Kinetic Study of the Inhibition of Linoleic Acid Oxidation in Aqueous Media by Phenolic Compounds

The antioxidant activity of several phenolic compounds has been evaluated in terms of the inhibition of the lipid oxidation. The extent of linoleic acid oxidation was monitored by the absorption of the conjugated diene hydroperoxydes formed as a result of oxidation. The antioxidant activity of phenolic compounds was evaluated in aqueous media consisting of negatively-charged micelles of sodium dodecyl sulfate (SDS) or positively-charged micelles of hexadecyl trimethylammonium bromide (HDTBr). 2,2′-azobis-(2-amidinopropane)-dihydrochloride (ABAP) was employed as the oxidation initiator. The assayed phenolic compounds showed good antioxidant efficiency, closely related to the presence of hydroxyl groups and the electron delocalization within the structure, which may stabilize the formed phenoxyl radicals. The kinetic analysis revealed that the oxidizability of linoleic acid was 10-fold higher in SDS than in HDTBr media, which could indicate that oxidation is favoured in negatively charged SDS micelles regarding to positive HDTBr medium. Furthermore, a greater antioxidant efficiency of phenolic compounds was found in HDTBr than in SDS micelles.     

Entire genome of a strain of human immunodeficiency virus type 1 with a deletion of nef that was recovered 20 years after primary infection: large pool of proviruses with deletions of env

We report the complete sequence analysis of the provirus harbored in a long-term nonprogressor (patient SG1) 20 years after the first infection with a human immunodeficiency virus type 1 strain lacking nef. The sequencing showed large deletions in the nef-nef and nef-U3 regions. Except for vpu, all of the other accessory genes were intact. The gag and pol genes did not show significant alterations. We found large deletions in env, spanning the V1, V2, V3, V4, and V5 regions. We believe that, when down-regulation of the class 1 major histocompatibility complex molecules is inhibited by the lack of nef function, the cells containing Env-defective molecules evade cytotoxic T lymphocyte killing and accumulate progressively.     

Dithiocarbamate fungicide thiram detection: comparison of bioluminescent and fluorescent whole-cell bioassays based on hsp22 stress promoter induction

Detection of toxic substances interfering with endocrine system is one of the major preoccupations of the European community. A whole-cell bioassay for pollution detection based on stress induction has been designed. Well characterized toxicants, cadmium chloride and thiram (a dithiocarbamate fungicide), were used to optimize the detection conditions such as time-course conditions, cell line and reporter gene to be used. HeLa cells containing the firefly luciferase (luc) reporter gene under the control of the Drosophila melanogaster hsp22 promoter were compared to liver cells (HepG2) containing the same stress gene promoter fused either to the luc or the EGFP (Enhanced-Green Fluorescent Protein) gene. The sensitivity of the obtained bioassay was found to be enhanced by the concomitant use of liver cells and EGFP reporter gene. The detection limits of the toxicants were then lowered from 1 to 0.1 microM and from 1 to 0.01 microM for CdCl(2) and thiram, respectively.     

Lipoprotein lipase: A bioinformatics criterion for assessment of mutations as a risk factor for cardiovascular disease

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. Decrease of the LPL enzymatic activity leads to elevated triglycerides (TG) and reduced high-density lipoprotein (HDL-C levels), both risk factors for cardiovascular disease (CVD). Therefore, mutations, which decrease the LPL activity, may confer susceptibility to CVD. Here, the informational spectrum method (ISM), a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied for identification of evolutionary highly conserved information encoded by the primary structure of LPL. It was demonstrated that mutations, which alter the LPL enzymatic activity also alter this information. On the basis of this finding, an efficient and simple bioinformatics criterion for assessment of the pathogenic effect of LPL nonsynonymous single nucleotide substitution as a risk factor of CVD has been proposed.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

HSP27 inhibits cytochrome c-dependent activation of procaspase-9.

We have previously shown that the small heat shock protein HSP27 inhibited apoptotic pathways triggered by a variety of stimuli in mammalian cells. The present study demonstrates that HSP27 overexpression decreases U937 human leukemic cell sensitivity to etoposide-induced cytotoxicity by preventing apoptosis. As observed for Bcl-2, HSP27 overexpression delays poly(ADP-ribose)polymerase cleavage and procaspase-3 activation. In contrast with Bcl-2, HSP27 overexpression does not prevent etoposide-induced cytochrome c release from the mitochondria. In a cell-free system, addition of cytochrome c and dATP to cytosolic extracts from untreated cells induces the proteolytic activation of procaspase-3 in both control and bcl-2-transfected U937 cells but fails to activate procaspase-3 in HSP27-overexpressing cells. Immunodepletion of HSP27 from cytosolic extracts increases cytochrome c/dATP-mediated activation of procaspase-3. Overexpression of HSP27 also prevents procaspase-9 activation. In the cell-free system, immunodepletion of HSP27 increases LEDH-AFC peptide cleavage activity triggered by cytochrome c/dATP treatment. We conclude that HSP27 inhibits etoposide-induced apoptosis by preventing cytochrome c and dATP-triggered activity of caspase-9, downstream of cytochrome c release.     

Surface mechanomyogram reflects the changes in the mechanical properties of muscle at fatigue

The contractile properties of muscle are usually investigated by analysing the force signal recorded during electrically elicited contractions. The electrically stimulated muscle shows surface oscillations that can be detected by an accelerometer; the acceleration signal is termed the surface mechanomyogram (MMG). In the study described here we compared, in the human tibialis anterior muscle, changes in the MMG and force signal characteristics before, and immediately after fatigue, as well as during 6 min of recovery, when changes in the contractile properties of muscle occur. Fatigue was induced by sustained electrical stimulation. The final aim was to evaluate the reliability of the MMG as a tool to follow the changes in the mechanical properties of muscle caused by fatigue. Because of fatigue, the parameters of the force peak, the peak rate of force production and the peak of the acceleration of force production (d2F/dt2) decreased, while the contraction time and the half-relaxation time (1/2-RT) increased. The MMG peak-to-peak (p-p) also decreased. The attenuation rate of the force oscillation amplitude and MMG p-p at increasing stimulation frequency was greater after fatigue. With the exception of 1/2-RT, all of the force and MMG parameters were restored within 2 min of recovery. A high correlation was found between MMG and d2F/dt2 in un-fatigued muscle and during recovery. In conclusion, the MMG reflects specific aspects of muscle mechanics and can be used to follow the changes in the contractile properties of muscle caused by localised muscle fatigue.     

Heat shock protein is a unique marker of growth arrest during macrophage differentiation of HL-60 cells

Prior to morphologic and functional maturation, terminally differentiating hematopoietic cells first exit the cell cycle and undergo growth arrest. Relatively little is known about which molecules regulate differentiation-induced growth arrest. In the present report, we sought to determine whether the mammalian low molecular weight heat shock protein (hsp28) was a candidate growth-regulatory molecule during human hematopoiesis. To this end, hsp28 protein expression was examined during phorbol ester (PMA)-induced macrophage differentiation of the human HL-60 promyelocytic leukemic cell line. Whereas hsp28 was constitutively expressed at relatively low levels in an unphosphorylated state, hsp28 was rapidly phosphorylated within 4 hr following PMA-induced differentiation, preceding increased hsp28 protein levels at 24–48 h. In contrast to other differentiative agents, hsp28 steady state mRNA and protein were regulated concordantly in response to macrophage differentiation. More importantly, these changes were transient, and occurred concomitant with the down-regulation of cellular proliferation and the onset of G₁ phase cell cycle arrest. In total, these observations implicate hsp28 as an intermediary in the myelomonocytic differentiative pathway of promyelocytic leukemic cells, and will shed light on the events regulating this process. © 1993 Wiley-Liss, Inc.