Hsp70 protects against UVB induced apoptosis by preventing release of cathepsins and cytochrome c in human melanocytes.

Stress-induced heat shock protein 70 (Hsp70) effectively protects cells against apoptosis, although the anti-apoptotic mechanism is still undefined. Exposure of human melanocytes to heat and subsequent UVB irradiation increased the level of Hsp70 and pre-heating reduced UVB induced apoptosis. Immunofluorescence staining of Hsp70 in combination with staining of lysosomes (Lamp2) or mitochondria (Mitotracker) in pre-heated UVB exposed cells showed co-localization of Hsp70 with both lysosomes and mitochondria in the surviving cell population. Furthermore, UVB induced apoptosis was accompanied by lysosomal and mitochondrial membrane permeabilization, detected as release of cathepsin D and cytochrome c, respectively, which were prevented by heat pre-treatment. In purified fractions of lysosomes and mitochondria, recombinant Hsp70 attached to both lysosomal and mitochondrial membranes. Moreover, in apoptotic cells Bax was translocated from a diffuse cytosolic location into punctate mitochondrial-like structures, which was inhibited by Hsp70 induction. Such inhibition of Bax translocation was abolished by transfection with Hsp70 siRNA. Furthermore, Hsp70 siRNA eliminated the apoptosis preventive effect observed after pre-heating. These findings show Hsp70 to rescue melanocytes from UVB induced apoptosis by preventing release of cathepsins from lysosomes, Bax translocation and cytochrome c release from mitochondria.

Modulation of macrophage structure and function by low level He-Ne laser irradiation.

Studies have shown that He-Ne laser irradiation can affect the biological functions of macrophages. The aim of the present study was to investigate the effect of He-Ne laser irradiation on the various functional parameters of macrophages and look for possible correlations in the effects to understand the mechanisms involved. Mice peritoneal macrophages were irradiated with a He-Ne laser (632.8 nm, approximately 10 W m(-2)) at energy densities ranging from 100 to 600 J m(-2) and the activities of lysozyme and cathepsin, phagocytosis, and cell spreading (markers of cell activation), as well as changes in NAD(P)H autofluorescence, were monitored. He-Ne laser irradiation was observed to lead to significant changes in all the parameters investigated. While lysozyme activity and spreading of the peripheral membrane were found to increase with the irradiation dose over the dose range investigated, the phagocytotic activity of macrophages, the activity of cathepsin, the observed decease in cell membrane fluidity and the observed increase in NAD(P)H level showed a peak at 200 J m(-2). Possible reasons for and the significance of the observed correlations are discussed.

UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin.

Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.

[Effects of ionizing radiation on the content of total collagen and its fractions and the activity of collagenolytic enzymes in rat tissues]. / Wplyw promieniowania jonizujacego na zawartosc kolagenu calkowitego i jego frakcji oraz aktywnosc enzymów kolagenolitycznych w tkankach szczura.

Changes in collagen fractions as well as in the activity of leucocytic collagenase and collagenolytic cathepsin of rats after 30 days of gamma-rays exposure were examined. A statistically significant decrease in total collagen content resulting from the reduction of salt--soluble (NSC) and acid--soluble (ASC) collagen fractions was found. An increased content of insoluble collagen fraction (ISC) may confirm the opinion about stimulative gamma-rays influence upon cross--links formation. An increase in collagenolytic enzymes activity may account for an elevated degradation of collagen; with a contribution of leucocytic collagenase and collagenolytic cathepsin. Perhaps the quantitative increase in insoluble collagen stimulates elevated biosynthesis of collagenolytic enzymes determining the process of protein degradation. Further investigations are needed to explain the radiobiological effects of gamma-rays on the connective tissue.

[Catalytic properties of cathepsin D from the brains of normal and irradiated animals]. / Kataliticheskie svoistva katepsina D iz mozga normal'nykh i obluchennykh zhivotnykh.

Biospecific sorbent, hemoglobin-biogel P-300, was used for purification of cathepsin D from the brain and spleen of a cat and from the brain of normal and irradiated rats. 800 R irradiation of rats in 7 days causes changes in the catalytic properties of cathepsin D: shift of the pH-optimum of the activity, increase in the enzyme affinity to the substrate (hemoglobin) and inhibitor (pepstatin), changes in the activation energy. These changes may be due to the destruction of the processes of posttranscriptional modification of the enzymes at the late stage of the radiation pathology. The temperature dependence of the enzyme reaction catalyzed by cathepsin D from different tissues expressed in the Arrhenius coordinates is characterized by changes in the activation energy in the high-temperature region beginning with the critical temperature (26-30 degrees C). The results obtained may be explained by the presence of cathepsin E admixtures in the purified cathepsin D preparation (in spleen) or by the presence of cathepsin D isoforms catalyzing hemoglobin hydrolysis with different activation energy.

The influence of protease inhibitor on hydroproteolytic activity and serotonin content in the intestine of rats after fractionated irradiation.

Hydroproteolytic activity and content of serotonin in the small intestine and colon were studied in rats X-irradiated on the whole body with fractionated doses of 5 X 150 R. Twenty-four hours after exposure to X-rays, lipase, acid phosphatase activities and cathepsin activity as well as serotonin content were assayed in the intestinal tissues. Irradiation altered the hydroproteolytic activity of the tissues, in a majority of cases by increasing hydrolytic activity and lowering proteolytic (catheptic) activity in all parts of the small intestine and decreased serotonin content in the first and third segments of the small intestine. Trasylol injected s.c. in doses of 10,000 units/kg before and after irradiation modified hydrolytic tissue activity of the studied segments of the intestine and lessened the fall in serotonin content of the intestines after irradiation.

Serotonin and proteolytic enzymes in brain of irradiated animals.

Early and late irradiation effects on biogenic amines (5-HT and NA) contents, intracellular hydrolytic enzyme activity and MAO in brain tissue was studied on mice and rats. In mice in the majority of cases (irradiated on the first day after birth) there occurs a remote effect of irradiation in the form of the increase in 5-HT content and different, species-dependent, changes in hydrolytic activity. Among early effects (24 hrs following the irradiation) the decrease in 5-HT and NA contents was stated in rats. These changes were accompanied by the increase in MAO and hydrolytic (proteolytic) activities.