Granule-mediated release of sphingosine-1-phosphate by activated platelets

Sphingosine-1-phosphate (S1P) is an intracellularly generated bioactive lipid essential for development, vascular integrity, and immunity. These functions are mediated by S1P-selective cell surface G-protein coupled receptors. S1P signaling therefore requires extracellular release of this lipid. Several cell types release S1P and evidence for both plasma membrane transporter-mediated and vesicle-dependent secretion has been presented. Platelets are an important source of S1P and can release it in response to agonists generated at sites of vascular injury. S1P release from agonist-stimulated platelets was measured in the presence of a carrier molecule (albumin) using HPLC-MS/MS. The kinetics and agonist-dependence of S1P release were similar to that of other granule cargo e.g. platelet factor IV (PF4). Agonist-stimulated S1P release was defective in platelets from Unc13d^Jinx (Munc13-4 null) mice demonstrating a critical role for regulated membrane fusion in this process. Consistent with this observation, platelets efficiently converted fluorescent NBD-sphingosine to its phosphorylated derivative which accumulated in granules. Fractionation of platelet organelles revealed the presence of S1P in both the plasma membrane and in α-granules. Resting platelets contained a second pool of constitutively releasable S1P that was more rapidly labeled by exogenously added sphingosine. Our studies indicate that platelets contain two pools of S1P that are released extracellularly: a readily-exchangeable, metabolically active pool of S1P, perhaps in the plasma membrane, and a granular pool that requires platelet activation and regulated exocytosis for release.     

Ellagic acid inhibits PKC signaling by improving antioxidant defense system in murine T cell lymphoma

Antioxidants protect the cells from the damaging effects of reactive oxygen species (ROS). Production of ROS during cellular metabolism is balanced by their removal by antioxidants. Any condition leading to increased levels of ROS results in oxidative stress, which participates in multistage carcinogenesis by causing oxidative DNA damage, mutations in the proto-oncogenes and tumor suppressor genes. Antioxidant defense system is required to overcome the process of carcinogenesis generated by ROS. Antioxidant enzymes are major contributors to endogenous antioxidant defense system. Protein kinase C (PKC) is generally involved in cell proliferation and its over expression leads to abnormal tumor growth. Out of three classes of PKC, classical PKC is mainly involved in cell proliferation and tumor growth. Classical PKC initiates signaling pathway and leads to activation of a number of downstream protein via activation of NF-κB. Therefore any agent which can promotes the endogenous antioxidant defense system should be able to down regulate PKC and NF-κB activation and thus may be useful in reducing cancer progression. To investigate this hypothesis we have tested the effect of antioxidant ellagic acid on antioxidant enzymes and PKC signaling in Dalton’s lymphoma bearing (DL) mice. DL mice were treated with three different doses of ellagic acid. The treatment significantly increases the activity and expression of antioxidant enzymes and down regulates the expression of classical isozymes of PKC as well as the activation of NF-κB, indicating that ellagic acid improves antioxidant defense system and PKC signaling via NF-κB which may contribute to its cancer preventive role.     

yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli

The outer membrane of Gram‐negative bacteria is an asymmetric lipid bilayer consisting of an essential glycolipid lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the inner leaflet. Here, we show that yciM, a gene encoding a tetratricopeptide repeat protein of unknown function, modulates LPS levels by negatively regulating the biosynthesis of lipid A, an essential constituent of LPS. Inactivation of yciM resulted in high LPS levels and cell death in Escherichia coli; recessive mutations in lpxA, lpxC or lpxD that lower the synthesis of lipid A, or a gain of function mutation in fabZ that increases the formation of membrane phospholipids, alleviated the yciM mutant phenotypes. A modest increase in YciM led to significant reduction of LPS and increased sensitivity to hydrophobic antibiotics. YciM was shown to regulate LPS by altering LpxC, an enzyme that catalyses the first committed step of lipid A biosynthesis. Regulation of LpxC by YciM was contingent on the presence of FtsH, an essential membrane‐anchored protease known to degrade LpxC, suggesting that FtsH and YciM act in concert to regulate synthesis of lipid A. In summary, this study demonstrates an essential role for YciM in regulation of LPS biosynthesis in E. coli.     

Long Term Effect of Curcumin in Restoration of Tumour Suppressor p53 and Phase-II Antioxidant Enzymes via Activation of Nrf2 Signalling and Modulation of Inflammation in Prevention of Cancer

Inhibition of carcinogenesis may be a consequence of attenuation of oxidative stress via activation of antioxidant defence system, restoration and stabilization of tumour suppressor proteins along with modulation of inflammatory mediators. Previously we have delineated significant role of curcumin during its long term effect in regulation of glycolytic pathway and angiogenesis, which in turn results in prevention of cancer via modulation of stress activated genes. Present study was designed to investigate long term effect of curcumin in regulation of Nrf2 mediated phase-II antioxidant enzymes, tumour suppressor p53 and inflammation under oxidative tumour microenvironment in liver of T-cell lymphoma bearing mice. Inhibition of Nrf2 signalling observed during lymphoma progression, resulted in down regulation of phase II antioxidant enzymes, p53 as well as activation of inflammatory signals. Curcumin potentiated significant increase in Nrf2 activation. It restored activity of phase-II antioxidant enzymes like GST, GR, NQO1, and tumour suppressor p53 level. In addition, curcumin modulated inflammation via upregulation of TGF-β and reciprocal regulation of iNOS and COX2. The study suggests that during long term effect, curcumin leads to prevention of cancer by inducing phase-II antioxidant enzymes via activation of Nrf2 signalling, restoration of tumour suppressor p53 and modulation of inflammatory mediators like iNOS and COX2 in liver of lymphoma bearing mice.     

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity. Complementation of the mutation with the sph2 gene partially restored production of hemolytic and sphingomyelinase activities. These results indicate that the sph2 gene product contributes to the hemolytic and sphingomyelinase activities secreted by L. interrogans and most likely dominates those functions under the culture condition tested.     

Improvisation and Evaluation of Laterosporulin Coated Titanium Surfaces for dental Applications: An In Vitro Investigation

Despite recent improvement in implant survival rates, there remains a significant demand for enhancing the long-term clinical efficacy of titanium (Ti) implants, particularly for the prevention of peri-implantitis. Bioactive substances such as antimicrobial peptides are emerging as effective alternatives for contemporary antimicrobial agents used in dental health care. Current research work was focused to use laterosporulins that are non-haemolytic cationic antimicrobial peptides from Brevibacillus spp. for coating commercially available Ti discs. The coated Ti surfaces were evaluated in vitro for biofilm formation by two dental plaque isolates Streptococcus gordonii strain DIGK25 and S. mutans strain DIGK119 as representatives of commensal and pathogenic streptococci respectively. The biofilm inhibition was ascertained with replicated experiments on hydroxyapatite discs and confirmed by florescence microscopy. The laterosporulin coated Ti discs showed significantly reduced biofilm formation by oral streptococci and displayed promising potential to enhance the antibacterial surface properties. Such improvised Ti surfaces may curb the menace of oral streptococcal biofilm formation on dental implants and the associated implant failures.     

Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway

Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.     

Evaluation of binding and antagonism/downregulation of brilanestrant molecule in estrogen receptor-α via quantum mechanics/molecular mechanics,molecular dynamics and binding free energy calculations

Abstract

The resistance to the endocrine therapy of breast cancer leads to the emergence of new class of drugs that downregulates the estrogen receptor action known as selective estrogen receptor downregulators (SERDs). The first approved SERD is fluvestrant; after this, there are several downregulators evolved and are in clinical trials, in which the brilanestrant (BRI) molecule shows nM range of binding affinity and efficacy. In the present study, to understand the binding nature of BRI molecule in the active site of ERα, the molecular docking analysis has been performed. Further, the QM/MM calculations were performed for the BRI–ERα complex to analyze the charge density distribution of intermolecular interactions. The molecular dynamics (MD) simulation was employed to understand the stability and binding mechanism of BRI molecule through root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF) and binding free energy calculations. From the MD simulation trajectory analysis, the alterations of Helix12 conformation and the key residue (Lys529), which is responsible for the ERα downregulation, have been identified. Further, the interaction between the H3 and H12 regions was identified for the antagonism of BRI molecule. The current study led us to understand the binding mechanism, antagonism and downregulation of BRI molecule, and this knowledge is essential to design novel SERDs for the treatment of endocrine-resistant positive breast cancer.

Communicated by Ramaswamy H. Sarma