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     

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.     

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)     

The effect of ovarian steroidogenesis on ovulation and fertilizability in the in vitro perfused rabbit ovary

The effects of aminoglutethimide phosphate (AGP) on ovulation, ovum maturation, fertilizability, and steroid production were studied with the use of an isolated perfused rabbit ovary preparation. AGP (10(-3) or 10(-4) M) was added to the perfusate of one ovary. The contralateral control ovary was perfused in medium alone. Thirty minutes later human chorionic gonadotropin (hCG) (50 IU) was added to the perfusate of all ovaries. No difference was observed in time of ovulation or ovulatory efficiency between controls and AGP-treated ovaries. The degree of ovum maturity and degeneration was also comparable in the two groups. Progesterone and estradiol production were significantly reduced by AGP treatment. A second experiment examined fertilizability of ova ovulated in vitro after perfusion with 10(-3) M AGP. AGP significantly reduced the rate of normal fertilization as observed 12 h after insemination. The percentage of inseminated ova with evidence of degeneration was greater in ova from AGP-treated ovaries than in those from controls, however, this difference was not significant. The study indicates that AGP affects neither hCG-induced ovulation nor meiotic resumption; however, fertilizability of ova from ovaries treated with AGP is impaired. These data suggest that the intrafollicular steroid environment may participate in cytoplasmic maturation of ovulated ova.     

Studies on calmodulin-binding proteins (CaMBPs) in the cilia of Tetrahymena

As a first step to elucidate the involvement of calmodulin in Ca2+-dependent regulation of ciliary motility, molecular species and properties of calmodulin-binding proteins (CaMBPs) in Tetrahymena cilia were investigated by a modified [125I]calmodulin overlay method. At least 36 kinds of CaMBPs were detected. All the CaMBPs bound to calmodulin in Ca2+-dependent and calmodulin-specific manners, but they showed different Ca2+-dependencies. Several of CaMBPs bound to calmodulin in the presence of 100 microM trifluoperazine, several did in the presence of 8 M urea, and a few of them were highly sensitive to trypsin digestion. Among these CaMBPs, we noticed a 95 000-dalton (D) CaMBP present in the outerdoublet microtubule fraction, which possessed some attributes of the calmodulin counterpart suggested from the results of our previous paper [12]. We discussed a possibility that this protein might correspond to one of the protein components of the interdoublet link.     

Control of K+ conductance by cholecystokinin and Ca2+ in single pancreatic acinar cells studied by the patch-clamp technique

Summary The effect of cholecystokinin (CCK) and internal Ca²⁺ on outward K⁺ current in isolated pig pancreatic acinar cells has been investigated using the patch-clamp method for whole-cell current recording under voltage-clamp conditions. CCK (2 × 10^–10 M) applied to the bath evoked a marked increase in the outward K⁺ current associated with depolarizing voltage steps, and this effect was fully reversible and acutely dependent on the presence of external Ca²⁺. When strongly buffered Ca²⁺-EGTA solutions were used inside the cells CCK failed to evoke an effect. Increasing the internal Ca²⁺ concentration ([Ca²⁺] _i ) from 5 × 10^–10 M to 10^–7 and 5 × 10^–7 M mimicked the effect of CCK. It would appear therefore that CCK controls K⁺ conductance in the acinar cells via changes in the internal free ionized Ca²⁺ concentration.     

13C- and 1H-nmr evidence for all-cis peptide bonds in cyclic tetrapeptide cyclo(L-Pro-Sar)2

Conformations of the cyclic tetrapeptide cyclo(L -Pro-Sar)₂ in solution were studied by ¹H- and ¹³C-nmr spectrometry and model building. The nmr data provide definite evidence that this cyclic peptide exists chiefly in two conformations, namely, a C₂-symmetric conformation and an asymmetric structure. The former was demonstrated to be predominant in polar solvents (100% in Me₂SO-d₆). This structure contains all cis-peptide bond linkages and all trans′ Pro C^α?CO bonds. It represents the first cyclic tetrapeptide in which all four peptide bonds have been found in the cis-conformation. As the polarity of the solvent decreases, the population of C₂-symmetric conformers decreases (88% in CD₃CN and 65% in CDCl₃). At the same time, a minor asymmetric conformer, characterized by cis-cis-cis-trans peptide bond sequences (two cis Sar-Pro bonds, one cis Pro-Sar bond, and one trans Pro-Sar bond), is seen to increase (9% in CD₃CN and 30% in CDCl₃). A proposed predominant conformation in solution for cyclo(L -Pro-Sar)₂ was compared with a crystal structure, as reported in an accompanying paper. Both structures show striking overall similarities.