Indranil Dey

Molecular architecture and biophysical properties of phospholipids during thermal adaptation in fish: An experimental and model study

Phospholipids from livers of carps (Cyprinus carpio L.) adapted to winter (5°C) and summer (25°C) temperatures were isolated, and the fatty acid composition of total phospholipids, as well as molecular species composition of diacyl phosphatidylcholines and ethanolamines, were determined. Order parameter of 5-doxyl stearic acid and steady-state fluorescence anisotropy of different anthroyloxy fatty acids—[2-, 12(N-9-anthroyloxy)stearic acid and 16(N-9-anthroyloxy)palmitic acid—embedded in native and synthetic (16∶0/16∶0, 16∶0/22∶6, 18∶0/22∶6, 18∶1/22∶6, 20∶4/20∶4, 22∶6/22∶6 phosphatidylcholines and 16∶0/18∶1, 18∶1/22∶6 phosphatidylethanolamines) phospholipid vesicles was also determined between −30 and 30°C and 5 and 30°C, respectively. There is an accumulation of 1-monoenoic, 2-polyenoic diacyl phosphatidylcholine and ethanolamine with a concomitant reduction of 1-stearoyl,2-docosahexaenoyl species in the cold-adapted state. Despite a 30% accumulation of long-chain polyunsaturated fatty acids in phospholipids in cold, there is only a 5°C downshift in the solid-gel to liquid-crystalline phase transition temperature (−8 vs. −13°C). Vesicles from total phospholipids of cold-adapted fish proved to be more disordered in all segments than from the warmadapted ones when assayed using 2,12-(N-9-anthroyloxy)stearic and 16-(N-9-anthroyloxy)palmitic acid. Vesicles made from purified phosphatidylcholines showed the same pattern, but they were more disordered than the corresponding total phospholipids. This could be modelled using mixed phospholipid vesicles made of synthetic 16∶0/22∶6 phosphatidylcholine (75%) and either 18∶1/22∶6 phosphatidylethanolamine (25%) vs. 16∶0/18∶1 phosphatidylethanolamine (25%) and comparison of the anisotropy parameters of 100% 16∶0/22∶6 and 100% 18∶1/22∶6 phosphatidylcholine vesicles. Mixing either 16∶0/18∶1 (25%) or 18∶1/22∶6 (25%) phosphatidylethanolamines to 18∶0/22∶6 (75%) phosphatidylcholine shifted down or up, respectively, the transition temperature of vesicles compared to 100% 18∶0/22∶6 vesicles assayed by electron spin resonance spectroscopy using 5-doxylstearic acid. It is concluded that it is not the gross amount of long-chain polyunsaturated fatty acids in phospholipids, but rather their specific combination withcis Δ9 monounsaturated fatty acids in the positionsn-1, especially in phosphatidylethanolamines, that is important in determining the physical properties of biomembranes in relation to adaptational temperature.

Identification and characterization of a cyclooxygenase-like enzyme from Entamoeba histolytica

The intestinal protozoan parasite Entamoeba histolytica remains a significant cause of morbidity and mortality worldwide. However, almost nothing is known about the molecules secreted by the parasite that modulate host immune responses or epithelial barrier function in the colon. Herein, we describe the isolation and characterization of a cyclooxygenase (COX)-like enzyme in E. histolytica that is responsible for the biosynthesis of prostaglandin (PG)E2. PGE2 produced by ameba was constitutive but highly dependent on exogenous arachidonic acid substrate. COX-like activity and the immunoreactive protein were localized to the nuclear fraction of E. histolytica. The COX-like protein (72 kDa) was microsequenced and cloned by reverse transcriptase PCR. Ameba COX showed little homology with COX-1/2 enzymes from different species at the nucleotide and amino acid levels. Surprisingly, the arachidonate-binding domain and heme-coordinating and catalytic sites, which are conserved in other species, were absent in ameba. Ameba COX expressed in Escherichia coli demonstrated COX-like enzyme activity in vitro by converting arachidonic acid into PGE2 but not into PGD2 or PGF2α. COX activity was inhibited with 1 mM aspirin but not with indomethacin or COX-1/2-specific inhibitors. Taken together, these studies reveal that E. histolytica produces PGE2, by means of a previously undescribed ancestral COX-like enzyme, which could play a major role in pathogenesis and immune evasion.

Role of phospholipid molecular species in maintaining lipid membrane structure in response to temperature

The compositions and physical states of the liver phospholipids of fish and phospholipids of shrimps adapted to relatively constant but radically different temperatures were investigated. There were no measurable differences in their gross fatty acid compositions of phospholipids from the species obtained from identical temperature. Saturated-to-unsaturated fatty acid ratio did not show any convincing difference. Docosahexaenoic acid (22:6) did not seem to participate in the process of adaptation. Cold adaptation was coincidental with oleic acid (18:1) accumulation, preferentially in the phosphatidylethanolamine. In the experiment with rats, it has been found that the fish oil fed rats showed a similarity in their liver phospholipid fatty acid composition like fish liver phospholipids. Determination of molecular species composition of phosphatidylcholine and phosphatidylethanolamine revealed a 4- to 5-fold and 10-fold increase in the level of 18:1/22:6 and 18:1/20:5 species respectively in favor of cold adaptation. Phospholipids from cold-adapted species showed a more fluid structure than that of warm-adapted species near the C-2 segment of the bilayer, but not in the deeper regions. Phospholipids from the rat livers did not show any change irrespective of diet. Phosphatidylcholines from rat liver, fish liver or shrimps did not show any difference among them in their fluidity irrespective of their environmental conditions or diet. An appropriate combination (75:25) of phosphatidylcholine from rat liver with phosphatidylethanolamines from cold adapted species showed a drastic fluidization at the C-2 segment, in comparison with their phosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 Produced by Entamoeba histolytica Binds to EP4 Receptors and Stimulates Interleukin-8 Production in Human Colonic Cells

ABSTRACT Entamoeba histolytica pathogenesis in the colon occurs in a stepwise fashion. It begins with colonization of the mucin layer, which is followed by stimulation of a proinflammatory response that causes nonspecific tissue damage that may facilitate parasite invasion of the underlying colonic mucosa. Unfortunately, the parasite and/or host factors that stimulate a proinflammatory response in the gut are poorly understood. In this study, we found that live E. histolytica or secretory or proteins (SP) and soluble ameba components (SAP) can markedly increase interleukin-8 (IL-8) mRNA expression and protein production in colonic epithelial cells. The IL-8-stimulating molecule produced by live amebae was identified as prostaglandin E2 (PGE2) as trophozoites treated with cyclooxygenase inhibitors inhibited the biosynthesis of PGE2 and eliminated IL-8 production induced by live parasites or ameba components. Moreover, using specific prostaglandin EP2 and EP4 receptor agonists and antagonists, we found that PGE2 binds exclusively through EP4 receptors in colonic epithelial cells to stimulate IL-8 production. Silencing of EP4 receptors with EP4 small interfering RNA completely eliminated SP- and SAP-induced IL-8 production. These studies identified bioactive PGE2 as a one of the major virulence factors produced by E. histolytica that can stimulate the potent neutrophil chemokine and activator IL-8, which can trigger an acute host inflammatory response. Thus, the induction of IL-8 production in response to E. histolytica-derived PGE2 may be a mechanism that explains the initiation and amplification of acute inflammation associated with intestinal amebiasis.

Fish erythrocytes as a tool to study temperature-induced responses in plasma membranes

The dorsal aorta of carp (Cyprinus carpio L.) was cannulated, and the fish were kept in thermostated aquaria at 5°C for 24h. The water temperature was then gradually increased to 25°C at a rate of 0.5°C/h, and then decreased to 5°C at the same rate. Blood was withdrawn at five-degree intervals to determine the fluidity of erythrocyte plasma membranes uponex vivo incorporation of the fluorescent dye, 3-[p-(6-phenyl-1,3,5-hexatrienyl)phenyl] propionic acid. Steady-state anisotropy (Rss) of the plasma membranes increased and decreased with the increase and decrease in water temperature, respectively. The sterol-to-phospholipid ratio remained unchanged throughout the thermal shifts. The fatty acid compositions of the total phospholipids, of phosphatidylcholine and of phosphatidylethanolamine remained virtually unchanged, except for the level of arachidonic acid, which increased in erythrocytes from fish at the higher temperature (25°C). The molecular species compositions of phosphatidylcholines and phosphatidylethanolamines also remained unaffected throughout the thermal shifts. The erythrocyte plasma membranes were more responsive to temperature shiftsin vivo thanin vitro when percent efficacy was compared. Thus, factors other than lipid changes are conceivably involved in the adaptation of erythrocyte plasma membranes to short-term thermal changes.

Induction of Monocyte Chemotactic Protein 1 in Colonic Epithelial Cells by Entamoeba histolytica Is Mediated via the Phosphatidylinositol 3-Kinase/p65 Pathway

ABSTRACT The role intestinal epithelial cells play in the pathogenesis of amebic colitis is poorly understood. Herein, we demonstrate that secreted and soluble ameba (Entamoeba histolytica) proteins (SAP) induce expression of the chemoattractant monocyte chemotactic protein (MCP) in the colonic epithelial cell lines Caco-2, T84, and LS174T. MCP-1 mRNA induction was both dose and time dependent, with peak induction occurring at 8 h and with 100 μg/ml of SAP. Significant increase in MCP-1 protein expression was observed after 12 h. SAP failed to activate any of the mitogen-activated protein kinase pathways or IκB kinase activity. Moreover, inhibiting the classical pathway of NF-κB activation did not affect SAP-induced MCP-1 expression. Instead, we find that SAP-induced MCP-1 expression is dependent on posttranslational modification of the NFκB p65 subunit. SAP induced phosphorylation of p65 and enhanced NF-κB transcriptional activity, which are phosphatidylinositol 3-kinase (PI3 kinase) dependent. Treatment with PI3 kinase inhibitor LY290004 significantly abrogated the activation of Akt, p65, and MCP-1 mRNA induction. We conclude that colonic epithelial cells play a role in the initiation of inflammation by secreting chemokines in response to soluble ameba components.

Temperature shifts induce adaptive changes in the physical state of carp (Cyprinus carpio L.) erythrocyte plasma membranes in vitro

Blood, freshly collected from warm- and cold-acclimated carp, Cyprinus carpio L., was cooled to 5°C for 4h or warmed to 25°C for 4h, respectively, and the fluorescence anisotropy of washed red blood cells was recorded using the fluorescent dye 3-(p-(6-phenyl-1,3,5-hexatrienyl) phenyl propionic acid [DPH-PA] (which is restricted to the outer leaflet of the plasma membrane) before and after the temperature shift. Despite individual variation, the plasma membrane of cold-exposed erythrocytes became more fluid while that of warm-exposed cells became more rigid following the temperature shift. This response was rapid and reversible. Cold-exposed cells from warm-acclimated fish became more fluid within 40–60 minutes and reverted to their original fluidity within the same time on warming, at a rate of 1°C/min; erythrocytes, from cold-adapted carp displayed an opposite change in fluidity over a similar time period. Cells from warm-acclimated, temperature down-shifted carp hyperfluidized their plasma membranes in the cold, whereas cells from cold-acclimated fish up-shifted in temperature showed no similar effect. These cells showed a complete adjustment of membrane physical state to the temperature. Total phospholipids obtained from warm-acclimated temperature down-shifted cells became more rigid than they were, when assayed at the acclimation temperature. In contrast, phospholipids obtained from cold-acclimated cells became more rigid when exposed to increasing temperatures. No significant changes occurred to the polar head groups, or to the fatty acid composition of the total phospholipids. It was concluded that the lipids play only a secondary role in the control of the physical state of plasma membrane in carp erythrocytes, and that some non-lipid components of these structures might be involved in these regulatory processes.