Active NDH-1 complexes from the cyanobacterium Synechocystis sp. strain PCC 6803

We identified eight bands by staining native gels for NADPH-nitrobluetetrazolium oxidoreductase activity after electrophoresis ofn-dodecyl-ß-D-maltoside-treated membranes of Synechocystissp. strain PCC 6803. Among them, bands A, C, D and E were attributedto the activity of NADPH dehydrogenase (NDH-1). Band A is ahighly active supercomplex of NDH-1 (about 1,000 kDa) that wasabsent in the     

Polylactosamines are not obligate receptors for invasion of Plasmodium falciparum malaria as shown in HEMPAS Variant II-gal- erythrocytes

A HEMPAS (hereditary erythroblastic multinuclearity with positiveacidified serum test) erythrocyte, atypical Variant II (referredto herein as Variant II-gal^-), lacking long-chain polylactosamineon both glycoproteins (Band 3 and 4.5) and glycosphingolipids,was characterized by the carbohydrate profile of the erythrocytemembrane according to Fukuda et al. (Blood, 73, 1331–1339,1989). Two laboratories previously reported that polylactosamineisolated from the erythrocyte protein Band 3 inhibited invasionof red blood cells by Plasmodium falciparum in malarial culture,suggesting a role for this carbohydrate in adhesion of the parasite.Therefore, HEMPAS erythrocyte Variant II-gal^- presented a uniqueopportunity to further examine this premise. Freshly drawn bloodsamples (normal and HEMPAS Variant II-gal^-) were separatelyincubated with P.falciparum from mannitol-synchronized cultures.The parasite was found to invade HEMPAS Variant II-gal^- erythrocytesat a 30% lower rate through two life cycles, as shown by microscopicevaluation of invasion and by [³H]hypoxanthine incorporationinto parasite. This observation, along with the published factthat glycophorin-deficient M^kM^k cells are also infectable, butat a lower rate, indicates that neither sialoglycoproteins norpolylactosamines are an obligate adhesive ligand for P.falciparum,although the possibility remains that either may still contributeto adhesive events during infection. HEMPAS malaria Plasmodium falciparum polylactosamine     

MICROGROWTH BANDS IN CHITONS: EVIDENCE OF TIDAL PERIODICITY

Microgrowth bands of typical molluscan appearance were clearlyrevealed by the acetate film technique in the sutural laminaeof the large chitons, Amaurochiton glaucus, Sypharochiton pelliserpentis,Cryptoconchus porosus and Acanthopleura granulata. In all speciesthere was a cyclical pattern, averaging close to 28 bands percycle suggesting that one growth increment was laid down pertide. Band widths were unusually small compared with those ofbivalves or gastropods, probably because the shell is dividedinto eight growing plates. Overall rates of growth calculatedfrom band widths were 1–2 mm per month, comparable withfield estimates. (Received 13 February 1984;     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

Changes in levels of Proteases and Their Inhibitors during Development of the Seeds of Job's Tears (Coix lacryma-jobi L. var. Ma-yuen Stapf) and Their Localization and Interactions

Changes in levels and localization of proteases and a trypsininhibitor (JBTI) in the developing seeds of Job's tears (Coixlacryma-jobi L. var. Ma-yuen Stapf) were followed, and theirinteractions were examined. The JBTI was induced from the middlestage of development (12 DAF, i.e., 12 days after flowering)and increased until the late stage of development (24 DAF),and was localized in the germ. Two groups of proteases (A, thosewith molecular weights of 55–70 kDa; and B, those withmolecular weights greater than 94 kDa) were detected by activestaining of substrate-containing SDS-polyacrylamide gels. Thegroup of larger proteases seems to consist of three members(B₁, B₂₎ B₃, with increasing molecular weights). Band A wasobserved only in the early stage of development (until DAF 9),band B₂ persisted during all stages, while bands B, and B₃ werepresent only during early and late stages, respectively. Thegroup A proteases and one of the group B proteases (probablyB₁) were inhibited by diisopropyl-fluorophosphate (DFP), bytrypsin inhibitors from soybean and rice, and by JBTI. The proteasesthat were present in the seeds of Job's tears at a late stageseemed to be localized in the germ. (Received September 9, 1988; Accepted April 19, 1989)     

Rodents infected with Schistosoma mansoni produce cytolytic IgG and IgM antibodies to the Lewis x antigen

Schistosoma mansoni is a blood fluke that produces glyco-conjugatescontaining the Lewis x antigen (Le^x) Galß1     

Differential expression of an endogenous mannose-binding protein R1 during muscle development and regeneration delineating its role in myoblast fusion

The role of the endogenous brain carbohydrate-binding proteinR1 in muscle cell development and regeneration was analysedboth in vivo and in vitro. In vivo, R1 was developmentally regulated,with an embryonic 65 000 subunit and a neonatal 67 000 subunit,being replaced progressively by a 135 000 adult form. LectinR1 was intracellularly localized at birth and in the prenatalperiod. During development and at the time of myoblast fusion,the antigen was progressively found at the surface, where itremained at low levels in the adult. In vitro, in pure myoblastcultures, only the embryonic form was present. The ultrastructuralstudies indicated that the lectin could participate in the membranefusion process during myoblast fusion. The specific role inmyoblast fusion, derived from the ultrastructural localizationof R1, was evidenced by a strong inhibitory effect of anti-R1 Fab fragments (10–100 µg/ml), relative to controlFab fragments. In vivo, the embryonic subunit pattern and subcellulardistribution of R1 reappeared in muscle cells after lesion ofthe adult muscle. This suggested that, as observed in vitro,R1 participated in vivo in the phenomenon of myoblast fusion.Similar modifications in subunit expression were observed inmuscles after denemation (the embryonic form of lectin R1 reappearingafter lesion), suggesting that R1 could be involved in the processof neuromuscular junction formation. Thus, it is proposed thatthe carbohydrate-binding protein R1 is an important recognitionmolecule for the formation of myotubes. Its potential involvementin a recognition process between axons and muscle cells duringneuromuscular junction formation is discussed. culture development fusion N-glycan glycoprotein lectin mannose myoblast     

N-Acetylglucosamine-binding proteins on Plasmodium falciparum merozoite surface

Plasmodium falciparum merozoite surface is specifically labelledwith a neoglycoprotein bearing N-acetylgluco-samine (GlcNAc)residues in a sugar-dependent manner, as shown by affinity cytochemistryin fluorescence and electron microscopy. To ascertain the natureof the sugar receptor, merozoite proteins were blotted and testedby a two-step method using biotinylated GlcNAc—bovineserum albumin (BSA) and streptavidin—peroxidase conjugate.Three parasite proteins were specifically revealed and designatedas Pf 120, Pf 83 and Pf 45 GlcNAc-binding proteins. These proteinsbind to a gel substituted with GlcNAc and are specifically elutedwith 300 mM GlcNAc. Using a rabbit antiserum raised againstPf 83, the Pf 120 GlcNAc-binding protein, in addition to Pf83, was labelled by Western blotting. Comparative analyses withan antibody against the Pf 83 MSP derived from the P.falciparummerozoite surface protein (Pf MSP) indicated that the Pf 83GlcNAc-binding protein is not related to the fragment of thePf MSP antigen. Similarly, the Pf 83 GlcNAc-binding proteinis not related to the apical membrane antigen 1 (AMA 1) whichalso has the same molecular mass. Therefore the Pf 120, Pf 83and Pf 45 GlcNAc-binding proteins which are located on the merozoitesurface and recognize GlcNAc residues could be involved in thebinding of merozoites to the glycoconjugates of the surfaceof the red blood cells. GlcNAc lectin neoglycoprotein Plasmodium falciparum red blood cell     

Effect of endurance training and seasonal fluctuation on coagulation and fibrinolysis in young sedentary men

Van den Burg, P. J. M., J. E. H. Hospers, M. Van Vliet, W. L. Mosterd, B. N. Bouma, and I. A. Huisveld. Effect of endurance training and seasonal fluctuation on coagulation and fibrinolysis inyoung sedentary men. J. Appl. Physiol.82(2): 613-620, 1997.The effect of 12 wk of submaximal trainingon hemostatic variables was studied in 20 young sedentary men (Tr) and19 nontraining matched controls (Con). After training, a morepronounced increase in factor VIII coagulant activity(P < 0.01), reflected in a decrease in activated partial thromboplastin time(P < 0.01) during maximal exercise,was seen. Both basal plasminogen activator inhibitor 1 antigen (PAI-1Ag) and activity (PAI-1 Act; P < 0.05), as well as basal and exercise-induced tissue-type plasminogenactivator antigen (t-PA Ag; P < 0.05), were decreased after training. The overall effect onfibrinolysis was reflected in an increase in the t-PA Act/t-PA Ag ratioin the Tr group. In contrast, during the same period (February-June),the Con group demonstrated an increase in basal PAI-1 Ag and PAI-1 Act(P < 0.05), together with anincrease in basal and exercise-induced t-PA Ag(P < 0.05). Both basal andexercise-induced t-PA Act were unchanged, but t-PA Act/t-PA Ag wasdecreased (P < 0.05) in the Congroup. We conclude that physical training promotes both coagulation andfibrinolytic potential during exercise and may reverse unfavorableseasonal effects on fibrinolysis.

     

Association between expression of the H histo-blood group antigen, alpha1,2fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus

RHDV (rabbit hemorrhagic disease virus) is a highly virulentcalicivirus that has become a major cause of mortality in wildrabbit populations (Oryctolagus cuniculus). It binds to thehisto-blood group antigen (HBGA) H type 2 which requires an1,2fucosyltransferase for its synthesis. In rabbit, three 1,2fucosyltransferasesgenes are known, Fut1, Fut2, and Sec1. Nonfunctional allelesat any of these loci could potentially confer resistance toRHDV, similar to human FUT2 alleles that determine the nonsecretorphenotype and resistance to infection by various NoV strains.In this study, we looked for the presence of H type 2 on buccalepithelial cells of wild rabbits from two geographic areas underRHDV pressure and from one RHDV-free area. Some animals withdiminished H type 2 expression were found in the three populations(nonsecretor-like phenotype). Their frequency markedly increasedaccording to the RHDV impact, suggesting that outbreaks selectedsurvivors with low expression of the virus ligand. Polymorphismsof the Fut1, Fut2, and Sec1 coding regions were determined amonganimals that either died or survived outbreaks. The Fut2 andSec1 genes presented a high polymorphism and the frequency ofone Sec1 allele was significantly elevated, over 6-fold, amongsurvivors. Sec1 enzyme variants showed either moderate, low,or undetectable catalytic activity, whereas all variant Fut2enzymes showed strong catalytic activity. This functional analysisof the enzymes encoded by each Fut2 and Sec1 allele suggeststhat the association between one Sec1 allele and survival mightbe explained by a deficit of 1,2fucosyltransferase expressionrather than by impaired catalytic activity.     

Inheritance of two endosperm protein loci in rice (Oryza sativa L.)

Summary Previous studies indicated two types of phenotypic protein markers as two minor bands of SDS-PAGE for rice storage protein. A variant derived from a Pakistani variety, Dular, was found to show a mobility variant with Band 11, a relatively faster-moving band as compared to Band 10, while most of the other cultivated rices exhibited Band 10 at a molecular weight of around 100–110 K. Band 11 was also observed in several wild rice species. How this variant occurred is not known. Another marker is characterized by the presence of either Band 56 (slower-migrating band) or Band 57 (faster-migrating band) in most cultivars at a molecular weight of about 28–27 K. Most indica varieties developed in Taiwan have Band 57 and japonica varieties have Band 56. Genetic analysis of F₁, F₂ and F₃ seeds from interstrain crosses indicated that Band 10 versus Band 11 and Band 56 versus Band 57 are due to codominant alleles at two loci. Tests of independent inheritance between these two loci (Band 10/11 versus Band 56/57) indicated that there is no linkage between them. Both of these two protein loci encode for endosperm proteins and mostly belong to the minor polypeptide subunits of the glutelin fraction of rice seed proteins. Studies on reciprocal crosses indicate dosage effects as exhibited in band patterns. Variations in band intensity were frequently observed when the maternal genotype was different.     

Antigenicity,storage, and aging: physiologic autoantibodies to cell membrane and serum proteins and the senescent cell antigen

Summary Normal human serum contains autoantibodies to a wide range of cellular and serum proteins. IgG autoantibodies to cell membrane proteins spectrin, syndein (Band 2.1), Band 3 degradation products, and the senescent cell antigen are among them. Physiologic autoantibodies to the senescent cell antigen, a 62 000 dalton glycopeptide derived from Band 3, initiate removal of senescent, damaged, and stored cells in vivo. The senescent cell antigen is one of the two Band 3 degradation products (Mr 66 000 and 62 000) observed in freshly prepared ghosts.Since the senescent cell antigen is observed on red cells aged in situ, data suggest that Band 3 undergoes proteolysis in situ. IgG eluted from blood stored for transfusion binds to the senescent cell antigen. The amount of IgG on red cells increases during storage suggesting accumulation of the senescent cell antigen. Autoantibodies to other cell and serum proteins are discussed as possible regulators of homeostasis. The effect of age on physiologic autoantibodies is reviewed.     

Antigenic determinants of senescent antigen of human erythrocytes are located in sialylated carbohydrate chains of Band 3 glycoprotein.

Naturally occurring IgG autoantibody against Band 3 glycoprotein of human erythrocyte membrane (anti-Band 3) recognizes the erythrocytes modified with oxidizing or SH-blocking agents as well as senescent erythrocytes. Location of the antigenic determinants of Band 3 this autoantibody recognizes was investigated by competitive inhibition studies of the antibody binding to the modified cells. Autologous IgG binds to the modified erythrocytes, and purified Band 3 totally inhibits the binding. This inhibitory activity of Band 3 was not affected by digestion of Band 3 with various proteases. Treatment of Band 3 with endo-beta-galactosidase that destroys the poly-N-acetyllactosaminyl sugar chain of Band 3 or with neuraminidase resulted in loss of the inhibitory activity. Oligosaccharides released from Band 3 by hydrazinolysis effectively inhibited the binding of autologous IgG and 125I-labeled purified anti-Band 3 to the modified cells, whereas the oligosaccharides depleted of acidic components did not. Endo-beta-galactosidase and neuraminidase destroyed the activity of the oligosaccharides, but alpha-L-fucosidase did not. Furthermore, human lactoferrin that contains sialylated two N-acetyllactosaminyl units also exhibited potent inhibitory activity, and the activity was destroyed by endo-beta-galactosidase and neuraminidase. These results indicate that the antigenic determinants of Band 3 are located in sialylated poly-N-acetyllactosaminyl sugar chains. Based on this finding, mechanism of appearance of the antigen on senescent erythrocytes recognized by anti-Band 3 (senescent antigen) was discussed.     

Ultrastructural localization of a diprenylated isoflavone in Rhizobium lupini-Lupinus albus symbiotic association

Polyclonal IgGs raised against the diprenylated isoflavone,2'-hydroxylupalbigenin were used in an immunocytochemical studyof the root and nodule tissues of white lupin (Lupinus albusL.). In the roots, the antigen was detected in the secondarywalls of both xylem vessels and pericycle cells. Examinationof nodules revealed the presence of the antigen in the innercortex, associated with a neoparietal material which is depositedas globules on the cell wall and occluding the intercellularspaces. The discrete location of the diprenylated isoflavonein specialized cytoplasmic organelles suggests that its compartmentationwithin the wall is mediated by membrane vesicles. In the infectedcells, more than half of the bacteroids exhibited a specificlabelling of their inner core associated with a central fibrillarsystem, and sometimes surrounding an electron-dense nucleoidregion. These results suggest that 2'-hydroxylupalbigenin, andpossibly other prenylated isoflavones, may play an importantrole as a biochemical factor in early symbiotic events otherthan nod gene induction or inhibition. Its putative biologicalfunctions in the post-infectional phases of symbiosis are discussed. Key words: Bacteroid, 2'-hydroxylupalbigenin, in situ localization, Lupinus albus, nodules     

华硬蜱和二棘血蜱的交叉免疫反应

本文首次比较了经中华硬蜱(Ixodes sinensis)叮咬三次后再经二棘血蜱(Haimaphysalis bispinosa)叮咬的家兔与仅经二棘血蜱叮咬的家兔的交叉免疫抗性。二棘血蜱叮咬被中华硬蜱致敏的家兔时,吸血增重为：143.12±32.67mg,但二棘血蜱在正常家兔体上寄生,初次吸血增重为：181.30±44.35mg,两者之间有显著性差异(P<0.01)。中华硬蜱和二棘血蜱唾液腺提取物(SGE)经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),显示两者分别有24条和22条电泳带,中华硬蜱主带有6条,分子量分别为142、105、94、66/65、64和56kD,而二棘血蜱主带有5条,分子量分别为：215、114、105、66/65和58kn,经中华硬蜱叮咬致敏的家兔血清和经二棘血蜱叫‘咬致敏的家兔血清作免疫印渍,均显示出105kD这一电泳带。该实验表明中华硬蜱和二棘血蜱叮咬家兔两者之间存在着交叉免疫反应,提示105kD蛋白质抗原可能是两者的共同抗原。     

Conformational conversion of PDE5 by incubation with sildenafil or metal ion is accompanied by stimulation of allosteric cGMP binding

Phosphodiesterase-5 (PDE5) is a dimer containing a cGMP-specific catalytic domain and an allosteric cGMP-binding subdomain (GAF A) on each subunit. PDE5 exhibits three conformational forms that can be separated by Native PAGE and are denoted as Bands 1, 2, and 3 in decreasing order of mobility. A preparation comprised mainly of Band 2 PDE5 was partially converted to Band 3 PDE5 by 1 h incubation with cGMP or the PDE5-specific inhibitors sildenafil, vardenafil, or tadalafil, but not with cAMP, milrinone (PDE3-specific), or rolipram (PDE4-specific). Band 2 PDE5 was converted almost entirely to Band 3 PDE5 by overnight incubation with sildenafil at 30 °C. This time-dependent conversion was accompanied by a 7-fold increase in allosteric cGMP-binding activity, suggesting that Band 3 PDE5 is a much more active form than Band 2 PDE5 for allosteric cGMP binding. Conversion of Band 2 PDE5 to Band 3 PDE5 occurred faster by pre-incubation with cGMP, which binds to both the allosteric and catalytic sites of PDE5, than with catalytic site-specific sildenafil. Overnight incubation of a Band 2/Band 3 PDE5 mixture with EDTA caused time-dependent conversion to Band 1 PDE5 (apoenzyme), and this conversion was accompanied by a 50% loss in cGMP-binding activity. After incubation with EDTA, addition of Mn⁺⁺ or Mg⁺⁺ caused reversion of Band 1 to a Band 2/Band 3 PDE5 mixture in which Band 3 PDE5 predominated. This reversion was accompanied by a 3-fold increase in allosteric cGMP-binding activity. The combination of results implied that physiological conversion of Band 2 to Band 3 PDE5 by cGMP and/or divalent metal ion occupancy of the catalytic domain would increase allosteric cGMP binding to the enzyme. This conversion would produce a greater negative feedback effect on cGMP action by increasing sequestration of cGMP at the allosteric cGMP-binding site of PDE5 and by increasing cGMP degradation at the catalytic site of the enzyme. This conversion would also increase PDE5 inhibitor binding to the enzyme.     

Studies on the Host-Specific Pathotoxins Produced by Helminthosporium maydis,race T

Two host-specific pathotoxins, Band 1- and Band 2-toxins, were isolated from the toxin complex obtained from the culture broth and mycelial mat of Helminthosporium maydis, race T, the fungus causing Southern corn blight disease. Chemical and spectrometric studies showed them to be polyketo-polyhydroxy compounds with C₄₁ carbon chains. Band 2-toxin is identical to Band 1-toxin except that Band 2 toxin has an additional hydroxyl group in place of one of the keto-group of Band 1-toxin.