Structures of the mating-type loci of Cordyceps takaomontana

Nucleotide sequences of the mating-type loci MAT1-1 and MAT1-2 of Cordyceps takaomontana were determined, which is the first such report for the clavicipitaceous fungi. MAT1-1 contains two mating-type genes, MAT1-1-1 and MAT1-1-2, but MAT1-1-3 could not be found. On the other hand, MAT1-2 has MAT1-2-1. A pseudogene of MAT1-1-1 is located next to MAT1-2.     

Urinary oligosaccharides of fucosidosis. Evidence of the occurrence of X-antigenic determinant in serum-type sugar chains of glycoproteins.

Urine of a fucosidosis patient contained a large amount of fucosyl oligosaccharides and fucose-rich glycopeptides. Six major oligosaccharides were purified by a combination of Bio-Gel P-2 and P-4 column chromatographies and paper chromatography. Structural studies by sequential exoglycosidase digestion and by methylation analysis revealed that their structures were as follows: Fucalpha1 leads to 6GlcNAc, Fucalpha1 leads to 2Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 2Manalpha1 leads to 3Manbeta1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 4GlcNAc, Galbeta1 leads to 4(Fucalpha1 leads to3)GlcNAcbeta1 leads to 2Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc, and Galbeta1 leads to 4(Fucalpha1 leads to 3)GlcNAcbeta1 leads to 4Manalpha1 leads to 6Manalpha1 leads to 6Manbeta1 leads to 4GlcNAc. In additon, the structure of a minor decasaccharide was found to be Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to [Galbeta1 leads to (Fucalpha1 leads to)GlcNAcbeta1 leads to Manalpha1 leads to]Manbeta1 leads to 4GlcNAc.     

The role of immune complexes in the activation of the first component of human complement

Immune complex-induced C1 activation and fluid phase C1 autoactivation have been compared in order to elucidate the immune complex role in the C1 activation process. Kinetic analyses revealed that immune complex-bound C1 activates seven times faster than fluid phase C1 spontaneously activates. The rate of spontaneous C1 activation increased after decreasing the solution ionic strength. In fact at one-half physiologic ionic strength (i.e., 0.08 M), the kinetics of spontaneous C1 activation were indistinguishable from the kinetics of activation of immune complex-bound C1 at physiologic ionic strength. The enhanced fluid phase C1 activation at low ionic strength resulted neither from C1 nor C1q aggregation, nor from selective effects on the C1r2S2 subunit; however, at the reduced ionic strength, the C1 association constant (defined for C1q + C1r2S2 in equilibrium C1qr2S2) did increase to 2.3 X 10(8) M-1, which is equal to that for C1 bound to an immune complex at physiologic ionic strength. Therefore, C1 can spontaneously activate in the fluid phase as rapidly as C1 on an immune complex when the strength of interaction between C1q and C1r2S2 is the same in both systems. In conclusion, under physiologic conditions, C1q and C1r2S2 are two weakly interacting proteins. Immune complexes provide a site for the assembly of a stable C1 complex, in which C1q and C1r2S2 remain associated long enough for C1q to activate C1r2S2. Thus, immune complexes enhance the intrinsic C1 autoactivation process by strengthening the association of C1q with C1r2S2.     

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.     

Structures of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma

Structures of the principal O-glycosides from the major cell surface sialoglycoprotein (ASGP-1) of the MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma have been determined. Oligosaccharitols were released by alkaline borohydride treatments of ASGP-1 and purified by gel filtration, DEAE-Sephadex ion exchange chromatography, and high performance liquid chromatography. On the basis of carbohydrate composition, methylation analysis, periodate oxidation, and exoglycosidase digestion, the five major oligosaccharides released by mild alkaline borohydride were assigned the following structures: Component II-3: (NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)Ga 1 NAcOH(3----1 betaGa 1 3----2 alpha NeuAc) III-2a: (Ga 1 beta 1----4G1cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) III-2c: (Ga 1 alpha 1----3Ga 1 beta 1----4G1cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) IV-1a: (Ga 1 beta 1----4G 1 cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1) IV-1c: (Ga 1 alpha 1----3Ga 1 beta 1----4G 1 cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1) Fucosylated derivatives of III-2a, IV-1a, and IV-1c were found in smaller amounts with the fucose tentatively assigned to the 2-position of the lactosamine galactose. Components II-3, III-2a, and the fucosylated derivative of III-2A were found in both MAT-B1 and MAT-C1 sublines. The alpha-galactosides were found in detectable quantities only in subline MAT-B1. Oligosaccharides from MAT-C1 cells were enriched in sialic acid when compared to those from MAT-B1 cells. These results suggest that the 13762 ascites sublines, which bear different oligosaccharides, will provide models useful for the investigation of mechanisms regulating the expression of structures of the larger O-linked oligosaccharides.     

Genetic history of Aleuts of the Komandor islands from results of analyzing variability of class II HLA genes

Variability of the HLA class II genes (alleles of the DRB1, DQA1, and DQB1 loci) was investigated in a sample of Aleuts of the Commanders (n = 31), whose ancestors inhabited the Commander Islands for many thousand years. Among 19 haplotypes revealed in Aleuts of the Commanders, at most eight were inherited from the native inhabitants of the Commander Islands. Five of these haplotypes (DRB1*0401-DQA1*0301-DQB1*0301, DRB1*1401-DQA1*0101-DQB1*0503, DRB1*0802-DQA1*0401-DQB1*0402, DRB1*1101-DQA1*0501-DQB1*0301, and DRB1*1201-DQA1*0501-DQB1*0301) were typical of Beringian Mongoloids, i.e., Coastal Chukchi and Koryaks, as well as Siberian and Alaskan Eskimos. Genetic contribution of the immigrants to the genetic pool of proper Aleuts constituted about 52%. Phylogenetic analysis based on Transberingian distribution of the DRB1 allele frequencies favored the hypothesis on the common origin of Paleo-Aleuts, Paleo-Eskimos, and the Indians from the northwestern North America, whose direct ancestors survived in Beringian/southwestern Alaskan coastal refugia during the late Ice Age.     

Determinants of the nucleolar targeting of protein phosphatase-1

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.     

Correlation of the Physical and Genetic Maps of the Centromeric Region of the Right Arm of Linkage Group III of Neurospora Crassa

We have cloned three linked genes serine-1 (ser-1), proline-1 (pro-1) and acetate-2 (ace-2) that lie near the centromere on the right arm of linkage group III (LGIIIR) of Neurospora crassa. The ser-1 gene was cloned by sib selection. A chromosomal walk that spans 205 kilobases (kb) was initiated from ser-1. Complementation analysis with clones isolated during the walk allowed identification of the pro-1 and ace-2 genes. Restriction fragment length polymorphism analysis has confirmed the localization of ser-1, pro-1 and ace-2 to the centromeric region of LGIIIR. Genetically, we measured 1% recombination between ser-1 and pro-1 and 2% recombination between pro-1 and ace-2. Physical distances for these intervals were 114 kb from ser-1 to pro-1 and 36 kb from pro-1 to ace-2. Thus, for the pro-1 to ace-2 interval we calculate a physical/genetic correlation of 18 kb/map unit (mu) whereas, in the immediately adjacent, centromere-proximal interval from ser-1 to pro-1, we calculate 114 kb/mu. This provides evidence for a centromere effect, a decrease in recombination frequency as one approaches the centromere.     

Mechanism of action of anti-C1-inhibitor autoantibodies: prevention of the formation of stable C1s-C1-inh complexes.

BACKGROUND: Acquired C1-inhibitor (C1-inh) deficiency is usually associated with the presence of circulating C1-inh autoantibodies. These autoantibodies have been shown previously to bind to two synthetic peptides corresponding to C1-inh amino acid residues 438-449 (peptide 2) and 448-459 (peptide 3) but not to peptide 1 (residues 428-440). MATERIALS AND METHODS: Affinity-purified C1-inh autoantibodies from two patients with acquired C1-inh deficiency were studied for their effects on the inhibition of C1s activity by C1-inh using SDS-PAGE and hydrolysis of a synthetic ester. RESULTS: Functional studies confirmed that the anti-C1-inh autoantibodies abrogated C1-inh activity, and their maximum effect was produced when the concentrations of C1-inh and autoantibody were approximately equimolar. The autoantibodies prevent the formation of the C1s-C1-inh complex, but they do not dissociate the preformed complex, suggesting that the autoantibodies act prior to the formation of the enzyme-inhibitor complex. In the presence of autoantibodies, C1s cleaves C1-inh, and a stable covalent bond between C1s and C1-inh does not form. Peptides 2 and 3, but not peptide 1 inhibited autoantibody activity, thus C1-inh inhibitory activity for C1s was expressed fully. CONCLUSIONS: Our data indicate that the anti-C1-inh autoantibodies convert C1-inh to a substrate by preventing the formation of the stable covalent protease-serpin complex. The data also suggest a possible therapeutic use for peptides 2 and 3 or their derivatives in the management of patients with type II acquired angioedema (AAE).     

几类异质小麦雄性不育系育性恢复性的细胞遗传学研究

系统调查了4类异质(粘果、易变、偏凸、二角山羊草细胞质)1BL/lRS、非1BL/1RS小麦雄性不育系与其恢复系杂种F减数分裂中期Ⅰ出现单价体细胞频率,以及后期Ⅰ出现落后染色体和染色体桥细胞频率,并对中、后期染色体变异率与杂种F自交结实率进行了相关分析.结果表明(1)1BL/1RS型杂种在中期Ⅰ、后期Ⅰ染色体变异率要明显高于非1BL/1RS杂种;(2)4类异源细胞质在非1BL/1RS杂种中有着明显提高单价体细胞频率的作用;(3)在1BL/1RS杂种中,1B@1BL/1RS杂合核型染色体联会松弛,对单价体频率的影响远大于异源细胞质的影响;(4)1BL/1RS型杂种自交结实率与中期出现单价体细胞频率不直接相关,而与后期出现落后染色体和染色体桥细胞的频率呈高度负相关;(5)非1BL/1RS型杂种在减数分裂中、后期染色体行为相对稳定,易恢复且恢复度高,很有实际利用价值.     

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.     

X-ray structure of the Ca2+-binding interaction domain of C1s. Insights into the assembly of the C1 complex of complement

C1, the complex that triggers the classical pathway of complement, is assembled from two modular proteases C1r and C1s and a recognition protein C1q. The N-terminal CUB1-EGF segments of C1r and C1s are key elements of the C1 architecture, because they mediate both Ca2+-dependent C1r-C1s association and interaction with C1q. The crystal structure of the interaction domain of C1s has been solved and refined to 1.5 A resolution. The structure reveals a head-to-tail homodimer involving interactions between the CUB1 module of one monomer and the epidermal growth factor (EGF) module of its counterpart. A Ca2+ ion is bound to each EGF module and stabilizes both the intra- and inter-monomer interfaces. Unexpectedly, a second Ca2+ ion is bound to the distal end of each CUB1 module, through six ligands contributed by Glu45, Asp53, Asp98, and two water molecules. These acidic residues and Tyr17 are conserved in approximately two-thirds of the CUB repertoire and define a novel, Ca2+-binding CUB module subset. The C1s structure was used to build a model of the C1r-C1s CUB1-EGF heterodimer, which in C1 connects C1r to C1s and mediates interaction with C1q. A structural model of the C1q/C1r/C1s interface is proposed, where the rod-like collagen triple helix of C1q is accommodated into a groove along the transversal axis of the C1r-C1s heterodimer.     

Structures of the Mating-Type Loci of Cordyceps takaomontana

Nucleotide sequences of the mating-type loci MAT1-1 and MAT1-2 of Cordyceps takaomontana were determined, which is the first such report for the clavicipitaceous fungi. MAT1-1 contains two mating-type genes, MAT1-1-1 and MAT1-1-2, but MAT1-1-3 could not be found. On the other hand, MAT1-2 has MAT1-2-1. A pseudogene of MAT1-1-1 is located next to MAT1-2.     

Functional model of subcomponent C1 of human complement

The domain organization of the zymogen subunits of the first component of human complement C1s, C1r2 and the complex C1s-C1r2-C1s was studied by electron microscopy. In the absence of Ca2+, monomeric C1s was visualized as a dumb-bell-shaped molecule consisting of two globular domains (center-to-center distance 11 nm) connected by a rod. One of the globular domains is assigned to the light chain (B-chain) of the activated molecule, which is homologous to trypsin and other serine proteases. The second globular domain and the rod are assigned to the heavy chain (A-chain) of CIs. The subunit C1r is a stable dimer in the presence or absence of Ca2+. This dimer C1r2 was visualized as composed of two dumb-bells of dimensions similar to those observed for C1s. These are connected near the junctions between the rod and one of the globular domains. This leads to the structure of an asymmetrical X with two inner closely spaced globules (center-to-center distance 7 nm) and two outer globules at a larger distance (14 nm). By comparison with fragment C1rII2, in which part of the A-chain is removed, the inner globular domains were assigned to the catalytic B-chains. This characteristic structure of C1r2 is readily recognized in the central portion of the thread-like 54 nm long C1s-C1r2-C1s complex formed in the presence of Ca2+. By affinity-labeling of C1s with biotin and visualization of avidin-ferritin conjugates in the reconstituted complex, it was demonstrated that C1s forms the outer portion of the complex. A detailed model of C1s-C1r2-C1s is proposed, according to which two C1s monomers bind to the outer globes of C1r2 by contacts between their heavy chains and those of C1r. According to this model the catalytic domains of C1r are located in the center and those of C1s at the very tips of the C1s-C1r2-C1s complex. On the basis of the structure of C1s-C1r2-C1s, we derived a detailed model of the C1 complex (composed of C1q and the tetrameric complex) and we discuss this model with a view to finding a possible activation mechanism of C1.(ABSTRACT TRUNCATED AT 400 WORDS)     

New evidence of the substrate specificity of endo-beta-N-acetylglucosaminidase D

The susceptibility of a variety of oligosaccharides to endo-beta-N-acetylglucosaminidase D was investigated. The oligosaccharides having the structures of Man alpha 1----6 (GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, derived from complex type triantennary sugar chains, released +/- Fuc alpha 1----6GlcNAcOT upon incubation with the enzyme at almost the same rate as Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. When the reaction products were reduced with NaB3H4 and analyzed by Bio-Gel P-4 column chromatography, a new radioactive peak was detected in both cases. This new radioactive oligosaccharide was confirmed to be Man alpha 1----6(GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAcOT in the former case and Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAcOT in the latter. These results indicated that endo-beta-N-acetylglucosaminidase D does not require the presence of a free hydroxyl group at the C-4 position of the alpha-mannosyl residue of the trisaccharide glycon: Man alpha 1----3Man beta 1----4GlcNAc beta 1----.     

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.     

Antagonistic interplay of Swi1 and Tup1 on filamentous growth of Candida albicans

Candida albicans is a polymorphic human opportunistic pathogen in which the Swi-Snf complex functions as an activator whereas Tup1 acts as a general repressor during the yeast-hyphae transition. In Saccharomyces cerevisiae, the interplay between the Swi-Snf complex and the Tup1-Ssn6 repressive complex regulates the balance between active and repressed chromatin structures of a number of genes. To study the interplay between Candida albicans Swi1 and Tup1 and their effects on morphogenesis, we analyzed phenotypes of swi1/swi1, tup1/tup1 and swi1/swi1 tup1/tup1 mutants under various growth conditions. The swi1/swi1 mutant failed to form true hyphae, whereas the tup1/tup1 mutant exhibited constitutive filamentous growth. Deletion of SWI1 in the tup1/tup1 mutant completely blocked hyphal growth under all the conditions examined. Under aerobic conditions, the swi1/swi1 tup1/tup1 mutant most resembled the swi1/swi1 mutant in phenotype, actin polarization and gene expression pattern. In invaded agar, the double mutant showed similar phenotypes as the swi1/swi1 mutant, while under embedded conditions, it grew as a pseudohypha-like form different from that of the wild-type strain, swi1/swi1 or tup1/tup1 mutants. These results suggest that Swi1 may play a dominant role by antagonizing the repressive effect of the Tup1 on hyphal development in C. albicans.     

Structural characterization of neutral oligosaccharides of human midcycle cervical mucin

It was previously shown that alkaline borohydride treatment of human midcycle cervical mucin releases a heterogeneous population of reduced neutral, sialylated, and sulfated oligosaccharides (Yurewicz, E. C., and Moghissi, K. S. (1981) J. Biol. Chem. 256, 11895-11905). Three major neutral oligosaccharides were isolated with approximate compositions of Fuc:Gal:GlcNAc:N-acetylgalactosaminitol (GalNAcol) = 0:2:1:1 (A1), 1:2:1:1 (A2), and 2:2:1:1 (A3). They comprised roughly 21%, 13%, and 8% of human cervical mucin oligosaccharide chains, respectively. In the present report, each was analyzed by periodate oxidation, methylation, and sequential degradation with glycosidases. A1 was shown to contain more than one component, but structural analyses clearly demonstrated the presence of one predominant (75%) tetrasaccharide. The proposed structure, Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAcol, has previously been found in human gastric, submaxillary, and ovarian cyst mucins in their carbohydrate-to-protein linkage regions. beta-Galactosidase from Aspergillus niger selectively cleaved the Gal beta 1-4GlcNAc linkage in the intact tetrasaccharide. Enzymatic hydrolysis of the Gal beta 1-3GalNAcol linkage required prior removal of the Gal beta 1-4GlcNAc beta 1-unit attached to 0-6 of GalNAcol. The data for A2 indicated a mixture of two oligosaccharides, Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNacol and Fuc alpha 1-2Gal beta 1-4GlcNac beta 1-6(Gal beta 1-3)-GalNacol, in an approximate molar ratio of 3 to 4:1, respectively. Two structures are consistent with the data obtained for A3: Fuc alpha 1-2Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNAc beta 1-6(Gal beta 1-3)GalNAcol and/or Gal beta 1-4,3(Fuc alpha 1-3,4)GlcNac beta 1-6(Fuc alpha 1-2Gal beta 1-3)GalNacol. The results indicate that A1 represents the "core" tetrasaccharide of the larger human cervical mucin oligosaccharides A2 and A3.     

Synthesis of a photoreactive, radiolabelled derivative of the oligosaccharide of GM1 ganglioside.

A photoreactive, radioiodinatable derivative of the oligosaccharide (GM1OS) of ganglioside GM1 was synthesized as follows: GM1OS was generated from GM1 by ozonolysis and alkaline fragmentation, and reductively aminated to GM1OSNH2 (1-amino-1-deoxymonosialogangliotetraitol). The latter compound was then reacted with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA) to form GM1OSNH-ASA [1-(4-azidosalicoylamido)-1-deoxymonosialogangliotetraitol], which was radioiodinated and further purified. To test the [125I]GM1OSNH-IASA [1-(4-iodoazidosalicoylamido)-1-deoxymonosialogangliotetraitol+ ++] as a probe for ganglioside-binding proteins, the derivative was incubated with cholera toxin, which specifically binds GM1, followed by photolysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The probe only labelled the B or binding subunit of cholera toxin, but not the A or adenylyl cyclase activating subunit. Labelling was inhibited by excess GM1OS, but not by the oligosaccharides from gangliosides GD1a and GD1b. [125I]GM1OSNH-IASA and analogous oligosaccharide derivatives may be valuable probes for detecting ganglioside-binding proteins.     

嗜热四膜虫异染色质蛋白Tcd1磷酸化位点的突变分析

四膜虫异染色质蛋白Tcd1在有性生殖时期特异表达,在大核基因组重排以及修复过程中发挥作用。磷酸化蛋白质组学分析表明,Tcd1存在3个磷酸化位点：S301,S303和S535。然而,Tcd1磷酸化修饰与其功能的关系并不清楚。本研究对TCD1基因的3个磷酸化位点进行了模拟磷酸化和模拟去磷酸化定点突变,获得模拟磷酸化突变基因TCD1S301D (TCD1S1D)、TCD1S301DS303D (TCD1S2D)与TCD1S301DS303DS535D (TCD1S3D) 和模拟去磷酸化的突变基因TCD1S301A (TCD1S1A)、TCD1S301AS303A (TCD1S2A)与TCD1S301AS303AS535A (TCD1S3A)。分别构建了不同突变体的过表达载体,转化四膜虫细胞并筛选获得不同突变体细胞株。Western印迹分析表明,Tcd1S1D、Tcd1S2D、Tcd1S3D与Tcd1S1A、Tcd1S2A和Tcd1S3A在四膜虫有性生殖期表达。免疫荧光定位分析发现,Tcd1S1D点状定位于细胞质中,Tcd1S2D在有性生殖初期点状定位于细胞质中,在新大核上形成均匀的定位,Tcd1S3D无法定位于亲本大核上,只是均匀定位于新大核上。Tcd1S2A和Tcd1S3A在新大核形成异常的块状定位,并且与异染色质蛋白Pdd1不能共定位。结果表明,Tcd1不同位点的磷酸化和去磷酸化修饰的动态变化决定了其在四膜虫细胞中的定位模式。