Microsatellite genotyping of Chromosome 14q13.2-14q13 in the vicinity of proteasomal gene<Emphasis Type="Italic"> PSMA6</Emphasis> and association with Graves’ disease in the Latvian population

The 270-kb Chromosome 14q13.2-14q13 region harboring the proteasomal alpha subunit 6 gene PSMA6 was analyzed for polymorphism of five microsatellite repeats in cases/controls and association with Graves disease. Four novel microsatellite markers were localized to the 14q13.2 region upstream of PSMA6. Dinucleotide repeats HSMS801, HSMS702, HSMS701 were identified in two introns of the gene KIAA0391; the most upstream trinucleotide HSMS602 marker was found in an intron of the C14orf24 gene. A polymorphism study performed on the Latvian population revealed 13 and 14 alleles for HSMS801 and HSMS702, respectively, seven alleles for HSMS701, and four alleles for HSMS602. Heterozygosity analysis revealed that all the four markers obey Hardy-Weinberg distribution. The previously described HSMS006 marker, represented by 12 alleles, is localized in intron 6 of the PSMA6 gene. No significant differences were observed between patients and controls in allele distribution of the HSMS702 and HSMS701 microsatellite repeats. However, the allele frequencies of HSMS006 and HSMS801 were significantly different between Graves disease and control subjects. The 181- and 185-bp alleles of HSMS006 and the 133-, 143-, and 149-bp alleles of HSMS801 were found more often, but the 189- and 191-bp alleles of HSMS006 were much less frequent in Graves disease patients compared with the controls. An additional 174-bp allele of the HSMS602 marker, absent in healthy subjects, was found in Graves disease patients.     

Polymorphism of 2-μm plasmids in industrial strains of Saccharomyces cerevisiae

Restriction fragment length polymorphism (RFLP) analyses of industrial Saccharomyces yeast DNA have identified eight 2-m plasmidsvariants that fall into two distinct types. Type-I plasmids are of unique form, whereas type-II plasmids exist in seven distinct RFLP forms. Only two different 2-m variants were observed in 35 bakers' strains analysed. One variant was the unique type-I whereas the second variant represents an ancestral form of the type-II plasmid. Sixteen of nineteen wine yeasts carried a distinctive type-II plasmid with a homeologous STB repeat whereas ale and lager yeasts had a wide range of type-II variants. Relative to nuclear and mtDNA, 2-m polymorphism is less diverse and not diagnostic for a specific strain. This 2-m DNA polymorphism is a convenient and useful addendum to nuclear and mtDNA RFLP analyses but cannot serve as the sole marker for strain identification. A tentative phylogeny of industrial S. cerevisiae yeasts is suggested with origins in bakers' yeast carrying the ancestral type-II form. Correspondence to: G. H. Rank     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR , -DQ , and -DQ cDNA probes in Southern blot analysis. Three subtypes are found using the DR cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, DW10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ and DQ cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ probe, probably due to DX polymorphism. DR RFLP divides the LB 14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.     

Karyological and serological studies in Rana lessonae,R. ridibunda and in their hybrid R. ‘esculenta’ (Amphibia,Anura)

The karyotypes of the three water frog forms: Rana lessonae, R. ridibunda and R. esculenta were analysed from bone marrow cell preparations of animals captured in several localities of the GDR. In the three forms chromosome morphology was similar (5 large and 8 small pairs), although differences in the relative length of most elements were found; R. esculenta chromosomes were always intermediate.One of the small pairs (Chr. 12) was found to be metacentric in R. lessonae and submetacentric in R. ridibunda. Most R. esculenta individuals examined had one meta-and one submetacentric 12th element, indicating the hybrid nature of this form. However 16.6% esculenta proved to be homozygous for either the metacentric or the submetacentric chromosome 12, while 13% lessonae individuals and 7.7% ridibunda were heterozygous for this element.By starch gel electrophoresis an analysis was undertaken of serum proteins from water frogs coming from regions in which the forms occur together (sympatric populations) and from zones in which only one of them lives (allopatric populations).In Rana lessonae, where only one allele had been previously described, two different alleles were found in animals coming from the GDR.If genetic polymorphism is excluded, between 6.1% and 9.1% individuals from sympatric lessonae and ridibunda populations showed introgression of an albumin allele. No gene introgression was found in allopatric lessonae populations from the Leningrad region or in ridibunda from Alma Ata and southern Bulgaria.     

Genomics of Type 1 Diabetes Mellitus and Its Late Complications

In ethnic Russians, MHC (HLA) was shown to be the major locus determining the predisposition to type 1 diabetes mellitus (T1DM). To map the regions linked to T1DM, families with concordant or discordant sib pairs were selected from the Russian population of Moscow. With these families, linkage to T1DM was demonstrated for the CTLA4 gene (IDDM12, 2q32.1–q33), which codes for a T-cell surface antigen, and the PDCD2 gene (IDDM8, 6q25–q27), which is homologous to the mouse programmed cell death activator gene. Using polymorphic microsatellites, we could also observe the linkage to T1DM of regions 3q21–q25 (IDDM9) and 10p12.2 (IDDM10). Complex analysis of linkage and association of the polymorphic markers from region 11p13 in the vicinity of the catalase gene (CAT) based on the case/control groups and two groups of families allowed us to reveal a new T1DM locus; the linkage to this locus was not reported earlier for other populations. Diabetic polyneuropathy (DPN) proved to be associated with polymorphic markers Ala(–9)Val of the SOD2 gene, Arg213Gly of the SOD3 gene, and T(–262)C of the CAT gene, and with a polymorphic microsatellite located in the promoter region of the NOS2 gene. It has been supposed that one of the main risk factors of DPN development in patients with type 1 diabetes is oxidative stress arising in hyperglycemia because of increased production of superoxide radicals in mitochondria and insufficient activity of antioxidative enzymes. Diabetic nephropathy (DN) showed no association with the antioxidative enzyme genes. However, the association was observed for the insertion/deletion (I/D) polymorphism of ACE and the ecNOS34a/4b polymorphism of NOS3, two genes involved in controlling vascular tonus, as well as for the I/D polymorphism of APOB and the 2/3/4 polymorphism of APOE, two genes involved in lipid transport. In addition, polymorphic microsatellites of chromosome 3q21–q25 proved to be closely associated with DN. The tightest association was established for D3S1550, carriers of allele 12 or genotype 12/14 having high risk of DN (OR = 4.85 and 6.25, respectively). Region 3q21–q25 perhaps contains a major gene determining DN development, while the other DN-associated genes mostly influence the progression of DN.     

Structural evidence that the small subunit found associated with the TL antigen is β 2-microglobulin

Comparative tryptic peptide mapping and partial amino-terminal primary sequence analysis of the light chain component associated with the TL antigens showed that the small subunit of TL was identical to the ₂m light chain associated with the H-2K or D product of the same strain. Peptide comparison of the ₂m from the Tla products of an A strain X-ray induced leukemia RADA1 (Tla ^a) and of a C57BL/6 strain X-ray induced leukemia ERLD (Tla ^b) showed differences to the extent of 25–35% in their peptides. This is consistent with previous results showing ₂m allelic variations between these mouse strains. The data prove the structural identity of the ₂m molecules from TL and H-2K, D antigens as well as reveal the strain specific polymorphism of the ₂m associated with these products.     

Seed protein and isozyme variations in Triticum tauschii (Aegilops squarrosa)

Sixty Triticum tauschii (Aegilops squarrosa, 2n=2x=14, DD) accessions were evaluated for the variability of high-molecular-weight (HMW) glutenins, gliadins and isozymes of seed esterase, -amylase and glucose-phosphate isomerase. Wide variability was observed for HMW-glutenins and gliadins. The implications of unique HMW-glutenin alleles for quality parameters are discussed. Isozyme evaluations indicated more variability for the Est-D ^t 5 locus as compared to the Est-D5 of bread-wheat. The polymorphism for -Amy-D ^t 1 was less than that of -Amy-D1. Similar to the bread-wheat situation, Gpi-D ^t 1 showed no polymorphism. The variability observed with the traits evaluated can be readily observed in T. turgidum x T. tauschii synthetic hexaploids (2n=6x=42, AABBDD) suggesting that T. tauschii accessions may be a rich source for enhancing the genetic variability of T. aestivum cultivars.     

Cloning and identification of the H-2D p gene

We have cloned six different class I genes from a B10.P sperm library. After cotransfection with the herpes simplex tk gene, one L-cell line was found to react with six H-2D^p-specific monoclonal antibodies. The cell line L12a did not react with K^p-specific monoclonal antibodies. This identification was confirmed by mapping a 2.5 kb Bam H 1 restriction fragment present in the 12a DNA clone to the D-TL region of H-2 ^p. Only a single 8.8 kb Barn H1 fragment can be assigned to K ^p by restriction fragment length polymorphism, while many others map to the D-TL interval. A restriction map of 12a is presented.     

Kinetic properties of bovine brain proteinl-isoaspartyl methyltransferase determined using a synthetic isoaspartyl peptide substrate

Proteinl-isoaspartyl methyltransferase, an enzyme enriched in brain, is implicated in the repair of age-damaged proteins containing atypical, isoaspartyl peptide bonds. We have investigated the kinetics of methylation using a synthetic peptide substrate having the structure Trp-Ala-Gly-Gly-isoAsp-Ala-Ser-Gly-Glu. Double-reciprocal plots of initial velocity versus concentration of S-adenosylmethionine (AdoMet) at different fixed concentrations of peptide gave straight lines converging at a positive 1/v value and a negative 1/AdoMet value. The product S-adenosylhomocysteine (AdoHcy) was a competitive inhibitor towards AdoMet and a linear mixed-type inhibitor towards peptide. These results are consistent with the rapid-equilibrium random sequential bi-bi mechanism previously proposed for the enzyme, but they also reveal the formation of the deadend, enzyme-peptide-AdoHcy, complex. The rate constants were:V _max=32–34 nmol/min/mg,K ^peptide=7.6–9.4 M,K ^AdoMet=1.9–2.2 M, =0.43–0.53,K ^AdoHcy=0.08 M, =2.9. The interaction factors and indicate that binding of enzyme to peptide increases its affinity for AdoMet and decreases its affinity for AdoHcy. Methylation was linear with time throughout the transfer of 2 mol of methyl groups/mol of enzyme. This absence of burst kinetics suggests that slow release of products cannot explain the low turnover number.Special issue dedicated to Dr. Paul Greengard     

Polymorphism and mapping of the class II genes in the rat: RT1.B,RT1.D,and RT1.H,a new DP-like region

The major histocompatibility complex of the rat (RTI) encodes the class II molecules involved with antigen presentation and cell to cell communication. The organization of these class II genes has been studied by Southern blot hybridization using genomic DNA from inbred and recombinant rat strains digested with various restriction endonuclease and hybridized under stringent conditions with probes for mouse class II and human class II genes. Analysis of the restriction fragment length polymorphisms has mapped the class II genes relative to each other. We have confirmed the order of the - and -chain genes in the RT1.B region, mapped the RT1.D region relative to RT1.B and showed that it has - and -chain loci, and identified a new HLA-DP-like locus, RT1.H, to the RT1.A side of RT1. B. The RT1. H and RT1.H genes map to the region around the recombination point in R22, and there appears to be a hot spot of recombination in RT1.H. The H and D genes have high levels of polymorphism; B , B ,and H have intermediate levels of polymorphism, and D has a low level of polymorphism.     

Human familial and sporadic breast cancer: analysis of the coding regions of the 17β-hydroxysteroid dehydrogenase 2 gene (EDH17B2) using a single-strand conformation polymorphism assay

17-Hydroxysteroid dehydrogenase (17HSD) is one of the key enzymes in estrogen metabolism, catalyzing the reversible reaction between estradiol and the less active estrogen, estrone. The gene encoding this enzyme, EDH17B2, has been mapped to chromosome 17, region q12–q21, in the vicinity of BRCA1, an as yet unidentified gene that appears to be involved in familial breast cancer and in familial ovarian cancer. The possibility that EDH17B2 gene is the same as BRCA1 was tested by screening for mutations in the coding regions of EDH17B2, using a polymerase chain reaction/single-strand conformation polymorphism method. An AG transition creating a new BstUI site at exon 6 was the only frequent sequence alteration found in the coding region of the gene. This mutation also led to an amino acid substitution of serine to glycine at position 312 (312^S312^G) in the 17HSD protein. Since the nucleotide change was detected both in specimens from patients with familial or sporadic cancer and in control samples, and at similar rates, this mutation appears to be of a polymorphic nature. In addition, a rare polymorphism located at intron 5 was detected. This CT substitution creates a BbvI site and is not thought to have any effect on 17HSD activity. The results indicate that there are no major alterations in the coding areas of EDH17B2 and thus studies testing the hypothesis that EDH17B2 may be the same as BRCA1 should be extended to the promoter and regulatory elements of EDH17B2.     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

The MHC haplotypes of the chicken

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as standard B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The standard haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Structure of the majorO-glycosidic oligosaccharide of monkey erythrocyte glycophorin

Sialic acids and the majorO-glycosidic oligosaccharide of glycophorin MK from monkey (Japanese monkey,Macaca fuscata) erythrocyte membranes were characterized.N-Glycolylneuraminic acid (neu5Gc) was found as the major sialic acid, which was confirmed by gas-liquid chromatography-mass spectrometry as the trimethylsilyl methyl ester. ThreeO-glycosidic oligosaccharide units were obtained from a tryptic glycopeptide that contained all of the carbohydrate units in glycophorin MK by mild alkaline borohydride/borotritide treatment. Carbohydrate analyses of the oligosaccharides revealed that they were composed of Neu5Gc, galactose andN-acetylgalactosaminitol in the molar ratios of 111 (trisaccharide), 211 (tetrasaccharide) and 111 (pentasaccharide). The content of oligosaccharide units was estimated to be 1125 for penta-, tetra- and trisaccharide, respectively, based on the yields, the molecular weight, and the number of oligosaccharide attachment sites in the amino-acid sequence. The tetrasaccharide was the major oligosaccharide and its structure was proposed to be Neu5Gc2-3Gal1-3[Neu5Gc2-6]GalNAcol.     

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E^d molecule prevents CIA development in susceptible H2 ^q mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F₁ mice, only H2Eb^d and H2Eb^s molecules showed protection. Using recombinant B10.RDD (Eb ^d/b) mice, we found that CIA protection was mediated by the first domain of the E^d molecule. Using peptides covering the third hypervariable region of the E chain, we found a perfect correlation between presentation of E peptides by the H2A^q molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E peptides for the H2A_q molecule.