Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa

The objectives of this cross-sectional observational study were: (i) to detect DNA damage and plasma membrane translocation of phosphatidylserine in purified sperm populations of high and low motility, and (ii) to analyse their relationship with the endogenous generation of reactive oxygen species. Ejaculates from infertile men were examined following gradient centrifugation. The main outcome measures were: sperm motion parameters (assessed with a computer analyser), generation of reactive oxygen species (measured by chemiluminescence), DNA damage (detected by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling and monoclonal antibody labelling of single-stranded DNA) and translocation of membrane phosphatidylserine (examined with annexin V staining). DNA fragmentation and membrane translocation of phosphatidyl-serine were observed in the fractions with low and high sperm motility in all patients. The fractions with low sperm motility had significantly higher proportion of cells with DNA damage and production of reactive oxygen species than the fractions with high sperm motility (P < 0.005). DNA fragmentation was significantly and positively correlated with the generation of reactive oxygen species (r = 0.42; P = 0.02). In conclusion: (i) spermatozoa from infertile men display translocation of membrane phosphatidylserine as diagnosed by annexin V positive staining; (ii) DNA damage (fragmentation and presence of single-stranded DNA) can be detected in ejaculated spermatozoa from infertile men in fractions with low and high sperm motility, and (iii) there is a relationship between DNA damage and oxidative stress.     

Protection against cerebral malaria by the low-molecular-weight thiol pantethine

We report that administration of the low-molecular-weight thiol pantethine prevented the cerebral syndrome in Plasmodium berghei ANKA-infected mice. The protection was associated with an impairment of the host response to the infection, with in particular a decrease of circulating microparticles and preservation of the blood-brain barrier integrity. Parasite development was unaffected. Pantethine modulated one of the early steps of the inflammation-coagulation cascade, i.e., the transbilayer translocation of phosphatidylserine at the cell surface that we demonstrated on red blood cells and platelets. In this, pantethine mimicked the inactivation of the ATP-binding-cassette transporter A1 (ABCA1), which also prevents the cerebral syndrome in this malaria model. However, pantethine acts through a different pathway, because ABCA1 activity was unaffected by the treatment. The mechanisms of pantethine action were investigated, using the intact molecule and its constituents. The disulfide group (oxidized form) is necessary to lower the platelet response to activation by thrombin and collagen. Thio-sensitive mechanisms are also involved in the impairment of microparticle release by TNF-activated endothelial cells. In isolated cells, the effects were obtained by cystamine that lacks the pantothenic moiety of the molecule; however, the complete molecule is necessary to protect against cerebral malaria. Pantethine is well tolerated, and it has already been administered in other contexts to man with limited side effects. Therefore, trials of pantethine treatment in adjunctive therapy for severe malaria are warranted.     

Antileukemic action of novel diamine Pt(II) halogenido complexes: Comparison of the representative novel Pt(II) with corresponding Pt(IV) complex

This study presents the synthesis, characterization, and antitumor action of five new Pt(II ) halogenido, chlorido, and iodido complexes with edda type of ligands. (S,S)‐ Ethylenediamine‐N,N′‐di‐2‐(3‐cyclohexyl)propanoic acid dihydrochloride and its methyl, ethyl, and n‐propyl esters were prepared according to the previously reported procedure. All investigated complexes were characterized by IR , ESI ‐MS (¹H, ¹³C, and HMBC ) NMR spectroscopy, and elemental analysis. Their cytotoxic action was investigated in four human tumor cell lines: promyelocytic (HL ‐60) and lymphocytic (REH ) leukemia, glioma (U251), and lung carcinoma (H460). Cell viability was assessed by acid phosphatase and LDH assay, while oxidative stress and cell death parameters were analyzed by flow cytometry. The results showed that novel Pt(II ) complexes exhibited antitumor action superior to precursor ligands, with iodido complexes being more efficient than corresponding chlorido complexes. Human promyelocytic cell line (HL ‐60) was the most sensitive to antitumor action of all investigated substances and was used for investigation of the underlying mode of antileukemic action. The investigated Pt(II ) complexes showed more potent antileukemic action than corresponding Pt(IV ) complex, through induction of oxidative stress and apoptosis, evidenced by caspase (8, 9, and 3) activation and phosphatidylserine externalization.     

Components of the phosphatidylserine endoplasmic reticulum to plasma membrane transport mechanism as targets for KRAS inhibition in pancreatic cancer

KRAS is mutated in 90% of human pancreatic ductal adenocarcinomas (PDACs). To function, KRAS must localize to the plasma membrane (PM) via a C-terminal membrane anchor that specifically engages phosphatidylserine (PtdSer). This anchor-binding specificity renders KRAS–PM localization and signaling capacity critically dependent on PM PtdSer content. We now show that the PtdSer lipid transport proteins, ORP5 and ORP8, which are essential for maintaining PM PtdSer levels and hence KRAS PM localization, are required for KRAS oncogenesis. Knockdown of either protein, separately or simultaneously, abrogated growth of KRAS-mutant but not KRAS–wild-type pancreatic cancer cell xenografts. ORP5 or ORP8 knockout also abrogated tumor growth in an immune-competent orthotopic pancreatic cancer mouse model. Analysis of human datasets revealed that all components of this PtdSer transport mechanism, including the PM-localized EFR3A-PI4KIIIα complex that generates phosphatidylinositol-4-phosphate (PI4P), and endoplasmic reticulum (ER)–localized SAC1 phosphatase that hydrolyzes counter transported PI4P, are significantly up-regulated in pancreatic tumors compared to normal tissue. Taken together, these results support targeting PI4KIIIα in KRAS-mutant cancers to deplete the PM-to-ER PI4P gradient, reducing PM PtdSer content. We therefore repurposed the US Food and Drug Administration–approved hepatitis C antiviral agent, simeprevir, as a PI4KIIIα inhibitor In a PDAC setting. Simeprevir potently mislocalized KRAS from the PM, reduced the clonogenic potential of pancreatic cancer cell lines in vitro, and abrogated the growth of KRAS-dependent tumors in vivo with enhanced efficacy when combined with MAPK and PI3K inhibitors. We conclude that the cellular ER-to-PM PtdSer transport mechanism is essential for KRAS PM localization and oncogenesis and is accessible to therapeutic intervention.

RAS proteins are small GTPases that switch between active GTP-bound and inactive GDP-bound states, regulating cell proliferation, differentiation, and apoptosis. RAS is regulated by guanine nucleotide exchange factors that promote GDP–GTP exchange, thereby activating RAS, and GTPase-activating proteins (GAPs), which stimulate intrinsic RAS GTPase activity to return it to its inactive state. Approximately 20% of human cancers express oncogenic RAS with mutations at residues 12, 13, or 61 (1), which prevent RASGAPs from stimulating GTP hydrolysis, rendering RAS constitutively active. The RAS isoforms, HRAS, NRAS, KRAS4A, and KRAS4B (hereafter referred to as KRAS), have near-identical G-domains, which are implicated in guanine nucleotide binding and effector interaction. However, they have different C termini and membrane anchors, which contribute to their differential signaling outputs (2). KRAS is the most-frequently mutated isoform in cancer and hence represents the major clinical concern, especially in pancreatic, colon, and non–small cell lung cancers (NSCLCs) in which mutant KRAS is expressed in ∼90%, ∼50%, and ∼25% of cases, respectively (3).RAS proteins must localize to the plasma membrane (PM) and organize into nanoclusters for biological activity (4–8), whereby RAS.GTP recruits its effectors to PM nanoclusters, leading to downstream pathway activation. KRAS interacts with the PM via its C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain (PBD) of six contiguous lysines (9–11). Together, the KRAS PBD sequence and prenyl group define a combinatorial code for lipid binding, resulting in a membrane anchor that specifically interacts with asymmetric species of phosphatidylserine (PtdSer) that contain one saturated and one desaturated acyl chain (8, 12–14). Since PtdSer binding specificity is hardwired into its anchor structure, KRAS–PM interactions are PtdSer dependent. KRAS that partitions into the cytosol following endocytosis is captured by PDEδ, which, upon interacting with ARL2, is released to the recycling endosome (RE) for forward transport back to the PM (15). Capture of KRAS by the RE is again PtdSer dependent; therefore, abrogating PtdSer delivery to the PM will reduce PM and RE PtdSer content, abrogating both KRAS PM binding and KRAS recycling back to the PM. In sum, KRAS–PM localization, nanoclustering, and signaling capacity are all exquisitely dependent on PM PtdSer levels.Previous attempts at preventing KRAS–PM localization to inhibit its function include the development of farnesyltransferase inhibitors (FTIs), which inhibit the first posttranslational processing step that generates the KRAS membrane anchor. FTIs were clinically unsuccessful since KRAS can alternatively be geranylgeranylated by geranylgeranyl transferase1 when cells are treated with FTIs, allowing for continued PM localization (2, 16, 17). We recently leveraged the dependence of KRAS on PM PtdSer to inhibit KRAS signaling by targeting the cellular machinery that actively maintains PM PtdSer levels (18). Genetic knockdown (KD) of ORP5 or ORP8, two lipid transporters that function at endoplasmic reticulum (ER)–PM membrane contact sites to transport PtdSer to the PM (Fig. 1), mislocalized KRAS from the PM and reduced nanoclustering of any remaining KRAS. Consequently, ORP5/8 KD decreased proliferation and anchorage-independent growth of multiple KRAS-dependent pancreatic cancer cell lines. In this study, we examine the effects of ORP5/8 genetic KD and knockout (KO) on tumor growth in vivo and provide compelling evidence that these proteins are essential for tumor maintenance in KRAS-dependent pancreatic cancer. ORP5/8 function by exchanging phosphoinositide-4-phosphate (PI4P) synthesized on the PM by PI4KIIIα for PtdSer synthesized in the ER (19, 20). We demonstrate both in vitro and in vivo that PI4KIIIα inhibitors can potently inhibit oncogenic KRAS function. One such inhibitor is simeprevir, a US Food and Drug Administration (FDA)–approved antiviral agent used for the treatment of hepatitis C, that may have potential for repurposing as a therapeutic for mutant KRAS-driven cancers.Open in a separate windowFig. 1.ORP5 and ORP8 transport PtdSer to the PM. ORP5 and ORP8 exchange ER PtdSer with PM PI4P. This is driven by a PI4P concentration gradient whereby PM PI4P levels are kept high by PI4KIIIα and low at the ER by SAC1P, which hydrolyzes PI4P. ORP, oxysterol-binding protein-related protein; PI4KIIIα, class III PI4 kinase alpha; and SAC1P, SAC1-like phosphatidylinositide phosphatase.     

Increased surface phosphatidylserine is an early marker of neuronal apoptosis

Neuronal apoptosis is the subject of intense investigation and is beginning to be understood in some molecular detail. In the present study, we show that PC12 cells, like certain other cell types, redistribute phosphatidylserine (PS) from the inner leaflet to the outer leaflet of the plasma membrane early in the process of apoptosis. The externalised PS can be readily visualised by incubating intact cells with a fluorescent derivative of the protein annexin V. When apoptosis is blocked with an inhibitor of interleukin-1β-converting-enzyme-like proteases, the increased annexin binding is also blocked. Fluorescent annexin V binding provides a rapid and convenient way to identify apoptotic neurones. J. Neurosci. Res. 48:563–570, 1997. © 1997 Wiley-Liss Inc.     

Clinical feature and anti-phospholipid antibody profiles of pregnancy failure in young women with antiphospholipid antibody syndrome treated with conventional therapy

Objective: To elucidate clinical feature and anti-phospholipid antibody (aPL) profiles, including lupus anticoagulant (LA), anti-cardiolipin (CL) antibodies and anti-phosphatidylserine/prothrombin (PS/PT) antibodies, of pregnancy failure in patients with antiphospholipid antibody syndrome (APS) already treated with conventional therapy.

Materials and methods: Thirty-four women with a history of pregnancy who were diagnosed with APS between 2008 and 2016 were included in the study. We defined the successful pregnancy group as women who gave birth to a healthy baby over 1500?g after 34 weeks of pregnancy under conventional treatment (heparin and/or low-dose aspirin). The unsuccessful pregnancy group was defined as women whose pregnancy outcomes did not meet the aforementioned criteria despite the conventional therapy. The clinical features and aPL profiles were compared between the two groups.

Results: Fifteen women were classified into the unsuccessful pregnancy group; seven women were in the successful pregnancy group. Having history of both thrombosis and pregnancy morbidity and LA positivity were significantly more prevalent in the unsuccessful pregnancy group than in the successful pregnancy group (p?<.05, respectively). In contrast, single positivity of anti-CL antibody was negatively associated with APS-associated pregnancy morbidity under the conventional treatment (p?<.01). The proportion of anti-PS/PT IgG-positive patients was significantly higher in the unsuccessful pregnancy group (p?=?.02, OR 18.7, 95% CI 1.50, 232.29) with high concordance rate with LA (97% consistence).

Conclusion: History of both thrombosis and pregnancy morbidity and the positivity of LA and/or anti-PS/PT-IgG, not but anti-CL-antibodies were correlated with APS-associated pregnancy morbidity refractory to conventional treatment. Clinical feature and aPL profiles might help us to make risk assessment for adverse pregnancy outcomes in patients with APS.     

Enniatin B-induced cell death and inflammatory responses in RAW 267.4 murine macrophages

The mycotoxin enniatin B (EnnB) is predominantly produced by species of the Fusarium genera, and often found in grain. The cytotoxic effect of EnnB has been suggested to be related to its ability to form ionophores in cell membranes. The present study examines the effects of EnnB on cell death, differentiation, proliferation and pro-inflammatory responses in the murine monocyte-macrophage cell line RAW 264.7. Exposure to EnnB for 24 h caused an accumulation of cells in the G0/G1-phase with a corresponding decrease in cyclin D1. This cell cycle-arrest was possibly also linked to the reduced cellular ability to capture and internalize receptors as illustrated by the lipid marker ganglioside GM1. EnnB also increased the number of apoptotic, early apoptotic and necrotic cells, as well as cells with elongated spindle-like morphology. The Neutral Red assay indicated that EnnB induced lysosomal damage; supported by transmission electron microscopy (TEM) showing accumulation of lipids inside the lysosomes forming lamellar structures/myelin bodies. Enhanced levels of activated caspase-1 were observed after EnnB exposure and the caspase-1 specific inhibitor ZYVAD-FMK reduced EnnB-induced apoptosis. Moreover, EnnB increased the release of interleukin-1beta (IL-1β) in cells primed with lipopolysaccharide (LPS), and this response was reduced by both ZYVAD-FMK and the cathepsin B inhibitor CA-074Me.In conclusion, EnnB was found to induce cell cycle arrest, cell death and inflammation. Caspase-1 appeared to be involved in the apoptosis and release of IL-1β and possibly activation of the inflammasome through lysosomal damage and leakage of cathepsin B.     

βIIPKC and εPKC isozymes as potential pharmacological targets in cardiac hypertrophy and heart failure

Cardiac hypertrophy is a complex adaptive response to mechanical and neurohumoral stimuli and under continual stressor, it contributes to maladaptive responses, heart failure and death. Protein kinase C (PKC) and several other kinases play a role in the maladaptative cardiac responses, including cardiomyocyte hypertrophy, myocardial fibrosis and inflammation. Identifying specific therapies that regulate these kinases is a major focus of current research. PKC, a family of serine/threonine kinases, has emerged as potential mediators of hypertrophic stimuli associated with neurohumoral hyperactivity in heart failure. In this review, we describe the role of PKC isozymes that is involved in cardiac hypertrophy and heart failure. This article is part of a special issue entitled “Key Signaling Molecules in Hypertrophy and Heart Failure”.     

Effects of sub-chronic clozapine and haloperidol administration on brain lipid levels

Abnormal lipid profiles have been reported in the central nervous system (CNS) in individuals with schizophrenia, although the etiology of these changes remains to be elucidated. While treatment with second-generation antipsychotics has been associated with alterations in peripheral lipid levels and changes in erythrocyte membrane composition, the relationship between peripheral and CNS lipid levels is complex and the effect of antipsychotics on CNS lipid regulation is not yet understood. In this study we investigated whether sub-chronic administration of the second-generation antipsychotic clozapine and the first-generation antipsychotic haloperidol alters brain membrane lipid composition in male Sprague–Dawley rats. The relationship between brain membrane lipid composition and plasma cholesterol concentrations was also assessed. Our results indicate that brain lipid composition and plasma cholesterol concentrations are not altered following administration of antipsychotics. No correlation was observed between plasma and brain membrane cholesterol levels. Our findings suggest that observed alterations in brain lipid profiles in individuals with schizophrenia are not a consequence of treatment with antipsychotic medications.     

Microvesiculation and cell interactions at the brain–endothelial interface in cerebral malaria pathogenesis

Cerebral malaria (CM) is still a major world health problem whose pathogenic mechanisms remain incompletely understood. After reviewing some particularities of anti-malarial immunity, we focus here on the neurovascular aspects of CM. We specifically address the central role of endothelial activation and alteration in disease pathogenesis. We discuss the respective roles of “mediator-induced” versus “host cell-induced” mechanisms of endothelial alteration. The former include cytokines, chemokines and their receptors, while the latter encompass cells located inside and outside the vessel, notably glial cells. We also present evidence for a pathogenic role for membrane microparticles (MP) in CM, based on studies in African patients and in a recognised mouse model. Intervention studies on MP production, via either gene knockout or pharmacological inhibition, can prevent the neurological syndrome and its associated mortality, suggesting potential new therapeutic avenues.