Modulation of the locomotion of polymorphonuclear leukocytes by a synthetic amphiphile, poly(ethyleneglycol) palmitate

To mimic in vitro the effect of biologically active amphipathic molecules on the locomotion of polymorphonuclear leukocytes (PMNL), the interaction and the consequences of the interaction between poly(ethyleneglycol) (PEG) esterified with palmitic acid (P-PEG) and PMNL, was studied. It was shown that P-PEG was more liable to hydrophobic interaction than PEG, and that P-PEG associated to a greater extent than PEG with PMNL. This association was inhibited by decreasing the interaction temperature from 37 to 4 °C, but it was insensitive to inhibitors of glycolysis. P-PEG decreased colchicine-facilitated capping by concanavalin A (ConA). P-PEG also decreased random locomotion of individual as well as population of PMNL. Neither cell viability nor the ability to respond by stimulated locomotion to an attractant was affected. The results indicate that P-PEG modulates PMNL locomotion by altering the membrane structure, e.g. by decreasing the lateral mobility of membrane constituents.     

Luminol dependent chemiluminescence and thiol group oxidation provoked by neutrophils is attributable to different oxidizing species

Luminol-dependent chemiluminescence and thiol group oxidation of glutathione and human serum albumin were measured in order to demonstrate whether the inhibition of polymorphonuclear leukocyte chemiluminescence by albumin was attributable to thiol group oxidation. We have shown that:

• 1 thiol groups on glutathione and albumin are oxidized by PMNL stimulated by soluble and phagocytic stimuli;
• 2 thiol group oxidation in albumin and glutathione did not correlate with the inhibitory effects of these substances on luminol-dependent chemiluminescence with respect to time course, magnitude, effects of known scavengers or extracellular activity. It was therefore concluded that thiol group oxidation was not the cause of albumin inhibition of luminol-dependent chemiluminescence;
• 3 a metastable oxidant was identified after PMNL activation which was capable of oxidizing thiol groups but unable to elicit chemiluminescence form luminol.

     

Bleached Porites compressa and Montipora capitata corals catabolize ��13C-enriched lipids

Corals rely on stored energy reserves (i.e., lipids, carbohydrates, and protein) to survive bleaching events. To better understand the physiological implications of coral bleaching on lipid catabolism and/or synthesis, we measured the δ¹³C of coral total lipids (δ¹³C_TL) in experimentally bleached (treatment) and non-bleached (control) Porites compressa and Montipora capitata corals immediately after bleaching and after 1.5 and 4 months of recovery on the reef. Overall δ¹³C_TL values in treatment corals were significantly lower than in control corals because of a 1.9 and 3.4‰ decrease in δ¹³C_TL immediately after bleaching in P. compressa and M. capitata, respectively. The decrease in δ¹³C_TL coincided with decreases in total lipid concentration, indicating that corals catabolized δ¹³C-enriched lipids. Since storage lipids are primarily depleted during bleaching, we hypothesize that they are isotopically enriched relative to other lipid classes. This work further helps clarify our understanding of changes to coral metabolism and biogeochemistry when bleached and helps elucidate how lipid classes may influence recovery from bleaching and ultimately coral survival.     

Cholera Toxin (Choleragen) - Polymorphonuclear Leukocyte Interactions: Effect on Migration in Vitro and FcγR - Dependent Phagocytic and Bactericidal Activity

PMNL leukocytosis is a feature common to many types of infectious and inflammatory diseases. How PMNL are recruited to tissues is not yet clear although it is a question that has considerable clinical importance. We investigated the function of PMNL which migrated through an artificial barrier (Chinese hamster ovary (CHO) cells, collagen and nylon cloth membrane) subjected to CT or choleragenoid treatment toward plain medium (the same RPMI in the upper and lower chamber) or medium containing chemotactic factor (fMLP or LPS or ZAS). CT treatment significantly (P<0.01) reduced the FcγR expression on the surface of PMNL. The PMNL functions, namely, migration, phagocytic activity and intracellular killing of staphylococci, also have been reduced significantly (P<0.01). FcγR expression and some functions of PMNL that migrate to chemoattractants were reduced, irrespective of the presence or absence of CT; however, the inhibitory effect of CT on PMNL function was observed only when PMNL migrate to the lower chamber without chemotactic factor. On the other hand choleragenoid treatment of CHO cells did not have any significant influence on PMNL function and FcγR expression. In conclusion, our experiments demonstrate that CT reduces EA_Fc rosetting and the FcγR-dependent phagocytic and bactericidal activity of bovine blood PMNL.     

The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such “alternative” NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C₁₂ (HDQ) and C₁₄ are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C₅ and C₆) displayed significantly higher IC₅₀ values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

The Glucose Concentration Modulates N-Formyl-Methionyl-Leucyl-Phenylalanine (fMet-Leu-Phe)-Stimulated Chemokinesis in Normal Human Neutrophils

The effects of glucose concentration on the chemokinetic effects of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) was evaluated for normal human neutrophils using a direct microscopic assay. fMet-Leu-Phe increased the rate of locomotion in the absence of glucose, but the chemokinetic effect of fMet-Leu-Phe was most potent at 5mM glucose and not further changed at 15 mM glucose. The chemokinetic effects of fMet-Leu-Phe and glucose were essentially the same in blood clot-isolated and gradient-isolated neutrophils. However, in gradient-isolated neutrophils, the rate of locomotion under different experimental conditions was strictly negatively correlated to the fraction of non-locomoting cells and the degree of adhesion to the substratum. These results indicate that the chemokinetic effects of fMet-Leu-Phe are regulated by the glucose concentration by inducing locomotor activity in otherwise non-locomoting cells and by improving adhesion to the substratum.     

Kinematics of nonstationary locomotion in the centipede <Emphasis Type="Italic">Scolopendra cingulata</Emphasis>: A single instant change of speed

The spatiotemporal parameters of leg movement in Scolopendra instantly changing the locomotion speed from V ₁ to V ₂ ≠ V ₁ are investigated. It is shown that (i) the principle of “in trail” leg placement is kept upon a change of speed; (ii) the continuity of the metachronal coordination is not disturbed; but (iii) for some time after changing speed the set of ipsilateral legs comprises (a) head-proximal legs creating a new trackway of steps with pace ℓ₂, and (b) distal legs still using the old trackway with pace ℓ₁. The two groups differ in kinematic parameters. Group (a) works in the stationary mode corresponding to speed V ₂, while group (b) works in a transitory mode. Consecutively, with a metachronal wave propagating backwards along the body but immobile relative to the ground, more and more legs from group (b) switch to the new stationary mode.     

Different influences of cytochalasin B on the activation of human neurophils settled onto petri dishes displayed by simultaneously detected native and luminol-dependent luminescence

Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells. The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli. PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N-formylmethionyl-phenylalaline. The production of reactive oxygen species were monitored by simultaneous detection of native, luminol-independent, luminescence (NL) and luminol-dependent luminescence (LDL) using a method of spectral discrimination. Different influences of CB on NL with respect to LDL as well stimuli-dependent influences of CB on the luminescence response of PMNL were observed. Especially phagocytosis-associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL. A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Lateral diffusion of plasma membrane receptors labelled with either platelet-derived growth factor (PDGF) or wheat germ agglutinin (WGA) in human polymorphonuclear leukocytes and fibroblasts

The aims of the present investigation were (a) to compare the lateral mobility of membrane receptors of human fibroblasts and polymorphonuclear leukocytes (PMNL) labelled with either platelet-derived growth factor (PDGF), or the lectin wheat germ agglutinin (WGA), and (b) to study effects of serum or PDGF on the mobility of these receptor molecules in human fibroblasts. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard (10%) or under serum-free conditions yielding normal and starved cells, respectively. The receptor mobility was studied in response to exposure to PDGF, or serum, in short time or prolonged incubations. Human polymorphonuclear leukocytes (PMNL) were adhered to microscope slides by clotting drops of blood. They were stained with rhodaminated PDGF or fluoresceinated WGA. The diffusion of labelled receptors was assessed with fluorescence recovery after photobleaching (FRAP). It was found that (a) fibroblasts grown at normal serum concentration had a lower diffusion coefficient (D=3×10^–10 cm² s^–1) for the PDGF-receptor and a slightly lower mobile fraction (R=60%) than starved cells (D=5×10^–10 cm²s^–1 and R=73%), (b) addition of serum to starved cells increased both D and R for the PDGF receptor to 12×10^–10 cm² s^–1 and 96%, respectively, (c) a similar pattern was obtained for WGA-labelled glycoconjugates indicating general membrane effects of serum-induced cell stimulation, and (d) in PMNL the PDGF receptor displayed motility characteristics (D=3–4×10^–10 cm² s^–1 and R=59%) similar to those in fibroblasts, possibly suggesting equivalent anchorage mechanisms in the membrane.     

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (C_m) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (π_o) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (f_FM,i) increased with reference to the total fluorescence (f_FM,t) by 7.4 × 10⁻⁴ min⁻¹. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of π_o by 200 mosmol kg⁻¹ caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time f_FM,i/f_FM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of C_m and total fluorescence of a protoplast (f_FM,p) showed that an increase in π_o evoked a decrease in C_m but no change in f_FM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area. Received: 4 May 1999 / Accepted: 19 August 1999     

The impact of bleaching on the metabolic contribution of dinoflagellate symbionts to their giant clam host

Bleaching (loss of symbiotic dinoflagellates) is known to significantly decrease the fitness of symbiotic marine invertebrates resulting in reduced growth, fecundity and survival. This report is the first to quantify the effects of bleaching on inorganic carbon (C_i) and ammonium flux, fixation and export of photosynthate to the host, in this case the giant clam Tridacna gigas. The 1998 bleaching event was found to decrease the zooxanthellae population 30‐fold when comparing bleached to non‐bleached clams. This resulted in significant increases in haemolymph C_i and decreases in haemolymph pH and glucose concentration, the predominant photosynthate exported from zooxanthellae in this symbiosis. There was also a decrease in the expression levels of host carbonic anhydrase, an enzyme involved in C_i transport to the zooxanthellae, and although host glutamine synthase levels were unaffected, the clams ability to assimilate ammonium was eliminated in bleached individuals, suggesting that photosynthate from the zooxanthellae is required for ammonium assimilation. In an artificial bleaching experiment haemolymph C_i (r² = 0.97), pH (r² = 0.94) and glucose levels (r² = 0.95) were correlated to zooxanthellae numbers during both bleaching and recovery. Recovery of the zooxanthellae population, was enhanced four‐fold by the addition of organic and inorganic nutrients, as were related haemolymph characteristics. These results highlight the profound physiological changes that occur in symbiotic organisms during and after a bleaching event.     

Studies of copper(II) and nickel(II) complexation by bis(1,4,7-triazacyclononane) ligands containing alkene and alkyne bridging groups

The copper(II) and nickel(II) complexes of three new 1,2-bis(1,4,7-triazacyclononane) ligands containing unsaturated four carbon bridging groups is studied by continuous variation UV-Vis spectroscopic and pH potentiometric equilibrium experiments. The cis-butene-2 (L_C) linked ligand may form monomeric MN₆-type complexes while the trans-butene-2 (L_T) and butyne-2 (L_Y) ligands are prevented by their stereochemistry from forming monomeric complexes and form oligomeric complexes. It is determined that the stability of the CuL_C²⁺ complex is not appreciably different from the oligomeric complexes of L_T and L_Y. Single-crystal X-ray structure determinations are made on three square pyramidal Cu₂L⁴⁺ complexes: [Cu₂L_CCl₄] (1), [Cu₂L_YCl₄] (2), and [Cu₂L_T(NO₃)₂(H₂O)₂](NO₃)₂ (3). The structure of [Ni₂(L_C)₂](ClO₄)₄ · 2H₂O (4) is a binuclear dimer that contains two nickel(II) ions sandwiched between two ligands, indicating that bis([9]aneN₃) ligands with four linker atom chains may form either monomeric or oligomeric structures.     

Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

Sequential bleaching in the presence of hydroxylamine and subsequent regeneration of the purple membrane of Halobacterium halobium was studied by concomitant monitoring of its absorption and circular dichroic spectra in order to ascertain its effects on protein interaction(s) (which may result in possible excitonic interaction between the retinal chromophores), chromophore-apoprotein interaction(s), and protein conformational stability in the membrane. It was concluded that (a) although experimental results are consistent with an exciton mechanism for the interaction between retinal π - π* (NV₁) transition movements in the purple membrane, no evidence for such a mechanism for interaction between retinaloxime transition moments is apparent in the case of the bleached membrane; (b) the bacteriorhodopsin molecules organized in clusters of three in the membrane appear to bleach simultaneously; (c) the retinaloxime produced on bleaching the purple membrane in the presence of hydroxylamine is strongly optically active, because of dissymmetry-inducing and/or -selecting constraints on the chromophore by a component of the membrane (most likely the apoprotein), and when the membrane is regenerated by the addition of retinal, these constraints are lost; and (d) evidence from ultraviolet absorption and circular dichroic spectra suggests that the membrane apoprotein undergoes appreciable conformational changes involving tertiary structure on bleaching with no significant secondary structure involvement. These results are compared with recently reported results from this laboratory on the effects of bleaching on the bovine rod outer segment disk membrane structure.     

Synthesis of (±)-8,16,26,34-Tetrahydroxy-6,10,14,18,24,28,32,36-octaoxohentetracontane as a Potent Synthetic Analog of Race T Toxin Produced by Helminthosporium maydis

A stereoisomeric mixture of (±)-8,16,26,34-tetrahydroxy-6,10,14,18,24,28,32,36-octaoxo-hentetracontane (C₄₁) was synthesized together with (±)-8,16-dihydroxy-6,10,14,18-tetraoxotri-cosane (C₂₃), utilizing bis addition of pentyl-l,5-dimagnesiumbromide to two moles of a C₁₈-alde-hyde.     

Alkyl esters from pinus radiata foliage epicuticular wax

Wax esters from the epicuticular wax of juvenile and mature-tree Pinus radiata foliage have been shown by capillary column GC-MS to consist mainly of short chain (C₆–C₁₂)alkanols esterified with long chain acids (C₂₄–C₃₂) and long chain alkanols (C₂₄–C₃₂) esterified with short chain acids (C₆-C₁₄) in a non-random manner. Mature-tree foliage wax esters also contained nonacosan-10-ol esterified with dodecanoic and tetradecanoic acids.     

Fluidity of Liposome Membranes Doped with Organic Tin Compounds: ESR Study

The kinetics of change in the fluidity of liposome membranes, obtained in the process of sonication of Egg Yolk Lecithin (EYL), with the admixture of organic tin compounds, was investigated. Five compounds were selected for the research: three differed in the length of hydrocarbon chains, (CH₃)₄Sn, (C₂H₅)₄Sn, and (C₃H₇)₃SnCl, whereas two differed in the number of aromatic rings, (C₆H₅)₂SnCl₂ and (C₆H₅)₃SnCl. The concentration of the compounds in proportion to EYL was 2 mol-%. Electron Spin (paramagnetic) Resonance (ESR) was applied using two spin probes TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and 16-DOXYL-stearic acid methyl ester (2-ethyl-2-(15-methoxy-15-oxopentadecyl)-4,4-dimethyl-3-oxazolidinyloxyl) localized at different sites within the membrane, to determine the spectroscopic parameters: partition (F) and rotation correlation time (τ), related to the membrane's fluidity. The ESR spectroscopic spectra of investigated samples were recorded from the moment of introducing the admixture to membranes for 180h. Analysing the obtained results, the following conclusions can be drawn: chain compounds slightly stiffened the membrane both on the inside (hydrophobic) layer and on the surface one, whereas ring compounds resulted in fluidization of the membrane—stronger in the case of the two-layer middle and weaker with reference to the surface layer.     

Isostructural unbranched alkyl‐chains as tools for stabilizing β‐turn structure

To investigate the structural role played by isostructural unbranched alkyl‐chains on the conformational ensemble and stability of β‐turn structures, the conformational properties of a designed model peptide: Plm‐Pro‐Gly‐Pda ( 1 , Plm: H₃C—(CH₂)₁₄—CONH—; Pda: —CONH— (CH₂)₁₄—CH₃) have been examined and compared with the parent peptide: Boc‐Pro‐Gly‐NHMe ( 2 , Boc: tert‐butoxycarbonyl; NHMe: N‐methylamide). The characteristic ¹³C NMR chemical‐shifts of the Pro C^β and C^γ resonances ascertained the incidence of an all‐trans peptide‐bond in low polarity deuterochloroform solution. Using FTIR and ¹H NMR spectroscopy, we establish that apolar alkyl‐chains flanking a β‐turn promoting Pro‐Gly sequence impart definite incremental stability to the well‐defined hydrogen‐bonded structure. The assessment of ¹H NMR derived thermodynamic parameters of the hydrogen‐bonded amide‐NHs via variable temperature indicate that much weaker hydrophobic interactions do contribute to the stability of folded reverse turn structures. The far‐UV CD spectral patterns of 1 and 2 in 2,2,2‐trifluoroethanol are consistent with Pro‐Gly specific type II β‐turn structure, concomitantly substantiate that the flanking alkyl‐chains induce substantial bias in enhanced β‐turn populations. In view of structural as well as functional importance of the Pro‐Gly mediated secondary structures, besides biochemical and biological significance of proteins lipidation via myristoylation or palmytoilation, we highlight potential convenience of the unbranched Plm and Pda moieities not only as main‐chain N‐ and C‐terminal protecting groups but also to mimic and stabilize specific isolated secondary and supersecondary structural components frequently observed in proteins and polypeptides. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 419–426, 2013.     

A short-chain alkyl derivative of Rhodamine 19 acts as a mild uncoupler of mitochondria and a neuroprotector

Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C₄R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C₁₂R1). Surprisingly, C₁₂R1 increased H⁺ conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C₄R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C₄R1. In experiments with HeLa cells, C₄R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C₄R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C₁₂R1. Thus, C₄R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.