Osteopontin facilitates ultraviolet B-induced squamous cell carcinoma development.

BACKGROUND: Osteopontin (OPN) is a matricellular glycoprotein that is markedly expressed in cutaneous squamous cell carcinomas (cSCCs) and in actinic keratoses implicating its role in photocarcinogenesis. OBJECTIVE: To determine whether OPN facilitates the development of cSCC and its function. METHODS: cSCCs development was compared between wild-type (WT) and OPN-null mice subjected to UVB irradiation for 43 weeks. UVB-induced OPN expression was determined by Western blot, immunoprecipitation, ELISA, and semi-quantitative RT-PCR. Epidermal layer and TUNEL analyses assessed if OPN mediates UVB-induced epidermal hyperplasia or suppresses UVB-induced apoptosis of basal keratinocytes, respectively. In vitro experiments determined whether OPN enhances cell survival of UVB-induced apoptosis and its potential mechanisms. Immunohistochemical analyses of epidermis assessed the expression of CD44 and focal adhesion kinase (FAK), molecules that mediate OPN survival function. RESULTS: Compared to female WT mice, OPN-null mice did not develop cSCCs. UVB irradiation stimulated OPN protein expression in the dorsal skin by 11h and remains high at 24-48h. OPN did not mediate UVB-induced epidermal hyperplasia; instead, it protected basal keratinocytes from undergoing apoptosis upon UVB exposure. Likewise, the addition of OPN suppressed UVB-induced OPN-null cSCC cell apoptosis, the activation of caspase-9 activity, and increased phosphorylation of FAK at Y397. Furthermore, the expression of CD44 and FAK in WT mice epidermis was greater than that of OPN-null mice prior to and during early acute UVB exposure. CONCLUSION: These data support the hypothesis that chronic UVB-induced OPN expression protects the survival of initiated basal keratinocytes and, consequently, facilitates cSCC develop.

Genistein and genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat.

Cataract-related loss of vision affects large numbers of people in today's aging populations and presents a healthcare burden to many nations. The role of dietary supplements within the lens is largely unknown, although benefits from dietary anti-oxidants are expected. In this study, the effects of genistein as its aglycone, a genistein-containing dietary supplement (Novasoy(®)200), and a genistein-containing food (soy protein isolate, PRO-FAM 932) on the development of lens opacity were examined in the hereditary cataractous ICR/f rat. These studies were carried out in a background diet of semi-purified, isoflavone-free AIN-76A with casein as its protein source. The amount of genistein for the experimental diets was standardized to its concentration (as genistein aglycone as well as simple and complex ß-glucoside conjugates) in the soy protein isolate supplement. Also tested was a high-dose genistein diet containing an 11-fold higher amount of genistein aglycone. The composition of each diet was verified by reverse-phase HPLC and blood plasma isoflavone concentrations were determined by LC-tandem mass spectrometry. The development of opacity in each lens was monitored and digitally recorded using slit-lamp examination over the course of the study. Each of the genistein-containing diets caused a significantly more rapid development of fibrous opacification in the anterior cortical region and development of apparent water clefts or vacuoles in the posterior subcapsular region than the AIN-76A control diet; however, the establishment of dense lens opacification was not significantly different between each of the diets. There was also no significant difference observed between the low-dose and high-dose genistein aglycone groups. These data suggest that genistein-containing dietary supplements accelerate the early stages of cataractogenesis in the male ICR/f rat, with no dose-dependent effects.

Isolation, identification, and complete genome sequence of an avian reticuloendotheliosis virus isolated from geese.

Naturally occurring lymphoreticular tumors in geese have been found from time to time in Taiwan, but their etiology has not been determined except through morphological descriptions. This study observed a reticuloendotheliosis virus (REV) infection occurring in a white Roman goose (Anser anser) farm in Yunlin, Taiwan in 2006. These geese showed growth-retarded and nodular lymphoma-like tumors in the liver, lung, kidney, and pancreas. Thirty blood samples were taken for REV detection and 21 (70%) of them contained REV genetic sequences using polymerase chain reaction (PCR). Virus isolation was attempted from 11 blood samples by inoculating the buffy coat onto DF1 cells. Nine (81%) REVs were isolated after three blind passages. The complete proviral sequence from one isolate was determined for phylogenetic analysis by direct sequencing using overlapping PCR products. The length of the provial genome is 8284 nucleotides. By comparing with other published REV complete sequences, the nucleotide percent identity ranged from 93.5% to 99.8% with most LTR varieties, ranging from 74.9% to 99.8%. The present isolated goose REV is most close to REV APC-566, a REV isolated from Attwater's Prairie chickens.

In vivo detection of secreted proteins from wounded skin using capillary ultrafiltration probes and mass spectrometric proteomics.

The identification of in vivo secreted proteins is a major challenge in systems biology. Here we report a novel technique using capillary ultrafiltration (CUF) probes to identify the secreted proteins involved in wound healing. CUF probes, which use semipermeable membrane hollow fibers to continuously capture secreted proteins, were used to sample skin wound fluids. To identify low-abundance proteins, we digested the CUF probe-collected wound fluid with trypsin and then directly subjected it to MS without using 2-DE separation. Two protein fragments with masses of 1565.7 and 1694.8 Da were identified by MS as peptides of thymosin beta10 and beta4, respectively. This is the first identification of thymosin beta10 as an in vivo constituent of the skin wound fluid. The LKKTETQ peptide, a common actin-binding domain of thymosin beta4 and beta10, significantly enhanced skin wound healing in vitro and in vivo. Our data suggest that the enhancement of wound healing by LKKTETQ may be mediated by purinergic receptors. The technique of using CUF probes linked to mass spectrometric proteomics represents a powerful method to identify in vivo secreted proteins, and may be applicable for identification of proteins relevant in various human diseases.

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements.

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.

In vivo protein sampling using capillary ultrafiltration semi-permeable hollow fiber and protein identification via mass spectrometry-based proteomics.

Here, we advanced a novel technique using capillary ultrafiltration (CUF) probes to collect in vivo secreted proteins in the subcutaneous tissue of mouse ear. We fabricated two kinds of CUF probe, one with and one without a semi-permeable membrane hollow fiber. Proteins collected by CUF probes were profiled and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MADLI-TOF-MS) and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS) without using two-dimensional gel electrophoresis (2-DE) separation. Five proteins including cofilin-1, futuin-A, complement C3, gelsolin, and apolipoprotein C-1 were identified from the sample collected by the CUF probe with a semi-permeable membrane hollow fiber. The presence of well documented secretory proteins supports the efficiency of CUF probes in sampling in vivo secreted proteins. We also found that hemoglobin collected by the CUF probe without a semi-permeable membrane hollow fiber completely masked protein identification by mass spectrometry. The presence of relatively large amounts of hemoglobin in this condition illustrates the necessity of the semi-permeable membrane hollow fiber to the technique of CUF probe in conjunction with mass spectrometry. Also, the technique represents a powerful method for the identification of in vivo secreted proteins and has potential application for in the detection of biomarkers for human diseases.

Surfactant sodium lauryl sulfate enhances skin vaccination: molecular characterization via a novel technique using ultrafiltration capillaries and mass spectrometric proteomics.

The skin is a highly accessible organ and thus provides an attractive immune environment for cost-effective, simple, and needle-free delivery of vaccines and immunomodulators. In this study, we pretreated mouse skin with an anionic surfactant, sodium lauryl sulfate (SLS), for a short period of time (10 min) followed by epicutaneous vaccination with hen egg lysozyme antigen. We demonstrated for the first time that pretreatment of skin with surfactant SLS significantly enhances the production of antibody to hen egg lysozyme. Short term pretreatment with SLS disorganized the stratum corneum, extracted partial lamellar lipids, induced the maturation of Langerhans cells, and did not result in epidermis thickening. To reveal the mechanism underlying these changes, particularly at the molecular level, we used a novel proteomic technique using ultrafiltration capillaries and mass spectrometry to identify in vivo proteins/peptides secreted in the SLS-pretreated skin. Two secretory proteins, named as calcium-binding protein S100A9 and thymosin beta4, were identified by this novel technique. These two proteins thus may provide new insight into the enhancing effect of surfactants on skin vaccination.

Proteomic characterization of skin and epidermis in response to environmental agents.

The skin and its outer epidermis layer in particular, prevent access of various environmental agents including potential allergens, irritants, carcinogens, ultraviolet radiation and microbes. Cells in the epidermis make a significant contribution to innate as well as adaptive immune reactions in skin. The skin immunity thus provides a biologic defense in response to hazardous environmental agents. Although proteomics has been utilized to establish skin proteomes and investigate skin responses to some environmental agents, it has not been extensively used to address the complexity of skin responses to various environments. This review summarizes cutaneous genes and proteins that have been characterized as related to skin exposure to environmental agents. In parallel, this review emphasizes functional proteomics and systems biology, which are believed to be an important future direction toward characterizing the skin proteome-environmental interaction and developing successful therapeutic strategies for skin diseases caused by environmental insults.

Mass spectrometric methods for the determination of flavonoids in biological samples.

There is an ever-increasing interest in the biological effects of the bioflavonoids, members of the large group of plant polyphenols. Because of the aromatic character of these compounds, they have been analyzed by several chromatographic methods. In the case of high-performance liquid chromatography, they are readily detected by their ultraviolet absorbance or electrochemical properties. More evidence that the bioflavonoids undergo extensive metabolism during uptake from the gut and distribution around the body and in specific tissues is accumulating. In addition, free radical products at sites of inflammatory processes react with bioflavonoids and their metabolites, generating important new compounds of as yet unknown properties. For these reasons, careful examination of the chemical nature of bioflavonoids and their products in biological systems is absolutely required. Combination of mass spectrometry with the various chromatographic methods has proved to be highly successful in this regard. This review of the literature on the bioflavonoids is focused on the methods that are currently available for their qualitative and quantitative analysis by mass spectrometry and covers the period 2001-2003. Emphasis is placed on the description and value of existing methods, followed by an examination of emerging technologies.

Review of the methods used in the determination of phytoestrogens.

Interest in analytical methods for plant estrogens (phytoestrogens) has risen sharply in the past 10 years. In this review, we examine the existing analytical methods based on separations by gas-liquid chromatography, high-performance liquid chromatography and capillary electrophoresis in addition to methods of detection by ultraviolet absorption, fluorescence, electrochemical oxidation/reduction and mass spectrometry. These methods are compared with other methods of phytoestrogen analysis utilizing immunoassay approaches. The advantages and disadvantages of each of these methods are highlighted and potential areas for further development identified.