Production of oxygen free radicals by Ehrlich ascites tumour cells: effect of lipids

Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and platelet activating factor (PAF) stimulated the generation of oxygen free radicals (nitro-blue tetrazolium reduction) in Ehrlich ascites tumour (EAT) cells. PAF was effective at an optimal concentration of 4 μM, but was inhibited by BN 52021, a specific PAF antagonist. Lyso-PAF was ineffective. Inclusion of different lipids during incubation prior to the addition of PAF, resulted in the activation/inhibition of free radical generation. Among the phospholipids at a concentration of 50 μg/ml, the order of activation was phosphatidylserine > phosphatidylglycerol > phosphoinositides > phosphatidylinositol > phosphatidylethanolamine. Phosphatidylcholine was not effective, while sphingolipids were inhibitory. In addition, Ehrlich ascites tumour cells grown in mice under marginal vitamin A deficiency, showed an augmented production of free radicals compared to control cells. This was suppressed by exogenous addition of vitamin A or superoxide dismutase. These results suggest that membrane lipids and dietary factors like vitamin A probably function as physiological modulators in regulating the free radical generation

Interference of phenol during quantification of a bacterial lipoprotein

Accurate protein estimation is an essential requirement for any biochemical investigation. The bacterial Braun liporotein (BLP) from E. coli (a Toll-2 receptor ligand) is purified via phenol extraction on the basis of selective extraction of the lipoprotein. The procedure leaves behind the major endotoxin lipopolysaccharide (LPS) that acts through the related Toll-4 receptor. However, as low as 0.00001% of phenol carried over during lipoprotein isolation interferes in the Lowry’s method of protein estimation. A simple gel filtration on sephadex G-50 efficiently separates lipoproteins from phenol thereby avoiding inaccurate protein estimation of the lipoprotein content and making it suitable ligand for Toll-2 receptor.Keywords: Lipoproteins; Lipopolysaccharide (LPS); Lowry's method; Phenol interferenc

Activated Polymorphonuclear Leukocytes Rapidly Synthesize Retinoic Acid Receptor-α: A Mechanism for Translational Control of Transcriptional Events

In addition to releasing preformed granular proteins, polymorphonuclear leukocytes (PMNs) synthesize chemokines and other factors under transcriptional control. Here we demonstrate that PMNs express an inducible transcriptional modulator by signal-dependent activation of specialized mechanisms that regulate messenger RNA (mRNA) translation. HL-60 myelocytic cells differentiated to surrogate PMNs respond to activation by platelet activating factor by initiating translation and with appearance of specific mRNA transcripts in polyribosomes. cDNA array analysis of the polyribosome fraction demonstrated that retinoic acid receptor (RAR)-α, a transcription factor that controls the expression of multiple genes, is one of the polyribosome-associated transcripts. Quiescent surrogate HL60 PMNs and primary human PMNs contain constitutive message for RAR-α but little or no protein. RAR-α protein is rapidly synthesized in response to platelet activating factor under the control of a specialized translational regulator, mammalian target of rapamycin, and is blocked by the therapeutic macrolide rapamycin, events consistent with features of the 5′ untranslated region of the transcript. Newly synthesized RAR-α modulates production of interleukin-8. Rapid expression of a transcription factor under translational control is a previously unrecognized mechanism in human PMNs that indicates unexpected diversity in gene regulation in this critical innate immune effector cell

Lysophosphatidic Acid Induces Neointima Formation Through PPARγ Activation

Neointimal lesions are characterized by accumulation of cells within the arterial wall and are a prelude to atherosclerotic disease. Here we report that a brief exposure to either alkyl ether analogs of the growth factor–like phospholipid lysophosphatidic acid (LPA), products generated during the oxidative modification of low density lipoprotein, or to unsaturated acyl forms of LPA induce progressive formation of neointima in vivo in a rat carotid artery model. This effect is completely inhibited by the peroxisome proliferator-activated receptor (PPAR)γ antagonist GW9662 and mimicked by PPARγ agonists Rosiglitazone and 1-O-hexadecyl-2-azeleoyl-phosphatidylcholine. In contrast, stearoyl-oxovaleryl phosphatidylcholine, a PPARα agonist and polypeptide epidermal growth factor, platelet-derived growth factor, and vascular endothelial growth factor failed to elicit neointima. The structure-activity relationship for neointima induction by LPA analogs in vivo is identical to that of PPARγ activation in vitro and disparate from that of LPA G protein–coupled receptor activation. Neointima-inducing LPA analogs up-regulated the CD36 scavenger receptor in vitro and in vivo and elicited dedifferentiation of cultured vascular smooth muscle cells that was prevented by GW9662. These results suggest that selected LPA analogs are important novel endogenous PPARγ ligands capable of mediating vascular remodeling and that activation of the nuclear transcription factor PPARγ is both necessary and sufficient for neointima formation by components of oxidized low density lipoprotein

Paf is a potent pyrogen and cryogen in rodents, but it does not mediate thermoregulatory responses to bacterial endotoxin

Concordance between lipopolysaccharide and platelet activating factor - mediated events have suggested that the latter likely mediates all effects induced by the former. In this issue of Temperature, Steiner and Romanovsky challenge this notion, showing that while platelet activating factor is a potent pyrogenic mediator, the thermoregulatory responses to lipopolysaccharide are instead induced by prostaglandins

Pituitary gonadotropins regulate spermatogonial differentiation and proliferation in the rat

The relative regulatory roles of the pituitary gonadotropins, luteinizing hormone and follicle stimulating hormone in the spermatogonial proliferation has been studied using specific antibodies against these hormones in the immature rats. Immunoneutralization of luteinizing hormone for 7 days resulted in significant reduction in tetraploid cells and total absence of haploid cells, while there was a relative increase in the diploid population. This was also accomopanied by a decrease in spermatogonial proliferation as indicated by a decrease in [H-3] thymidine incorporation into DNA by purified spermatogonia. Administration bf follicle stimulating hormone als for 7 days also caused a significant decrease in the rate of spermatogonial proliferation. Withdrawal of follicle stimulating hormone led to a significant reduction in tetraploid and haploid cells However interestingly, it failed to totally abolish the appearance of these cells. Administration of testosterone (3mg/day/rat) for 2 days along with the gonadotropin a/s could partially reverse the effect on spermatogonial proliferation. It is concluded that (i) both luteinizing hormone and follicle stimulating hormone are involved in spermatogonial proliferation, (ii) lack of testosterone consequent of the neutralization of luteinizing hormone prevented the entry of spermatogonial cells into meiosis, (iii) testosterone may be involved in spermatogonial proliferation providing a mitotic signal and (v) both follicle stimulating hormone and testosterone act synergistically and lack of any one of the hormones results in impairment of spermatogonial proliferation

Oxidized glycerophosphocholines as biologically active mediators for ultraviolet radiation-mediated effects

Ultraviolet light radiation (UVR) has profound effects upon human skin. Yet, the exact targets for UVR are unclear. Inasmuch as UVR is a known pro-oxidative stressor, one potential target for UVR could be oxidatively modified glycerophosphocholines (GPC). Importantly, recent studies demonstrate that these oxidized GPCs (ox-GPC) are potent agonists for the platelet-activating factor receptor and peroxisome proliferator-activated receptor gamma. This review discusses these new biologically active lipids and their down-stream receptor targets that provide a unique system of biosensors for detecting and responding to UVR photo-oxidation

Proteinaceous sperm motility inhibitory factor from the female Indian garden lizard Calotes versicolor

Female sperm storage is an intriguing adaptation exhibited by a wide array of both vertebrates and invertebrates. The mechanisms underlying female sperm storage have remained elusive. Using the Indian garden lizard Calotes versicolor as a model organism, we investigated the role of low and high molecular weight factors in this phenomenon. Previously, we demonstrated three distinct phases of the reproductive cycle in this animal with live, motile spermatozoa recovered from the uterovaginal region during the reproductive phase. In the present study, we analysed the uterovaginal contents using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and identified an abundant protein band corresponding to ~55 kDa regardless of the phase of the reproductive cycle. Analysis of the purified protein by liquid chromatography–tandem mass spectrometry suggested a unique protein without any homology to the National Center for Biotechnology Information database. Exogenous addition of this protein to washed spermatozoa derived from the epididymis reversibly inhibited sperm motility in a concentration- and time-dependent manner, suggesting it plays a key role in sperm storage. These studies are likely to offer new avenues to unravel the secrets of female sperm storage seen across the animal taxa and may have novel applications not only in reproductive biology, but also in general cell storage and preserving endangered animal species