Yoram Soroka

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin

Background:  Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

Background:  Ultraviolet (UV) irradiation is a major cause of skin damage, of long‐term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV‐induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non‐invasive procedures.

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas‐mediated apoptosis. Here, we describe the induction of the Fas‐dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV‐IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV‐IgG‐mediated apoptotic death and culture lesions. Together with immunosenescence, this phenomenon may explain the late onset of pemphigus disease.

Ovarian protein synthesis in the prawn Macrobrachium rosenbergii: Does ovarian vitellin synthesis exist?

Summary During the reproductive cycle oocytes of Macrobrachium rosenbergii females grow in diameter from 20 μm to 650 μm, while the gonado-somatic index increases from 0.2 to 8.0. SDS-PAGE polypeptide profiles of the cytosolic fraction from ovaries in various stages were studied. Specific vitellin bands were identified by immunoblotting. In vitro synthesis of protein was measured by TCA precipitation and autoradiography of SDSPAGE separated polypeptides. Incorporation of [35S]-methionine and cysteine was highest in animals having an oocyte diameter of 50–150 pm and lower in pre-vitellogenic ovaries (20–50 μm). The lowest synthesis was found in late-vitellogenic ovaries (300–500 μm). Most of the incorporation was found in non-vitellin polypeptide bands. These results suggest the existence of an extraovarian source of vitellogenin in M. rosenbergii.

Aged keratinocyte phenotyping : Morphology, biochemical markers and effects of Dead Sea minerals

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging. We showed that the expression of 16 biochemical markers was altered in aged cultured cells and in tissues, including caspases 1 and 3 and beta-galactosidase activities, immunoreactivities of p16, Ki67, 20S proteasome and effectors of the Fas-dependent apoptotic pathway. Aged cells diversity, and individual variability of aging markers, call for a multifunctional assessment of the aging phenomenon, and of its modulation by drugs. As a test case, we have measured the effects of Dead Sea minerals on keratinocyte cultures and human skin, and found that they stimulate proliferation and mitochondrial activity, decrease the expression of some aging markers, and limit apoptotic damage after UVB irradiation.

High resolution SEM imaging of gold nanoparticles in cells and tissues.

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.

The hepatopancreas as a site of yolk protein synthesis in the prawn Macrobrachium rosenbergii

Summary Our previous study failed to show vitellin synthesis in the ovary of the prawn Macrobrachium rosenbergii (Sagi et al., 1995); thus the role of the hepatopancreas as a possible site of synthesis was evaluated. Extracts of hepatopancreas and hemolymph of a secondary-vitellogenic female exhibited higher levels of yolk protein than those from a primary-vitellogenic female. Clear vitellin immuno-cross-reactivity was observed in hepatopancreas sections from a secondary-vitellogenic female while no such reaction was found in a male hepatopancreas. Furthermore, vitellin-immuno-cross-reactive polypeptides released into the culture medium of the hepatopancreas of a secondary-vitellogenic female were similar to those found in the hemolymph and ovary (92 and 105kDa). The most prominent immuno-reactive polypeptide in the hepatopancreas extract was a relatively low-molecular-weight species (42kDa). De novo synthesis of cross-reactive-vitellin polypeptides (34, 38 and 42kDa) was detected in the hepatopancreas of a secondary-vitellogenic female. Synthesis of these polypeptides were not detected in the secondary-vitellogenic ovary or in the male hepatopancreas. The appearance of similar polypeptides following incubation of a secondary-vitellogenic ovarian extract with a glycosidase suggests that these polypeptides could be subunits of a core protein of vitellogenin, which was synthesized in the hepatopancreas and then released to the hemolymph following post-translational modifications. Our findings thus suggest the hepatopancreas to be a likely site of synthesis of a yolk protein precursor in M. rosenbergii.

Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests. Nanoparticles were more toxic than micro-sized particles, and their effects were stronger when supplied in growth medium than in topical application. Still topically applied CuO nanoparticles induced inflammatory cytokine secretion and necrosis, especially in epidermis deprived of its protective cornea. Since nanoparticle penetration was not seen, we propose that they may adhere to skin surface, react with the local acidic environment, and generate soluble ions that make their way to inner sites. This work illustrates the abilities of skin organ culture to evaluate the biological effects of topically-applied materials on skin in vitro.

Non-invasive skin biomarkers quantification of psoriasis and atopic dermatitis: cytokines, antioxidants and psoriatic skin auto-fluorescence.

BACKGROUND Psoriasis and atopic dermatitis (AD) are challenging to treat due to the absence of suitable monitoring procedure and their recurrences. Alteration of skin hydrophilic biomarkers (SHB) and structural elements occur in both disorders and may possess a distinct profile for each clinical condition. OBJECTIVE To quantify skin cytokines and antioxidants non-invasively in psoriatic and in AD patients and to evaluate skin auto-fluorescence in psoriatic patients. METHODS A skin wash sampling technique was utilized to detect the expression of SHB on psoriatic and AD patients and healthy controls. Inflammatory cytokine (TNFα, IL-1α and IL-6) levels, total antioxidant scavenging capacity and uric acid content were estimated. Additionally, measurement of the fluorescent emission spectra of tryptophan moieties, collagen cross-links and elastin cross-links were performed on psoriatic patients and healthy controls. RESULTS Our findings demonstrate significant alterations of the SHB levels among psoriasis, AD and healthy skin. Differences were also observed between lesional and non-lesional areas in patients with psoriasis and AD. Ultra-structural changes were found in psoriatic patients both in lesional and non-lesional areas. CONCLUSION Employing non-invasive measurements of skin wash sampling and skin auto-fluorescence might serve as complementary analysis for improved diagnosis and treatment of psoriasis and AD. Furthermore, they may serve as an additional monitoring tool for various diseases, in which skin dysfunction is involved.

Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected. The observed velocities in situ (500pmoles/min/mg protein) were comparable to the best values of proteasome activity in crude cellular extracts. These features altogether allowed to identify the in situ activity as that of proteasome. To characterize proteasome complexes present in human keratinocytes, we analyzed cellular lysates by ultracentrifugation and gel filtration: most proteasome activity was associated with PA700-bound, presumably 26S, particles. PA28 activator was detected only when cells were treated by gamma interferon. Proteasome activities were determined using the in situ method in keratinocytes at different stages of replicative senescence. Only a slight decrease of proteasome activity per cell was seen at intermediate passages, followed by a slight increase in senescent cells. In the same time, the amount of total proteins increased notably with cellular ageing. Thus, proteasome activity decreased relatively to total proteins, but not relatively to cell numbers. Flow cytometry confirmed that the size of aged keratinocytes increased with the ageing marker beta-galactosidase.