Cold adaptation of eicosapentaenoic acid-less mutant of Shewanella livingstonensis Ac10 involving uptake and remodeling of synthetic phospholipids containing various polyunsaturated fatty acids

An Antarctic psychrotrophic bacterium, Shewanella livingstonensis Ac10, produces cis-5,8,11,14,17-eicosapentaenoic acid (EPA), a long-chain polyunsaturated fatty acid (LPUFA), as a component of membrane phospholipids at low temperatures. The EPA-less mutant generated by disruption of the EPA synthesis gene becomes cold-sensitive. We studied whether the cold sensitivity could be suppressed by supplementation of various LPUFAs. The EPA-less mutant was cultured at 6°C in the presence of synthetic phosphatidylethanolamines (PEs) that contained oleic acid at the sn-1 position and various C20 fatty acids with different numbers of double bonds from zero to five or cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) at the sn-2 position. Mass spectrometric analyses revealed that all these fatty acids became components of various PE and phosphatidylglycerol species together with shorter partner fatty acids, indicating that large-scale remodeling followed the incorporation of synthetic PEs. As the number of double bonds in the sn-2 acyl chain decreased, the growth rate decreased and the cells became filamentous. The growth was restored to the wild-type level only when the medium was supplemented with phospholipids containing EPA or DHA. We found that about a half of DHA was converted into EPA. The results suggest that intact EPA is best required for cold adaptation of this bacterium.     

Anaerobic ammonium oxidation (anammox) irreversibly inhibited by methanol

Methanol inhibition of anaerobic ammonium oxidation (anammox) activity was characterized. An enrichment culture entrapped in a polyethylene glycol gel carrier was designed for practical uses of wastewater treatment. Batch experiments demonstrated that anammox activity decreased with increases in methanol concentration, and relative activity reached to 29% of the maximum when 5 mM methanol was added. Also, batch experiments were conducted using anammox sludge without immobilization. Anammox activity was evaluated by quantifying ¹⁴N¹⁵N (²⁹N) emission by combined gas chromatography-quadrupole mass spectrometry, and the anammox activity was found to be almost as sensitive to methanol as in the earlier trials in which gel carriers were used. These results indicated that methanol inhibition was less severe than previous studies. When methanol was added in the influent of continuous feeding system, relative activity was decreased to 46% after 80 h. Although the addition was halted, afterwards the anammox activity was not resumed in another 19 days of cultivation, suggesting that methanol inhibition to anammox activity was irreversible. It is notable that methanol inhibition was not observed if anammox activity was quiescent when substrate for anammox was not supplied. These results suggest that methanol itself is not inhibitory and may not directly inhibit the anammox activity.     

Effect of Heavy Metal Ions on the Growth and Iron-oxidizing Activity of Thiobacillus ferrooxidans

Effect of heavy metal ions on the growth and the iron-oxidizing activity of Thiobacillus ferrooxidans were investigated.

Cupric, zinc, cadmium, and chromium ions had no effect on the growth and the iron-oxidizing activity of cell suspensions or cell-free extracts of the bacterium in high concentrations (10^?3~10^?2M). Lead ion delayed the start of the growth slightly in 10^?3 M, but it did not inhibit the iron-oxidizing activity of the cells in the concentration. Tin and molybdenum oxide ions inhibited both of them in the concentration above 10^?3 M.

Mercuric mercurous, and silver ions had the most harmful effect. In the concentration of 10^?3 .M, each of the cations inhibited almost completely both the growth and the iron-oxidizing activity of the cells.

In the experiments with cell-free extracts it was observed that the activity of cytochrome oxidase (cytochrome a₅₉₇) operating in the iron-oxidizing system of the bacterium was specifically inhibited with mercuric ion in the concentration above 5 × 10^?4 M.     

Identification of the Female Sex Pheromone of the Cabbage Webworm

Gibberellins A₄ and A₃₆ were identified from flowering and vegetative apices of ten month-old sugarcane (Saccharum spp. hybrids) plants. The identifications were based on retention times, relative to authentic standards, on sequential silica gel partition column chromatography→bioassay→C₁₈ reverse phase high performance liquid chromatography→bioassay→ capillary gas chromatography (GC), and on GC-selected ion monitoring (SIM), the relative intensities of six characteristic ions being monitored in comparison with authentic standards.     

Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

Effect of Ca channel blockers on glycerol levels in Dunaliella tertiolecta under hypoosmotic stress

The role of Ca²⁺ in glycerol dissimilation under hypoosmotic stress in the halotolerant alga Dunaliella tertiolecta was investigated using a pharmacological approach. A stretch-activated Ca²⁺ channel blocker, GdCl₃, inhibited glycerol dissimilation under hypoosmotic stress. However, addition of voltage-dependent Ca²⁺ channel blockers and inhibitors of mitochondrial and endoplasmic reticulum Ca²⁺ channels did not affect the glycerol dissimilation under hypoosmotic stress. The results of the present study suggest that the influx of Ca²⁺ from the extracellular space via the stretch-activated Ca²⁺ channels localized in the plasma membrane is required for the transduction of osmotic signal of D. tertiolecta.     

Effect of ischemic preconditioning on cerebral blood flow after subsequent lethal ischemia in gerbils

Ischemic tolerance, the phenomenon where a sublethal ischemic preconditioning protects the brain against a subsequent lethal ischemia, has been widely studied. Studies have been done on cerebral blood flow levels prior to the lethal ischemia, but the hemodynamic pattern after global ischemia with ischemic preconditioning has not been reported. Sequential changes in regional cerebral blood flow (rCBF) in gerbil hippocampus after 5 min global ischemia with or without 2 min ischemic preconditioning were studied to determine if ischemic preconditioning affects rCBF. Four different treatments were given: (1) sham-operated, (2) 2 min ischemia, (3) non-preconditioned, and (4) preconditioned. Groups (1) and (2) (both groups n = 5) were given a 24-h recovery period and the rCBF was measured for baseline values. 24 h after sham-operation (3) and 2 min ischemia (4), gerbils were subjected to 5 min ischemia followed by 1 h, 6 h, 1-day or 7-day reperfusion periods (all groups n = 5). Although no regional difference was observed in the recovery pattern of rCBF, the values of rCBF were significantly higher in the preconditioned group throughout whole brain regions including hippocampus. These results indicate that ischemic preconditioning facilitated the recovery of rCBF after 5 min global ischemia. It needs further study to determine whether the protecting effects of preconditioning relate to the early recovery of rCBF or not. However, our results could be interpreted that the early recovery of rCBF may lead to benefits for cell survival in the CA1 neuron, probably facilitating other protecting mechanisms.     

Interactions of Synphilin-1 with phospholipids and lipid membranes

Synphilin-1 is an alpha-synuclein binding protein that is involved in the pathogenesis of Parkinson's disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610-640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson's disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function.     

Mechanical mutability in connective tissue of starfish body wall

Stiffness changes in response to mechanical and chemical stimulation were studied in muscle-free dermal samples from the body wall of the starfish Linckia laevigata. The ultrastructural study showed that the dermis was packed with collagen fibrils between which only a small number of cells were observed. Muscles were found only in the walls of coelomic extensions leading to papulae. Stress-strain tests were performed on isolated dermis containing no muscles. The tangent modulus was 27.5 MPa at 0.04% strain rate in the stress-strain tests. It was increased to 40.7 MPa by mechanical stimulation, which also increased the tensile strength and breaking-strain energy density. Dynamic mechanical tests showed that the increase in stiffness in response to mechanical stimulation was transient. Acetylcholine (10(-6)-10(-3) mol l(-1)) and artificial seawater with an elevated potassium concentration (KASW) stiffened the dermis. Mechanical stimulation caused a 12% mass loss. KASW also caused mass loss, which was inhibited by anesthesia. These results clearly showed that the stiffness changes in the starfish dermis were based on a non-muscular mechanism that was similar to that of other echinoderm connective tissues with mechanical mutability.     

Ultrastructure of the kidney of a South American caecilian,Typhlonectes compressicaudus (Amphibia,Gymnophiona)

Summary The ultrastructure of the distal nephron, the collecting duct and the Wolffian duct was studied in a South American caecilian, Typhlonectes compressicaudus (Amphibia, Gymnophiona) by transmission and scanning electron microscopy (TEM, SEM). The distal tubule (DT) is made up of one type of cell that has a well-developed membrane labyrinth established both by interdigitating processes and by interlocking ramifications. The processes contain large mitochondria, the ramifications do not. The tight junction is shallow and elongated by a meandering course. The connecting tubule (CNT) is composed of CNT cells proper and intercalated cells, both of which are cuboidal in shape. The CNT cells are characterized by many lateral interlocking folds. The intercalated cells have a dark cytoplasm densely filled with mitochondria. Their apical cell membrane is typically amplified by microplicae beneath which a layer of globular particles (studs) is found. The collecting duct (CD) is composed of principal cells and intercalated cells, again both cuboidal in shape. The CD epithelium is characterized by dilated intercellular spaces, which are often filled with lateral microfolds projecting from adjacent principal cells. The apical membrane is covered by a prominent glycocalyx. The intercalated cells in the CD are similar to those in the CNT. The Wolffian duct (WD) has a tall pseudostratified epithelium established by WD cells proper, intercalated cells and basal cells. The WD cells contain irregular-shaped dense granules located beneath the apical cell membrane. The intercalated cells of the WD have a dark cytoplasm with many mitochondria; their nuclei display a dense chromatin pattern.Research fellow of the Alexander von Humboldt Foundation