Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La. Another possible mechanism for estradiol influence of lupus erythematosus

A strong association between anti-SS-A/Ro and anti-SS-B/La antibodies and skin lesions has been well documented in subacute cutaneous lupus erythematosus and neonatal lupus erythematosis in which 70 to 80% of patients are female. In order to better understand the mechanisms of the influence of sex hormones on cutaneous lupus, we designed immunopathological in vitro experiments to evaluate the effects of estradiol and other sex steroids on the binding of SS-A/Ro- and SS-B/La-specific antibodies to cultured human keratinocytes from neonates. Cultured human keratinocytes incubated with antisera specific for SS-A/Ro or SS-B/La Ag were fixed with either acetone or paraformaldehyde and then analyzed in indirect immunofluorescent assays or by FACS analysis to detect cell surface IgG binding as an indirect measure of SS-A/Ro and SS-B/La Ag expression on the cell surface of keratinocytes. Estradiol (10(-5) to 10(-7) M) augmented binding of antiserum probes on the surface of cultured keratinocytes, with 10(-7) M estradiol showing the highest induction of cell surface binding of antisera specific for SS-A/Ro plus SS-B/La Ag (24.5% of cells were positive). In contrast, dihydrotestosterone, testosterone, and progesterone showed no augmentation. The augmentation by estradiol was partially inhibited by the antiestrogen nafoxidine. Estradiol augmented the relative incidence and absolute number of small or cuboidal cells binding antibodies specific for SS-A/Ro and SS-B/La Ag, whereas the number and incidence of larger differentiated cells binding anti-SS-A/Ro and anti-SS-B/La decreased significantly in cell cultures stimulated with estradiol. Flow cytometric analysis utilizing monospecific anti-SS-A/Ro or anti-SS-B/La sera showed that estradiol induced binding of anti-SS-A/Ro in 13.1% of cultured keratinocytes, of anti-SS-A/La in 14.4%, and of sera specific for both Ag in 21.4%. This direct association between estradiol and the augmentation of binding to the cell surface of human keratinocytes of IgG from antisera specific for SS-A/Ro and SS-B/La Ag may be a trigger factor of immunologic damage in lupus and may be important in the different sex rates observed in skin manifestation of subacute cutaneous and neonatal lupus erythematosis.     

Structural characterization of lipid A component of Erwinia carotovora lipopolysaccharide

The chemical structure of the lipid A component of lipopolysaccharide excreted into the liquid medium by the plant pathogenic enterobacterium Erwinia carotovora FERM P-7576 was characterized. It consists of a -1, 6-linked glucosamine disaccharide which carries ester-and amide-bound fatty acids and phosphate similar to the lipid A from other gram-negative bacteria. The lipid A preparation was not uniform in the number and composition of the fatty acids linked to the disaccharide. Four prominent lipids A were involved, they were composed of five to seven residues of fatty acid. Among them the major component was hexa-acyl lipid A, in which the hydroxyl group at position 3 and the amino group of the non-reducing glucosamine unit carry 3-dodecanoyl-oxytetradecanoyl residues. Positions 2 and 3 of the reducing glucosamine unit were substituted by 3-hydroxytetradecanoic acid. In the hepta-acyl lipid A, an additional hexadecanoic acid was linked to the hydroxyl group of the 3-hydroxytetradecanoyl residue at position 2 of the hexa-acyl lipid A. Two penta-acyl lipids A were the homologs of the hexa-acyl lipid A with decreasing acylation. Dodecanoic acid was missing from one, and 3-hydroxytetradecanoic acid from another. 3-Dodecanoyloxytetradecanoyl residue at position 3 differentiates E. carotovora lipid A from that of other gram-negative bacteria.Abbreviations LPS lipopolysaccharide - GlcN glucosamine - KDO 3-deoxy-d-manno-octulosonic acid - FAB-MS fast atom bombardment mass spectrometry - u atomic mass unit     

Germination ecology of coastal plants in relation to salt environment

Responses of seed germination to salinity were examined using 37 species collected from salt marshes, cliffs, and fore (unstable) and hind (stable) sand dunes along Japanese coasts. For comparison, seed germination of nine inland species was also examined. The soil salinities in salt marshes ranged from 150 to 300 mmol/L NaCl, whereas those in fore and hind dunes ranged from 0 to 150 mmol/L NaCl, with a few exceptions. Cliff soils showed relatively high salinities up to 300 mmol/L NaCl. Ciff and foredune soils that encountered a typhoon and storm showed high salinities >300 mmol/L NaCl. Salt tolerance in seed germination of coastal plants was ordered by comparing the responses of percentage and rate of germination to salinity conditions up to 200 mmol/L NaCl, being in the order of salt marsh>cliff>foredune≅hind dune≅inland. Thse results indicate that salt tolerance in seed germination of coastal plants is closely related to the salinity conditions of their habitats. Germination experiments under favorable conditions showed that a high percentage of the seeds of salt marsh species germinate rapidly, those of diff species germinate slowly and those of foredune species exhibit a low percentage and low rate of germination. It seems that these germination characteristics contribute to the success of germination at the ‘safe site’ and the subsequent survivorship of emerged plants in their natural habitats.     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.     

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice ( Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester-linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate-limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Effect of Xyloglucan Nonasaccharide on Cell Elongation Induced by 2,4-Dichlorophenoxyacetic Acid and Indole-3-acetic Acid

Xyloglucan nonasaccharide (XG9) is recognized as an inhibitorof 2,4-D-induced long-term growth of segments of pea stems.In the presence of 10^–5 M 2,4-D, inhibition by 10^–9M XG9 of elongation of third internode segments of pea seedlingswas detected within 2 h after the start of incubation, in someexperiments. Analysis by double-reciprocal (Lineweaver-Burk)plots of elongation in the presence of various concentrationsof 2,4-D, with or without XG9, gave parallel lines, indicatingthat XG9 inhibited 2,4-D-induced elongation in an uncompetitivemanner. XG9 did not influence the 2,4-D-induced cell wall loosening.Thus, XG9 does not fulfill the proposed definition of an "antiauxin". XG9 at 10^–11 to 10^–6 M did not influence IAA-inducedelongation of segments from pea third internodes, azuki beanepicotyls, cucumber hypocotyls, or oat coleoptiles. Inhibitionof IAA-induced elongation by XG9 was not observed even whenthe segments from pea or azuki bean were abraded. Furthermore,fucosyl-lactose at 10^–11 to 10^–4 M did not affectthe IAA-induced elongation of segments of pea internodes orof azuki bean epicotyls. XG9 may be incapable of inhibitingthe IAA-induced cell elongation (especially in oat) or, alternatively,the endogenous levels of XG9 may be so high that exogenouslyapplied XG9 has no inhibitory effect on IAA-induced elongation. (Received February 28, 1991; Accepted May 25, 1991)     

Protein glycosylation.

Glycan structures can modulate the biological properties and functions of glycoproteins. This has been shown by investigation of the biological activities and glycan structures of several recombinant glycoproteins. Glycan structures of glycoproteins differ according to the species and tissue producing them, and selection of an appropriate host-cell type can generate recombinant glycoproteins with new characteristics.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.