Francesco Cajone

Oxidative stress induces a subset of heat shock proteins in rat hepatocytes and MH1C1 cells

Lipoperoxidative damage caused by exposure of isolated hepatocytes or cultivated hepatoma cells to ADP-iron or to 4-hydroxynonenal induces the synthesis of some proteins which are different under these two conditions but are always a subset of the proteins induced in each type of cells upon heat-shock (heat-shock proteins). For at least one of these proteins (hsp 31), induced by 4-hydroxynonenal, the increase is dose-dependent and the effect of heat and the chemical seems to be additive. Lipoperoxidation may be implicated in the induction of some of the heat shock proteins, but reproduces only incompletely the response of protein synthesis typical of heat-shock conditions.

Structural and functional changes in polysomes from ischemic livers

Abstract Some aspects of the state of aggregation and functioning of polysomes have been investigated in preparations obtained from rat liver lobes subjected to ischemia. The incroporation of amino acid into protein is reduced in postmitochondrial supernatant fractions, microsomes, and C-ribosomes from ischemic livers. In the presence of cell sap from ischemic livers, normal C-ribosomes, but not normal microsomes, show a reduced incorporating capacity, which is not affected by a fivefold increase of the amount of cell sap in the incubation mixture. The analysis of the polysomal patterns shows that ischemia causes a progressive disaggregation of polyribosomes, with a reduction in the number of heavy aggregates and an increase of dimers and monomers. This disaggregation is best seen in the ribosomes recovered from the fraction bound to the membranes of the endoplasmic reticulum. The determination of RNA -used as a reference basis of the various subcellular fractions—shows that ribosomes are also reduced in number in preparations from ischemic livers. Bound ribosomes are more affected than the free forms. On the whole, ribosomes obtained from ischemic livers seem to be reduced in number, less aggregated, and less efficient in carrying out protein synthesis.

Structural requirements of aldehydes produced in LPO for the activation of the heat-shock genes in Hela cells

The ability of various aldehydes, some of which are produced in lipid peroxidation, to effect heat-shock gene expression and heat-shock proteins synthesis was evaluated in HeLa cells. Only (E)-4-hydroxyalk-2-enals were active both in racemic and homochiral form. Between the reported primary metabolic products of (E)-4-hydroxynon-2-enal, only the glutathione conjugates were active, whereas (E)-4-hydroxynon-2-enoic acid and 2-nonen-1,4-diol were inactive. Also, unnatural (E)-5-hydroxynon-2-enal and (E)-5-hydroxyhex-2-enal were active, whereas (E)-6-hydroxynon-2-enal was inactive. Thus, it was established that the active aldehydic compounds must possess an (E)-2 double bond and an hydroxy group in a position suitable for the formation of a cyclic hemiacetal in a possible adduct of these aldehydes with proteins. An irreversible binding to proteins could be the first step of the mechanism by which these compounds exert their biological activity.

State and function of liver polysomes during recovery from ischemia

Abstract The reestablishment of blood supply to the liver lobes which have suffered ischemia is followed by an increase in protein synthesis only if ischemia did not last longer than 60 minutes. The increase is accompanied by the recovery of a normal polyribosomal pattern. Treatment of the rats with actinomycin D does not prevent this restoration. In the early phase of the reestablishment of circulation there is a sudden formation of monomers. These monosomes respond well to polyuridylic acid (poly-U) in vitro . The monosome formation can be prevented by treatment of the animal with cycloheximide. Treatment with cycloheximide also prevents the disappearance of the polysomal shoulder during ischemia. The results support the hypothesis that the lowering of the polysomal shoulder and the formation of monosomes are not the effect of ribonuclease action, but the consequence of an interference with recycling of ribosomes on mRNA. The recovery of a normal polysomal pattern after ischemia does not depend on the synthesis of new mRNA.

Hyperthermia inhibits cell proliferation and induces apoptosis: Relative signaling status of P53, S100A4, and Notch in heat sensitive and resistant cell lines

The effects of hyperthermia on the expression of p53, the apoptosis‐associated genes Bax and Bcl‐2, Notch and S100A4 have been studied in the HepG2 cell line and the HUT cell line derived from HepG2, adapted for growth in hyperthermic conditions. Hyperthermia inhibits cell proliferation and induces apoptosis. HepG2 and HUT cells differed in respect of anchorage to growth surface, degree of proliferation and apoptosis and expression of p53, Bax, Bcl‐2, Notch, and S100A4 genes. The induction of apoptosis and the inhibition of cell proliferation occurred independently of p53, and independently also of involvement of the apoptosis family genes Bax and Bcl‐2. We demonstrate novel and marked differences between transient heat shock and heat adaptation in respect of pathways of signaling and generation of phenotypic effects in vitro. Different signaling patterns have been identified here. Pathways of signaling by S100A4, by its interaction with and sequestration of p53, and by Notch also seem differentially operational in the induction of apoptosis, and both appear to be activated as alternative pathways in the context of hyperthermia signaling independently of p53. J. Cell. Biochem. 103: 212–220, 2008.

In vitro activation of heat shock transcription factor by 4-hydroxynonenal

In the activation of eukaryotic heat shock genes, the acquisition of a binding ability to specific DNA sequence by a transcriptional activator, heat shock factor (HSF), is believed to be a crucial step. The induction of this new DNA binding activity of HSF is also obtained in a cell-free system (in vitro activation) by hyperthermia or at physiological temperature by calcium ions, low pH, urea, or non-ionic detergent. We report here the in vitro activation of HSF by treating at 0 degrees C a HeLa cell-free system with the aldehyde 4-hydroxynonenal (HNE), a highly cytotoxic product of lipid peroxidation. The in vitro activation of HSF by HNE occurred only if some components of the cell-free system were not sedimented at 100,000 x g. The reason for this is unclear but the release of active HSF from nuclei of unshocked cells and the involvement of Ca2+ contained in the mitochondria and ER have been excluded. Although HNE is known to be a sulfhydryl blocking agent, the results obtained with N-ethylmaleimide suggest that different mechanisms might be involved in the in vitro activation of HSF by HNE.

Further studies on ribosomal damage in liver ischemia

Abstract In the ribosomal populations obtained from 120-minute ischemic livers the number of particles which react with labeled puromycin (active ribosomes) is reduced; this confirms the results obtained by density gradient centrifugation. The endogenous incorporation of phenylalanine is lowered in unfractionated ribosomal preparations from 120-minute ischemic livers: on the contrary, polyuridylic acid (poly-U) directed phenylalanine incorporation is normal, and the + poly-U/-poly-U ratio doubles in ischemic ribosomes. Both isolated polysomes of definite chain length (n = 4,5) and isolated monosomes from 120-minute ischemic livers synthesize less polyphenylalanine than their normal counterparts. Together with the results of some side experiments, these observations seem to indicate a percentage increase in the number of monosomes, a slowing down of the ribosomal run-off and an intrinsic defect of the native monosomes from ischemic liver cells: the latter result could explain the hampered reutilization of monomers in the ribosomal cycle. The Mg2+ requirements for maximal incorporation do not change in ischemic ribosomes. All the multiple defects caused by ischemia in the protein-synthetic machinery accumulate in liver slices, where the derangement in protein synthesis is more severe than in any other preparation tested up to now.

Enzymatic resolution of the ethyl acetals of (R)- and (S)-4-hydroxyalk-2-ynals

Abstract 4-hydroxyalk-2-ynals diethylacetals have been efficiently resolved by enatioselective acetylation mediated by Pseudomonas fluorescens lipase affording the acetylated ( R )-enantiomers in preference, independent of the length and bulk of the alkyl substituent at the carbinol group. Reduction with LiAlH 4 of unreacted or acetylated isomers affords the corresponding ethylenic acetals with the hydrogen deriving from the hydride in the β position in respect to the allylic hydroxyl.

Synthesis of all four isomers of (E)-4,5-dihydroxydec-2-enal using osmium-catalysed asymmetric dihydroxylation

Abstract The enantioselective synthesis of the four possible isomers of ( E )-4,5-dihydroxydec-2-enal, a a cytotoxic product formed in peroxidised liver microsomal lipids, is accomplished via a Sharpless AD reaction alone or associated as appropriate with a regioselective epimerisation of one of the introduced hydroxy groups.

The first synthesis of threo- and erythro-(E)-4,5-dihydroxydec-2-enals carbonyls related to the peroxidation of liver microsomal lipids

Abstract The first synthesis of threo and erythro (E)-4,5-dihydroxydec-2-enals, aldehydes related to the lipid peroxidation is accomplished by reaction of α-benzoyloxheptanal with the Grignard reagent of 3,3-diethoxy-1-propyne and lithium aluminum hydride reduction. Acetonization of the diol system allows a simple chromatographic separation of the diastereoisomers, the geometry of which was established by 1 H and 13 C NMR experiments and was confirmed by an independent synthesis of (E,4R,5R)-4,5-O-isopropylidene-4,5-dihydroxydec-2-enal from D-mannitol.