the frequencies of six restriction fragment length polymorphisms (RFLPs) of type-I collagen genes in a random sample of 100 subjects. Alpha 1 gene (COL1A1) DNA polymorphisms, FG2/MspI, 2FC6/RsaI, and NST70/RsaI, had polymorphism information content (PIC) values of 0.35, 0.32, and 0.26, respectively. Alpha 2 gene (COL1A2) RFLPs, NJ3/EcoRI, Hf32/RsaI, and Hf32/MspI had PIC values of 0.36, 0.35, and 0.25, respectively. The combined haplotype PIC values were 0.71 at the COL1A1 locus and 0.73 for COL1A2. Two COL1A1 and two COL1A2 RFLPs were more polymorphic than in the English population, making them better markers for the analysis of Italian families affected by osteogenesis imperfecta and some other inherited collagen diseases.     

Expression of a new disialyllacto structure in the lipopolysaccharide of non-typeable Haemophilus influenzae

The structure of lipopolysaccharide (LPS) expressed by non-typeable Haemophilus influenzae (NTHi) strains 1008 and 1247 has been investigated by mass spectrometry and NMR analyses on O-deacylated LPS and core oligosaccharide material. Both strains express the conserved triheptosyl inner core, [l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-Lipid A] with PCho→6)-β-d-Glcp (GlcI) substituting the proximal heptose (HepI) at O-4. Strain 1247 expresses the common structural motifs of H. influenzae; globotetraose [β-d-GalpNAc-(1→3)-α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and its truncated versions globoside [α-d-Galp-(1→4)-β-d-Galp-(1→4)-β-d-Glcp-(1→] and lactose [β-d-Galp-(1→4)-β-d-Glcp-(1→] linked to the terminal heptose of the inner core and GlcI. A genetically distinct NTHi strain, 1008, expresses identical structures to strain 1247 with the exception that it lacks GalNAc. A lpsA mutant of strain 1247 expressed LPS of reduced complexity that facilitated unambiguous structural determination of the oligosaccharide from HepI. By CE-ESI-MS/MS we identified disialylated glycoforms indicating disialyllactose [α-Neu5Ac-(2→8)-α-Neu5Ac-(2→3)-β-d-Gal-(1→4)-β-d-Glcp-(1→] as an extension from GlcI which is a novel finding for NTHi LPS.     

Control of the activity of plant plasma membrane MgATPase by the viscosity of the aqueous phase

The activity of plant plasma membrane (PM) MgATPase (EC 3. 6. 1. 35) was studied in PM vesicles purified from spring wheat (Triticum aestivum L. cv. Drabant) roots, winter wheat (Triticum aestivum L. cv. Martonvásári-8) roots, and soybean (Glycine max L. cv. Williams) hypocotyls by aqueous polymer two-phase partitioning. MgATPase from spring wheat roots was assayed at 23°C (a) in the absence and presence of Triton X-100, (b) in the presence of either 1 mM or 3 mM MgATP^2?, and in the presence of increasing concentration of sucrose from 10 mM upto 1. 2 M. Activity of MgATPase in PM vesicles from winter wheat roots was measured at 21°C at sucrose concentrations of 15 mM–1. 1 M in the presence of 3 mM MgATP^2? and absence of Triton X-100 only. MgATPase activity from soybean hypocotyls was studied (a) in the absence and presence of Triton X-100, (b) both at 21 and 31°C, in the presence of 3 mM MgATP^2? and varying concentrations of sucrose between 10 mM and 1. 1 M. In all cases, independently of the assay conditions and the source of PM, the MgATPase activity decreased with increasing sucrose concentration. Latency of the MgATPase activity depended only slightly on the concentration of sucrose. The Q₁₀ value for the MgATPase activity from soybean hypocotyls (and thereby the Arrhenius activation energy of the enzymatic reaction) was independent of the sucrose concentration and of the presence of Triton X-100. At optimal assay conditions, the ATP-hydrolyzing activity of plant PM MgATPase (v) was inversely proportional to the m-th power of the viscosity of aqueous phase (η) as predicted by the modified Kramers'theory of enzymatic catalysis: v ∝ (1/η)^m, where m is an empirical parameter between 0 and 1. For the activity of MgATPase in the three species studied, m varied between 0. 5 and 1. 1 in good agreement with the theory. We thus conclude that (a) the activity of integral membrane enzyme-proteins may be controlled not only by the property(ies) of the membrane lipid phase but also by the viscosity of the aqueous phase in the vicinity of such enzymes, and (b) the determination of vesicle sidedness based on enzyme latency may need a minor revision. Our interpretation is in agreement with the molecular dynamics approach of enzymatic catalysis worked out for soluble enzymes.     

Mapping of the resistance genes of the R plasmid NR1.

Summary The drug resistance genes on the r-determinants component of the composite R plasmid NR1 were mapped on the EcoRI restriction endonuclease fragments of the R plasmid by cloning the fragments using the plasmid RSF2124 as a vector. The sulfonamide (Su) and streptomycin/spectinomycin (Sm/Sp) resistance genes are located on EcoRI fragment G of NR1. The expression of resistance to mercuric ions (Mer) requires both EcoRI fragment H and I of NR1. The expression of chloramphenicol (Cm) and fusidic acid (Fus) resistance requires EcoRI fragments A and J of NR1. The kan fragment of the related R plasmid R6-5 can substitute for EcoRI fragment J of NR1 in the expression of Cm and Fus resistance. The structural genes for Cm and Fus resistance appear to be a part of an operon whose expression is controlled by the same promoter.     

Structural studies of the lipopolysaccharide from Haemophilus parainfluenzae strain 20

Haemophilus parainfluenzae is a Gram-negative bacterium that colonizes the upper respiratory tract of humans and is a part of normal flora. In this study, we investigated the lipopolysaccharide (LPS) expressed by H. parainfluenzae strain 20. Using NMR and MS techniques on LPS, oligosaccharide samples and lipid A, the structures for O-antigen, core oligosaccharide and lipid A could be established. It was found that the biological repeating unit of the O-antigen is →4)-α-d-GalpNAc-(1→P→6)-β-d-Glcp-(1→3)-α-d-FucpNAc4N-(1→, in which d-FucpNAc4N is 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose. This sugar is in β-configuration when linked to O-4 of the glucose residue of β-d-Galp-(1→2)-l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp-(1→5)-[PPEtn→4]-α-Kdo-(2→6)-lipid A. LPS from a wbaP mutant of H. parainfluenzae strain 20 did not contain an O-antigen, consistent with the wbaP gene product being required for expression of O-antigen in fully extended LPS.     

Inter and intra-species-related differences in the regulation of the cardiac autonomic system

The heart rate response to isoproterenol (HR-Iso), density and affinity (k_d) of β-adrenergic (β-AR) and muscarinic (M₂) receptors were compared among three rodents with different generation-life histories of confinement and of high altitude exposure. The European guinea pig (Cavia porcellus) (EGp), a laboratory animal that arrived in Europe after the Spanish Conquest of South America and the Peruvian guinea pig (C. porcellus) (PGp), a semi-wild animal that came from the altiplano to sea level at least 25 generations ago, were used for intra-species comparison. Wistar rats (WR) were used for inter-species comparison as representative of a typical sea level laboratory animal. The HR-Iso was lower in EGp than in the PGp. The PGp showed the highest β-AR density (P<0.0005) and the highest β-AR k_d values (P<0.0005) when compared to both EGp and WR groups (β-AR B_max (fmol mg⁻¹ prot), WR, 19±4; Egp, 34±10; PGp, 74±15. β-AR k_d (pM), WR, 24±10; Egp, 17±7; PGp, 39±14). In contrast, PGp showed lower M₂ receptor density values than the EGp (P<0.0005). The WR had the highest M₂ receptor densities (M₂ B_max (fmol mg⁻¹ prot), WR, 188±15; Egp, 147±9; PGp, 118±6 and M₂ k_d (pM), WR, 65±12; Egp, 67±6; PGp, 92±2). The inter and intra-species differences found may be related to their respective history of confinement rather than to their history of exposure to high altitude.     

Investigation of the Backcross Progeny of Barley–Wheat Hybrids by the RAPD and RAMPO Methods

The genomes of alloplasmic wheat lines were analyzed by PCR-based methods: random amplified polymorphic DNA (RAPD) and random amplified microsatellite polymorphism (RAMPO). Lines L-16(1) and L-17(2) were obtained by three backcrosses and line L-79(10), by four backcrosses of the barley–wheat hybrid Hordeum vulgare (2n = 14) (variety Nepolegayushchii) × Triticum aestivum (2n = 42) (variety Saratovskaya 29) with different common wheat varieties. These lines proved to be euploid (2n= 42). The aneuploid line L-9 (2n = 43 +t) was obtained after a second backcross of the hybridH. geniculatum All. (2n = 28) × T. aestivum (2n = 42) (Pyrotrix 28) with the variety Pyrotrix 28. The RAPD patterns of L-16(1) and L-17(1) contained fragments present only in the patterns of the parental wheat varieties and, in addition, fragments absent from the latter. This fragment from the pattern of L-16(1) was cloned. Analysis of its primary structure showed that the difference between L-16(1) and the parental wheat genotypes may be related to a mutation that had occurred during the development of the alloplasmic line at the binding site of an arbitrary primer. The genomes of plants of the lines L-79(10) and L-9 contain, in addition to the RAPD fragments of wheat, those characteristic of barley. RAMPO revealed higher polymorphism level among wheat varieties than that detected by RAPD. The hybridization patterns of the lines L-16(1), L-17(1), and L-79(10) contained fragments specific for wheat, and the patterns of L-9 contained both wheat and barley fragments.     

Genetic control of high stearic acid content in the seed oil of the sunflower mutant CAS-3

A sunflower mutant, CAS-3, with about 25% stearic acid (C18:0) in the seed oil was recently isolated after a chemical-mutagen treatment of RDF-1-532 seeds (8% C18:0). To study the inheritance of the high C18:0 content, CAS-3 was reciprocally crossed to RDF-1–532 and HA-89 (5% C18:0). Significant reciprocal-cross differences were found in one of the two crosses, indicating possible maternal effects. In the CAS-3 and RDF-1–532 crosses, the segregation patterns of the F₁, BC₁, and F₂ populations fitted a one-locus (designated Es1) model with two alleles (Es1, es1) and with partial dominance of low over high C18:0 content. Segregation patterns in the CAS-3 and HA-89 crosses indicated the presence of a second independent locus (designated Es2) with two alleles (Es2, es2), also with partial dominance of low over high C18:0 content. From these results, the proposed genotypes (C18:0 content) of each parent were as follows: CAS-3 (25.0% C18:0) =es1es1es2es2; RDF-1–532 (8.0% C18:0) =Es1Es1es2es2; and HA-89 (4.6% C18:0) =Es1Es1Es2Es2. The relationship between the proposed genotypes and their C18:0 content indicates that the Es1 locus has a greater effect on the C18:0 content than the Es2 locus. Apparently, the mutagenic treatment caused a mutation of Es1 to es1 in RDF-1–532. Received: 20 September 1998 / Accepted: 1 February 1999     

Enantiopurity Determination of the Enantiomers of the Triple Reuptake Inhibitor Indatraline

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in ¹H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)‐indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high‐performance liquid chromatography (HPLC) method based on a modified β‐cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25–1000 μM for the (1R,3S)‐enantiomer and 1.25‐750 μM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)‐enantiomer and up to 99.67% ee for the (1S,3R)‐enantiomer. Chirality 25:923–933, 2013. © 2013 Wiley Periodicals, Inc.     

Toward positional cloning of Vgt1, a QTL controlling the transition from the vegetative to the reproductive phase in maize

Vgt1 (Vegetative to generative transition 1) is a quantitative trait locus (QTL) for flowering time in maize (Zea mays L.). Vgt1 was initially mapped in a ca. 5-cM interval on chromosome bin 8.05, using a set of near-isogenic lines (NILs) in the genetic background of the late dent line N28, with the earliness allele introgressed from the early variety Gaspé Flint. A new large mapping population was produced by crossing N28 and one early NIL with a ca. 6-cM long Gaspé Flint introgression at the Vgt1 region. Using PCR-based assays at markers flanking Vgt1, 69 segmental NILs homozygous for independent crossovers near the QTL were developed. When the NILs were tested in replicated field trials for days to pollen shed (DPS) and plant node number (ND), the QTL followed a Mendelian segregation. Using bulk segregant analysis and AFLP profiling, 17 AFLP markers linked to the QTL region were identified. Statistical analysis indicated a substantial coincidence of the effects of Vgt1 on both DPS and ND. Vgt1 was mapped at ca. 0.3 cM from an AFLP marker. As compared to DPS, the higher heritability of ND allowed for a more accurate assessment of the effects of Vgt1. The feasibility of the positional cloning of Vgt1 is discussed.     

Co-segregation of the maize dwarf mosaic virus resistance gene,Mdm1, with the nucleolus organizer region in maize

The mdm1 locus on the short arm of chromosome six confers resistance in maize to five strains of the maize dwarf mosaic virus (MDMV), an aphid transmitted potyvirus. The location of mdm1 in relation to RFLP and morphological loci on the short arm of chromosome six was determined using BC₁ and F₂ mapping populations. The following map order and distance in cM was obtained from the F₂ population; jc1270-2.5-npi245-1.6-umc85/po1-0.5-mdm1/nor-0.5-bnl6.29A-0.5-npi235-0.8-npi101A-4.3-numc59. No recombination between mdm1 and the nucleolus organizer region (nor) was detected, as determined using a probe from the intergenic spacer region of the rDNA repeat. In order to resolve the relationship between mdm1 and the nor, and to recover recombinants around mdm1, a highresolution map within the polymitotic1 (po1) yellow kernel1 (y1) interval was generated using [po1 y1 tester (po1 mdm1 y1) x Pa405 (Po1 Mdm1 Y1)] F₂ plants. The recessive po1 allele imparts a male-sterile phenotype when homozygous and since po1 and y1 are closely linked, the majority of fertile plants from white endosperm (y1/y1) F₂ kernels will arise though a recombination event between the Pa405 Po1 allele and the y1 allele of the po1 y1 tester. Plants from 7,650 white (y1/y1) F₂ kernels were examined (15,300 chromosomes) and a total of 626 F₂₃ recombinant families was recovered. Analysis of these recombinants revealed that mdm1 cosegregates with the nor. This lack of recombination between mdm1 and the nor suggests that: either (1) mdm1 is located in the region flanking the nor and recombination is suppressed within that region, or (2) mdm1 is located within the nor.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.