Optical trapping microrheology in cultured human cells

We present the microrheological study of the two close human epithelial cell lines: non-cancerous HCV29 and cancerous T24. The optical tweezers tracking was applied to extract the several seconds long trajectories of endogenous lipid granules at time step of 1μs. They were analyzed using a recently proposed equation for mean square displacement (MSD) in the case of subdiffusion influenced by an optical trap. This equation leads to an explicit form for viscoelastic moduli. The moduli of the two cell lines were found to be the same within the experimental accuracy for frequencies 102 - 105 Hz. For both cell lines subdiffusion was observed with the exponent close to 3/4, the value predicted by the theory of semiflexible polymers. For times longer than 0.1s the MSD of cancerous cells exceeds the MSD of non-cancerous cells for all values of the trapping force. Such behavior can be interpreted as a signature of the active processes and prevents the extraction of the low-frequency viscoelastic moduli for the living cells by passive microrheolog

Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction in Atherosclerotic Lesion

This is the final version of the article. Available from Hindawi Publishing Corporation via the DOI in this record.The infiltration of red blood cells into atheromatous plaques is implicated in atherogenesis. Inside the lesion, hemoglobin (Hb) is oxidized to ferri-and ferrylHb which exhibit prooxidant and proinflammatory activities. Cystathione gamma-lyase-(CSE-) derived H 2 S has been suggested to possess various antiatherogenic actions. Expression of CSE was upregulated predominantly in macrophages, foam cells, and myofibroblasts of human atherosclerotic lesions derived from carotid artery specimens of patients. A similar pattern was observed in aortic lesions of apolipoprotein E-deficient mice on high-fat diet. We identified several triggers for inducing CSE expression in macrophages and vascular smooth muscle cells including heme, ferrylHb, plaque lipids, oxidized low-density lipoprotein, tumor necrosis factor-α, and interleukin-1β. In the interplay between hemoglobin and atheroma lipids, H 2 S significantly mitigated oxidation of Hb preventing the formation of ferrylHb derivatives, therefore providing a novel function as a heme-redox-intermediate-scavenging antioxidant. By inhibiting Hb-lipid interactions, sulfide lowered oxidized Hb-mediated induction of adhesion molecules in endothelium and disruption of endothelial integrity. Exogenous H 2 S inhibited heme and Hb-mediated lipid oxidation of human atheroma-derived lipid and human complicated lesion. Our study suggests that the CSE/H 2 S system represents an atheroprotective pathway for removing or limiting the formation of oxidized Hb and lipid derivatives in the atherosclerotic plaque.The research group is supported by the Hungarian Academy of Sciences (11003). This work was supported by Hungarian Government Grants (OTKA) K112333 (József Balla), K109843 (Péter Nagy), and K116024 (Viktória Jeney) and Marie Curie International Reintegration Grant PIRG08-GA-2010-277006 (Péter Nagy). Péter Nagy is a János Bolyai Research Scholar of the Hungarian Academy of Sciences. Viktória Jeney was supported by Zoltán Magyary Fellowship (TÁMOP 4.2.4.A/2-11/1-2012-0001). László Potor was supported by János Apáczai-Csere Fellowship (TÁMOP 4.2.4.A/2-11/1-2012-0001). The project was cofinanced by the European Union and the European Social Fund (ESF) GINOP-2.3.2-15-2016-00043 IRONHEARTH and EFOP-3.6.2-16-2017-00006 LIVE LONGER

Hydrogen Sulfide Abrogates Hemoglobin-Lipid Interaction In Atherosclerotic Lesion

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Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from M. marinum (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs

N-acetylcysteine reduces oxidative stress in sickle cell patients

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSβ0-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress