Purine phosphoribosyltransferase activities in guinea pig epidermis

Purine phosphoribosyltransferase activities in normal and experimental hyperkeratotic epidermis of guinea pig skin were demonstrated quantitatively by a new microassay method. The ratio of HGPRTase with hypoxanthine as a substrate to APRTase activity in normal and hyperkeratotic epidermis was found to be 0.94 and 0.60, respectively. The HGPRTase and APRTase activities expressed as micromoles per gram wet weight per min. were increased in experimental hyperkeratotic epidermis and it is suggested that the salvage pathway for purine nucleotide biosynthesis is activated in experimental hyperkeratotic epidermis. The pH optimum of these enzymes and their stability in the frozen state were also demonstrated.     

Pyrrolidone carboxylic acid synthesis in guinea pig epidermis

To establish the in vivo mechanism of synthesis and accumulation of epidermal pyrrolidone carboxylic acid (PCA), enzymes potentially capable of PCA synthesis have been quantified and located within the guinea pig epidermis. Intermediates in the synthesis of [3H]PCA from a pulse of [3H]glutamine have been identified and quantified to determine which of the several possible metabolic routes occurs in vivo. PCA appears to be synthesized from substrate derived from the breakdown within the stratum corneum of protein synthesized several days earlier. The predominant route is probably via the nonenzymic cyclization of free glutamine liberated from this protein. In view of the high activity of gamma-glutamyl cyclotransferase in the stratum corneum, a minor contribution to PCA formation by the action of the enzyme on gamma-glutamyl peptides cannot be excluded.     

Some ultrastructural observations on keratohyalin granules of guinea pig epidermis

Summary Keratohyalin in the cavy back and plantar epidermis was examined by transmission electron microscopy. These cytoplasmic aggregates are made up of numerous fine particles uniformly stained by osmium, probably the protein component. Phospholipid in the interstices is removable in lipid solvents before osmium post-fixation. After glutaraldehyde fixation without osmium, tissue stained in uranyl acetate showed keratohyalin aggregates composed of mixtures of darkly stained and weakly stained groups of fine particles which indicate heterogeneity in composition. More weakly stained aggregates of keratohyalin composed of these particles were found in the interiors of keratinized cells in the lower part of the stratum corneum not previously demonstrated at this level. Oxidation in peracetic acid followed by staining in uranyl acetate showed dark areas in keratohyalin aggregates, which it is thought contain cystine. Keratohyalin aggregates are not surrounded by a membrane and they are not organelles. Their heterogeneous composition suggests different derivations and destinies of component substances, some of which may be synthetic and others autolytic products. It is suggested that keratohyalin is a cytoplasmic coacervate of protein, phospholipid, and bound calcium precipitated under unstable physiochemical conditions associated with keratinization involving extensive cytolysis.

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Zusammenfassung Das Keratohyalin der Meerschweinchenrücken- und Sohlenepidermis wurde transmissionselektronenmikroskopisch untersucht. Die cytoplasmatischen Komplexe bestehen aus zahlreichen feinen Partikeln, die, wahrscheinlich Proteinanteile, sich mit Osmium homogen färben lassen. In den Spalträumen gelagerte Phospholipide können mit fettlösenden Substanzen entfernt werden. Glutaraldehydfixierung ohne Osmium und anschließende Färbung mit Uranylacetat zeigte, daß Keratohyalin aus einer Mischung von dunkel gefärbten und schwach gefärbten Gruppen feiner Partikel bestehen, die auf die Heterogenität dieser Komplexe hinweisen. Schwächer gefärbte Keratohyalinkomplexe wurden in den verhornten Zellen der unteren Stratum corneum-Abschnitte erstmals nachgewiesen. Essigsäureoxydation mit nachfolgender Uranylacetatfärbung zeigte innerhalb der Keratohyalinkomplexe dunkle Areale, die vermutlich Cystin enthalten.Keratohyalinkomplexe sind nicht membrangebunden und stellen keine Organellen dar. Ihre heterogene Zusammensetzung läßt unterschiedliche Herkunft der Einzelsubstanzen vermuten, von denen einige neu synthetisiert werden, andere als Folge von Autolyse entstehen. Es wird angenommen, daß Karatohyalin ein Komplex von Proteinphospholipid und gebundenem Calcium darstellt, das unter unstabilen physikochemischen Bedingungen im Verlaufe der Keratinisation und Cytolyse präcipitiert wird.

     

193 nm excimer laser selective ablation of in vivo guinea pig epidermis

We have previously shown that 193 nm excimer laser irradiation cleanly and effectively ablates avascular tissue with minimal thermal damage to surrounding adjacent structures. In this study, the 193 nm excimer laser is used to remove guinea pig epidermis in vivo. The epidermis can be totally ablated with thermal damage extending only superficially into the dermis. Reepitheliazation of the ablated area takes place in 1 week or less. This technique may be applicable to the removal of benign epidermal lesions.     

Changes of membrane fluidity and Na+, K+-ATPase activity during cellular differentiation in the guinea pig epidermis

Summary The dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na⁺, K⁺-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be assiciated with a decrease in plasma membrane fluidity and with a decline of Na⁺, K⁺-ATPase activity.     

Mathematical assessment of the spatial distribution of Langerhans cells in guinea pig epidermis.

Two mathematical indexes, Hopkins-Skellam index (HSI) and Morisita index (MI), were applied to assess the distribution of ATPase-stained epidermal Langerhans cells (ELC) in the guinea pig skin. To our knowledge, this is the first report in which the degree of regularity has been expressed numerically by computation based on theoretical equations. The regularity of ELC in the normal skin was confirmed by the value of HSI (P < 0.0001). In the topical steroid applied skin, the number of ELC decreased significantly but the value of HSI was similar to that of the normal skin; while in the ultraviolet B (UVB) exposed skin, both number of ELC and regularity of ELC distribution decreased significantly. The graph of MI for the UVB exposed skin clearly showed that the distribution of ELC had local clumps. The two indexes, HSI and MI have been quite useful for determining the regularity of ELC. These indexes may have a wide application to other cells.     

Elemental changes in guinea pig epidermis at repeated exposure to sodium lauryl sulfate.

Epidermal hyperplasia is the response of the epidermis to external harmful stimuli. The control and regulation of this hyperplasia is not completely understood. It has been proposed that changes in the cellular sodium/potassium ratio are of importance in the regulation of cell proliferation. To evaluate if such a change in the elemental content of epidermal cells can be one factor to consider at irritant contact dermatitis, we performed a quantitative assessment of sodium lauryl sulfate (SLS)-induced contact reactions in the guinea pig. SLS was applied 1, 2 or 3 times and biopsies were obtained at 24 and 84 h after the last application. It was found that repeated exposures to SLS induced a hyperplasia of epidermis at 24 h persisting at 84 h. At 24 h there were significant changes in the sodium and potassium content of the keratinocytes. At 84 h there was still an increased potassium level in the cells and the sodium/potassium ratio was significantly decreased in epidermis exposed three times to SLS. This implies that changes in cellular sodium/potassium ratios occur in epidermal hyperplasia following irritant stimuli.     

The in vivo effects of lymphokines on mitotic activity and keratinization in guinea pig epidermis

Lymphokines may alter epidermal growth and differentiation contributing to changes such as acanthosis and hyperkeratosis. The main in vivo effects of lymphokines on epidermal mitotic activity were therefore investigated. Guinea pigs were injected intradermally with antigen-stimulated lymphocyte culture supernatants and a partially purified lymphokine preparation in phosphate buffered saline (PBS) 18, 24, 36 and 48 hr prior to biopsy. Control sites were injected with unstimulated supernatants and PBS respectively and the mitotic activity determined by use of a stathmokinetic agent. Both lymphokine injected areas and controls showed significantly increased mitotic indices compared to untreated skin which was apparent only at 24 hr. However mitotic activity in lymphokine lesions was significantly higher than in control lesions. There was no difference in the effect on mitotic activity between PBS and unstimulated culture supernatants. Lymphokine lesions at 24 hr also exhibited marked epidermal edema and acanthosis compared to minimal changes in controls. A variable patchy parakeratosis developed between 18 and 24 hr in areas injected with partially purified lymphokine but not in control sites or after injection with unpurified supernatants. The lymphokine-induced inflammatory infiltrate was mild and consisted mainly of neutrophils not differing significantly from that of the control lesions. This strongly suggests that lymphokines induce an alteration in epidermal kinetics and keratinization by a direct effect on keratinocytes and not indirectly via the dermal inflammatory infiltrate.     

Elemental redistribution and ultrastructural changes in guinea pig epidermis after dinitrochlorobenzene (DNCB) exposure

Energy dispersive x-ray microanalysis and transmission electron microscopy were used to study ultrastructural and chemical changes occurring in the keratinocytes at the primary irritant dinitrochlorobenzene (DNCB) reaction. The results of the microanalysis showed a similarity between dose- and time-dependent effects of DNCB and were in accordance with the ultrastructural findings. With increasing cell injury (as judged by the ultrastructure) there was an increasing loss of phosphorus, potassium, and magnesium from the cells and an increase of cellular calcium content. Sodium and chloride were only moderately changed. Results recorded when the skin was exposed to a weak DNCB dose were in accordance with a stimulation of the basal keratinocyte cell population.     

The effect of systemic treatment with 5-fluorouracil on the epidermis of the guinea pig

Summary The thymidine labelling index and the mitosis rate (blocked by colcemid) in the basal cell layer of the guinea pig were determined before and during treatment with 5-fluorouracil (1 mg i.p. in 2 day's interval/100 g body weight). Additionally, the epidermal area and thickness and the number and size of cells were measured. 5-Fluorouracil equally reduces the thymidine labelling index and the mitosis rate with the full effect after treatment of at least 22 days. From these findings it is concluded that the S-phase — calculated from the end of the thymidine synthesis — is not altered. Furthermore atrophy of the epidermis runs parallel with the reduction of the ³H thymidine labelling index and the mitosis rate.     

Epidermal hyperplasia induced in guinea pig flank skin by intradermal injection of Sudan red.

Studies of dermal-epidermal interactions were conducted with guinea pig flank skin and intradermal injections of the irritant, Sudan IV dye in olive oil. These injections led to epidermal hyperplasia in areas overlying the irritant and the effect was most significant when the irritant was placed in the upper dermis. Basal cell mitotic activity and thymidine uptake reached a peak by 24 h and thereafter dropped rapidly. Maximal epidermal thickness (4.3 times the control) resulting from an increase in cell number occurred within 2-4 days. Despite the very short period of increased cell growth, epidermal thickness returned to control values only after a 24-day period. A similar growth response could not be induced by saline injections. A single topical application of the irritant showed a qualitatively and quantitatively different epidermal response. These experiments indicate that an intradermal irritant can lead to epidermal hyperplasia and a long-lasting epidermal thickening.     

Sebaceous hyperplasia within epidermis after scald

A 16-year-old girl with polycystic ovarian syndrome presented with numerous rufous papules arising within a large depigmented macule that developed following a severe scald injury on the back. Histopathology revealed that many mature sebaceous glands were growing in the middle and bottom of the epidermis with slight acanthosis. On the basis of patient history, clinical manifestation and histopathology, we suggested that this patient's skin lesion be diagnosed as 'sebaceous hyperplasia within epidermis after scald'.     

Increased biosynthesis of lipoxygenase products by UVB-irradiated guinea pig epidermis: evidence of a cyclooxygenase inhibitor

The present studies demonstrate that incubation of arachidonic acid (AA) with a 20,000 g homogenate (containing both microsomal and cytoplasmic fractions) from UVB-irradiated guinea pig epidermis (24-72 h) resulted in decreased transformation of the [14C]AA into the cyclooxygenase products (PGD2, PGE2, and PGF2 alpha) while the incorporation of 14C into lipoxygenase products (15-HETE and 12-HETE) increased. An investigation into the selective inhibition of the cyclooxygenase pathway revealed that the in vitro transformation of [14C]AA into [14C]-cyclooxygenase products by the 100,000 g particulate fraction prepared from normal unirradiated guinea pig epidermis was inhibited by the 100,000 g cytoplasmic extract prepared from a 24-h postirradiated guinea pig epidermis. These latter data imply that an endogenous inhibitor of the cyclooxygenase pathway is generated and released into the cytoplasm during UVB irradiation and it is likely that this selective inhibition of the cyclooxygenase pathway may contribute at least in part to the increased lipoxygenase products in the 24-h postirradiated skin specimens and possibly the recognized prolonged UVB-induced inflammatory process.     

Sphingomyelinase in pig and human epidermis.

The enzyme sphingomyelinase (sphingomyelin phosphorylcholine phosphohydrolase E.C.3.1.4.12) which hydrolyzes sphingomyelin to ceramide (N-acylsphingosine) and phosphorylcholine was identified in the subcellular fractions of pig and human epidermis. The enzyme has an optimum pH of 4.5 to 5 and is activated by Triton X-100 (0.1% w/v). Approximately two-thirds of the enzyme activity in both the pig and human epidermal homogenates was in the soluble subcellular fraction and more than half of the enzyme activity in the subcellular particulate fraction was solubilized by freeze-thawing. The pH optimum suggests that epidermal sphingomyelinase is probably a lysozomal enzyme. The enzymes in both pig and human epidermis exhibited Michaelis-Menten kinetics. The soluble sphingomyelinase in pig epidermis had an apparent Km, 4.5 X 10(-5) M and that in human epidermis an apparent Km 7.7 X 10(-5) M. The pig epidermal sphingomyelinase had no special requirement for either divalent or heavy metal ions and was not inhibited by sulfydryl group-blocking agents but it was moderately inhibited by dithiothreitol. No evidence was found in either pig or human epidermis for the presence of a phospholipase C (E.C.3.1.4.3) which hydrolyzes phosphatidylcholine to diglyceride and phosphorylcholine but there was suggestive evidence of another catabolic pathway for phosphatidylcholine.