Culture condition ofPseudomonas aeruginosa F722 for biosurfactant production

Pseudomonas aeruginosa F722 produces a biosurfactant (BS) during its degradation of carbon and hydrocarbon compounds. The culture conditions for upgrading the biosurfactant productivity were investigated. The concentration of the biosurfactant produced byP. aeruginosa F722 was 0.78 g/L in C-medium; however, this increased to 1.66 g/L in BS medium, which was experimentally adjusted to optimal conditions. NaNO₂ was found to be most effective for microbial growth, with an O.D_600nm of 1.18 for 0.1% NaNO₂. Microbial growths, according to the O.D_600nm were 2.53, 2.68, 2.89, and 2.87 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Clear zone diameters (cm), indicating biosurfactant activity, were 9.0, 8.8, 5.7, and 8.5 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Microbial growth was not consistent with the biosurfactant activity. The best biosurfactant activity was found with a C/N ratio of 20. Under optimal culture condition, the average surface tension decreased from 70 to 30 mN/m after 5 days. With aeration of 1.0 vvm, the biosurfactant produced increased to 1.94 g/L (up to 20%) compared to that of 1.66 g/L with no aeration. With aeration, the velocities of glucose degradation during both the log and stationary growth phases increased from 0.25 and 0.18 h⁻¹ to 0.33 and 0.29 h⁻¹, respectively, and the time for the culture to arrive at the maximum clear zone diameter became shorter, from 80 down to 60 h with no aeration.     

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.     

The throat flora and its mitogenic activity in patients with Kawasaki disease

The etiology of Kawasaki disease (KD) remains unknown, although some infectious organism has been suggested as the cause. Recent studies suggest that some bacterial toxins with superantigen activity are involved in its pathogenesis, but no specific bacterial toxin has yet been identified. Throat swabs for bacterial culture were obtained from 21 patients with KD and 20 with other febrile illnesses as controls. Mitogenic activity in culture supernatants obtained from individual bacterial strains was measured by lymphocyte proliferation assay. Sixty-one bacterial strains were isolated from KD patients, and 62 strains from control patients. There was no apparent difference in bacterial species in the throat flora between KD patients and febrile controls. Moreover, total and individual mitogenic activity of strains from KD patients was no greater than that of strains from febrile controls. The bacterial superantigen activity of throat flora may not play a major role in the pathogenesis of KD.     

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time-dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser-15 and Ser-85 in smooth muscle of aorta in vivo. The AVP-induced phosphorylation of HSP27 at Ser-15 and Ser-85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser-15 and Ser-85. The AVP-induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen-activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of gibberellins in Arabidopsis

Gibberellins (GAs) are diterpene plant hormones essential for many developmental processes. Although the GA biosynthesis pathway has been well studied, our knowledge on its early stage is still limited. There are two possible routes for the biosynthesis of isoprenoids leading to GAs, the mevalonate (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in plastids. To distinguish these possibilities, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C-labeling was achieved by blocking the endogenous pathway chemically or genetically during the feed of a (13)C-labeled precursor specific to the MVA or MEP pathways. Gas chromatography-mass spectrometry analyses demonstrated that both MVA and MEP pathways can contribute to the biosyntheses of GAs and campesterol, a cytosolic sterol, in Arabidopsis seedlings. While GAs are predominantly synthesized through the MEP pathway, the MVA pathway plays a major role in the biosynthesis of campesterol. Consistent with some crossover between the two pathways, phenotypic defects caused by the block of the MVA and MEP pathways were partially rescued by exogenous application of the MEP and MVA precursors, respectively. We also provide evidence to suggest that the MVA pathway still contributes to GA biosynthesis when this pathway is limiting.     

Role of CD13/aminopeptidase N in rat lymphocytic alveolitis caused by thoracic irradiation

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.     

Mechanomyogram from the different heads of the quadriceps muscle during incremental knee extension

To investigate the time- and frequency-domain responses of mechanomyograms (MMGs) during the progressive fatigue induced by intermittent incremental contractions, a surface MMG was obtained from the three muscle heads of the quadriceps muscle in seven subjects while they performed isometric knee extensions lasting 7.6 min. Isometric intermittent incremental contractions started at 1% of the maximal voluntary contraction (MVC) for 3 s, with a 3-s relaxation period in between each contraction, and the contraction level was increased by 1% of MVC for every contraction (by 10% of MVC per min) up to exhaustion. Separate contractions with sufficient rest periods were also conducted to serve for the MMG characteristics without fatigue. The integrated MMG (iMMG) was linearly related to force in all of the muscles when fatigue was not involved. With regard to the incremental contractions, the relationship exhibited an ascending-descending shape, but the behavior was not the same for the individual muscle heads, especially for the rectus femoris muscle. A steep increase in the median frequency of MMG from around 60% of MVC corresponded to a decrease in iMMG. These results suggest that analysis of MMG in the time- and frequency-domain during an incremental protocol is a useful way of characterizing the motor unit recruitment strategy and fatigue properties of individual muscles. Accepted: 19 March 1998     

Mechanism of Inhibition of Cytoplasmic Streaming by Auxin in Root Hair Cells of Hydrocharis

It has been reported that auxin accelerates cytoplasmic streamingat low concentrations and inhibits it at high concentrationsin several plant cells. In the present study, the mechanismof inhibition of cytoplasmic streaming by naphthalene aceticacid (NAA) at high concentrations was analyzed in root haircells of Hydrocharis. Because the effective concentration ofNAA inhibiting cytoplasmic streaming decreased when the extracellularpH (pHo) was lowered, it was hypothesized that cytoplasmic streamingis inhibited by NAA via acidification of the cytoplasm. Thispossibility was supported by the fact that acetic acid, pro-pionicacid and decanoic acid also inhibited cytoplasmic streamingat low pHo. Acidification of the cytoplasm disturbed the orientationof actin filaments (AFs) and disrupted cortical microtubules(MTs). The effects of NAA were reversible; both cytoplasmicstreaming and organization of the cytoskeleton were recoveredupon removal of NAA. During the recovery, tracks of cytoplasmicstreaming in the subcortical region temporarily showed a helicalpattern along the longitudinal direction of the cell. Fluorescencestaining of cytoskeletons revealed that both AFs and MTs alignedobliquely to the longitudinal axis of the cell. The helicalstreaming returned to the original reverse fountain streamingafter several hours. The simultaneous changes in the organizationof both cytoskeletons supported our previous report that theorganization of AFs is regulated by MTs. ¹Author for correspondence. Fax, 81-7915-8-0175. e-mail: tomy-@sci.himeji-tech.ac.jp     

Role of isozyme group-specific sequence 4 in the isozyme-specific properties of human aldolase C

To assess which regions of the aldolase C molecule are required for exhibiting isozyme-specific kinetic properties, we have constructed nine chimeric enzymes of human aldolases A and C. Kinetic studies of these chimeric enzymes revealed that aldolase C absolutely required its own isozyme group-specific sequences (IGS), particularly IGS-4, for exhibiting the characteristics of aldolase C which differ significantly from those of isozymes A and B (Kusakabe T, Motoki K, Hori K. Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli. J Biochem (Tokyo) 1994;115:1172–7). Whereas human aldolases A and B required their own isozyme group-specific sequences-1 and -4 (IGS-1 and -4) as the main determinants of isozyme-specific kinetic properties (Motoki K, Kitajima Y, Hori K. Isozyme-specific modules on human aldolase A molecule. J Biol Chem 1993;268:1677–83; Kusakabe T, Motoki K, Sugimoto Y, Takasaki Y, Hori K. Human aldolase B: liver-specific properties of the isoenzyme depend on type B isozyme group-specific sequence. Prot. Eng. 1994;7:1387–93), the present studies indicate that the IGS-1 is principally substitutable between aldolases A and C. The kinetic data also suggests that the connector-2 (amino acid residues 243–306) may modulate the interaction of IGS units with the α/β barrel of the aldolase molecule.     

Optical resolution by preferential crystallization of (2RS,3SR)-2-amino-3-chlorobutanoic acid hydrochloride

(2RS,3SR)-2-Amino-3-chlorobutanoic acid hydrochloride [(2RS,3SR)-ACB · HCl] was found to exist as a conglomerate based on the melting point, infrared spectrum, and solubility. Optical resolution by preferential crystallization of (2RS,3SR)-ACB · HCl was achieved to yield both (2R,3S)- and (2S,3R)-ACB · HCl of 80–100% optical purities. The obtained (2R,3S)- and (2S,3R)-ACB · HCl were recrystallized, taking into account the solubility of (2RS,3SR)-ACB · HCl, to give efficiently optically pure (2R,3S)- and (2S,3R)-ACB · HCl. Treatment of the purified (2R,3S)- and (2S,3R)-ACB · HCl with triethylamine gave optically pure (2R,3S)- and (2S,3R)-2-amino-3-chlorobutanoic acid, respectively. Chirality 9:656–660, 1997. © 1997 Wiley-Liss, Inc.