Localization of vitamin a by autofluorescence during induced metaplastic changes in cultures of skin

Summary A technique was devised for following the uptake and location of vitamin A in organ cultures. Explants of 12- and 13-day embryonic mouse upper lip skin were grown for 3,6 or 9 days in biological medium to which was added 0,4.1 or 6.9 μg per ml of retinyl acetate. This form of vitamin A caused glandular morphogenesis of vibrissa follicles, and keratinization in epidermis and follicles was completely suppressed in 12-day explants and partially suppressed in 13-day explants. Frozen sections at 16 μm showed the white, non-fading fluorescence of keratin and the green, rapidly-fading fluorescence due to vitamin A which was captured by high-speed photography. Although more concentrated within lipid droplets in the dermis, the vitamin penetrated both the epidermis and the hair follicles. The ability to obtain permanent photographic records of the fading fluorescence makes this a useful method for analyzing vitamin A distribution as well as keratin distribution. This work was supported by the National Research Council of Canada (Operating Grant to M. H. Hardy and Postgraduate Scholarship to R. Van Exan) and by the Ontario Ministry of Agriculture and Food.     

Pangenomic changes induced by DHEA in the skin of postmenopausal women

The objective of this study was to explore, for the first time, the changes in the pangenomic profile induced in human skin in women treated with dehydroepiandrosterone (DHEA) applied locally.Sixty postmenopausal women participated in this phase II prospective, randomized, double-blind and placebo-controlled study. Women were randomized to the twice daily local application of 0% (placebo), 0.3%, 1% or 2% DHEA cream. Changes in the pangenomic expression profile were studied using Affymetrix Genechips.Significant changes (p < 0.05) in sixty-six DHEA-responsive probe sets corresponding to 52 well-characterized genes and 9 unknown gene sequences were identified. A dose-dependent increase in the expression of several members of the collagen family was observed, namely COL1, COL3 and COL5 as well as the concomitant modulation of SPARC, a gene required for the normal deposition and maturation of collagen fibrils in the dermis. Several genes involved in the proliferation and differentiation of keratinocytes were also modulated. In addition, topical DHEA reduced the expression of genes associated with the terminal differentiation and cornification of keratinocytes.Our results strongly suggest the possibility that DHEA could exert an anti-aging effect in the skin through stimulation of collagen biosynthesis, improved structural organization of the dermis while modulating keratinocyte metabolism.     

Localization of nucleophosmin in nuclear matrix and changes in its expression during the differentiation of human neuroblastoma induced by retinoic acid

In this article, we selectively extracted the nuclear matrix and intermediate filament system of human neuroblastoma SK‐N‐SH cells pre‐ and post‐treated with retinoic acid (RA). The distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with several products of related genes were investigated. Results from two‐dimensional gel electrophoresis and MALDI‐TOF showed that NPM was a component of the nuclear matrix and its expression in SK‐N‐SH cells post‐treated with RA was down‐regulated. Immunofluorescent microscopy observations further showed that NPM was localized in the nuclear matrix of SK‐N‐SH cells, and its expression level and distribution were altered after treatment with RA. The colocalization of NPM with c‐myc, c‐fos, p53, and Rb in SK‐N‐SH cells was observed under a laser scanning confocal microscope, but the colocalization region was changed by RA. Our results prove that NPM is a nuclear matrix protein, which is localized in nuclear matrix fibers. The colocalization of NPM with its related genes and oncogenes affect the differentiation of SK‐N‐SH cells. The expression of NPM and its distribution in the process of cell differentiation deserve more intensive investigation. J. Cell. Biochem. 111: 67–74, 2010. © 2010 Wiley‐Liss, Inc.     

Modifications of proteoglycans produced by human skin fibroblast cultures during replicative senescence

The properties of proteoglycans (PGs) produced by normal human skin fibroblast were investigated with increasing passage. The increase of subculture number was associated with a constant increase in PG molecular size, which was particularly evident in cell layer extracts. In the cell layer, the ratio of DS-PGs/HS-PGs was markedly higher in early passage cultures. Moreover, the cell layer from young cells contained lower amounts of radioactivity incorporated into the most hydrophobic PG populations, suggesting that the PG core protein might also undergo significant modification with increasing subcultures. There was no significant difference in energy charge value between early and late passage cultures, whereas the NAD/NADH ratio was found to decrease markedly in senescent cells.     

Sex steroid induced morphological changes in primary uterine cell cultures

The ultrastructure of primary cultures of endometrial cells after treatment with diethystilbestrol, progesterone, and hydrocortisone was examined using the method of electronmicroscopic stereology. Cells treated with diethylstilbestrol had the appearance of a rapidly dividing culture having large euchromatic nuclei and prominent nucleoli with a cytoplasm containing many free ribosomes. Progesterone appeared to convert the cells to a more secretory type with large amounts of RER and smaller nucleoli. Mitochondrial enlargement was induced by sex hormones but not by hydrocortisone.     

Detection of mitochondrial dysfunction in vitro by laser‐induced autofluorescence

Mitochondrion plays a significant role in a variety of biological functions. Because of their diverse character and location in the cellular systems, mitochondria commonly get exposed to various extrinsic and intrinsic cellular stresses. The present study reports a novel approach to detection of mitochondrial dysfunction based on tryptophan autofluorescence of its proteins in mouse liver, using laser‐induced fluorescence (LIF) as a tool. Mitochondria, isolated from the mouse liver, were initially tested for purity and integrity using lactate dehydrogenase and succinate dehydrogenase (SDH) assays. Mitochondrial stress was induced by treating the isolated mitochondria with heavy metals at 10 and 0.01 mM for sodium arsenite and mercuric chloride, respectively. Upon treatment with the heavy metal, tryptophan autofluorescence quenching was recorded at 281 nm excitation. The functional integrity of the mitochondria treated with heavy metals was evaluated by measuring SDH and cytochrome c oxidase activities at various concentrations of mitochondria, which showed impaired activity as compared to control upto a concentration of 6.25 μg. A significant shift was also observed in the autofluorescence of proteins upto the level below 1 μg, suggesting their conformational change and hence altered structural integrity of mitochondria. Circular dichroism spectroscopy data of the mitochondrial proteins treated with heavy metals further validates their conformational change as compared to untreated control. The present study clearly shows that the LIF can be a novel detection tool to detect altered structural integrity of cellular mitochondria upon stress, and it also possesses the potentiality to combine with other interdisciplinary modalities.     

Hemostatic changes induced by exercise during oral contraceptive use

Exercise-induced changes in hemostatic measurements were studied in 25 women. Twelve of the subjects were not using oral contraceptives and the remainder were using Demulen (ethynodiol diacetate (1 mg) and ethinyl estradiol (0.05 mg)). Exercise on a treadmill induced similar changes in both groups, but during the use of Demulen the levels of fibrinogen and plasminogen were higher, antithrombin level was lower, and the recalcified clotting and dilute whole blood lysis times were shorter in group 2 than in the corresponding samples obtained from the nonpill users.     

Quantification of organelle changes in plant suspension cultures during growth

Stereological techniques were used to quantify ultrastructural changes which occurred during maturation of cultured Paul's Scarlet rose cells. The volume and ultrastructural composition of young, dividing, unsynchronized 5-day-old cells were compared to that of mature, nondividing 14-day-old cells. The volume of the 14-day-old cells was 4-fold greater than that of the 5-day-old cells, primarily due to vacuole expansion. Numerous quantitative changes occurred in the organelle composition during cell maturation, but distinctive differences were observed in the magnitude and direction of change among the different types of organelles. There was an overall decline in the plastid population as measured by both percent of cell volume and numbers of plastids per cell. The percent of cell volume and numbers of lipid bodies increased, whereas the percent volume of the mitochondria remained relatively constant while the number per cell declined.     

Hormonal changes during forced moult induced by progesterone in domestic hen

Forced moulting has been induced in domestic hens by progesterone treatment (5 mg/day) for 25 days. Moult happened between the 11th and 19th day after the first treatment. Endocrine changes were followed during the moult by blood sampling in one week intervals. At the time of the last sampling, new egg laying cycle was initiated in all birds. Plasma progesterone concentration increased significantly in response to the treatment then tended to decrease. Oestrone and testosterone levels were the lowest during the period when feather loss was most intensive and increased in the course of feathering. This increase was significant in the case of oestrone. The level of 17-beta-oestradiol did not vary during moult induced by progesterone treatment. Plasma concentration of thyroxine significantly increased during feather loss, showing a maximum in the second and/or third week after the beginning of the treatment, while it decreased when feather growth had begun. Plasma triiodothyronine as well as corticosterone levels were the highest during the latest phase of moult, at the time of feather outgrowing. It has been supposed that moulting would be initiated in response to the synergistic effect on feather follicles of progesterone and thyroxine, which was stimulated by the progesterone treatment. The atrophic stage of the ovary suggested that progesterone was probably of adrenal origin. It was assumed that triiodothyronine and oestrone were responsible for controlling feather outgrowth.     

Biological changes in suspension cultures of Taxus cuspidata induced by dimethyl sulphoxide and ethanol

The biological changes in suspension cultures of Taxus cuspidata caused by dimethyl sulphoxide (DMSO) and ethanol, two commonly used solvents for water-insoluble elicitors, were investigated. The activities of peroxidase (POD) and superoxide dismutase (SOD) changed remarkably after the addition of small amount (0.4% (v/v)) of DMSO compared to those of the control culture at 4 h, however, they were less affected by small amount (0.4% (v/v)) of ethanol within 20 h. The biomass, cell viability, contents of intra/extracellular proteins did not change obviously when the amounts of DMSO and ethanol were below 1% (v/v) and 0.4% (v/v), respectively, but they varied significantly when the contents of DMSO and ethanol were 4% (v/v) and 1% (v/v), respectively. Obvious DNA fragmentation occurred in the case of ethanol at 2% (v/v), while no DNA fragments were observed in the case of DMSO at the same concentration level. It is inferred that DMSO below 1% (v/v) is a better solvent for investigating the effects of water-insoluble elicitors at a long-term contact, while ethanol less than 0.4% (v/v) is more suitable for a short-term contact.     

Portable spectroscopic system for in vivo skin neoplasms diagnostics by Raman and autofluorescence analysis

The present paper studies the applicability of a portable cost‐effective spectroscopic system for the optical screening of skin tumors. in vivo studies of Raman scattering and autofluorescence (AF) of skin tumors with the 785 nm excitation laser in the near‐infrared region included malignant melanoma, basal cell carcinoma and various types of benign neoplasms. The efficiency of the portable system was evaluated by comparison with a highly sensitive spectroscopic system and with the diagnosis accuracy of a human oncologist. Partial least square analysis of Raman and AF spectra was performed; specificity and sensitivity of various skin oncological pathologies detection varied from 78.9% to 100%. Hundred percent accuracy of benign and malignant skin tumors differentiation is possible only with a combined analysis of Raman and AF signals.      

Factors influencing skin autofluorescence of patients with peritoneal dialysis

Skin autofluorescence (SAF) measurement is a simple, noninvasive method to assess tissue advanced glycation end products (AGE). In patients with end-stage renal disease and in those on hemodialysis AGE production is increased. Less is known about those treated with peritoneal dialysis (PD). In this study we tested if SAF is influenced by clinical and treatment characteristics in PD patients.This cross-sectional study included 198 PD patients (of those, 128 were on traditional glucose-based solutions and 70 patients were partially switched to icodextrin-based PD). SAF measurements were done with a specific AGE Reader device. The impact of patients' age, gender, current diabetes, duration of PD, cumulative glucose exposure, body mass index, smoking habits and use of icodextrin on SAF values were tested with multiple regression analysis.Our analysis revealed that patients' age, current diabetes and icodextrin use significantly increase patients' SAF values (p = 0.015, 0.012, 0.005, respectively). AGE exposure of PD patients with diabetes and on icodextrin solution is increased. Further investigation is required whether this finding is due to the icodextrin itself or for a still unspecified clinical characteristic of PD population treated with icodextrin.