Young Rat Skin Research Articles

Release of Amino Acids, Fluorescamine-Reactive Substances and Substance P from the Epidermis of the Living Animal

The present study demonstrates a procedure for extraction and determination of stratum corneum amines in the living animal. A nonleaky well, containing 10 mM Tris-HCl buffer, pH 7.0, was constructed on the shaved backs of anesthetized animals. It was found that Ser, Ala, Gly and Pro are mainly released from the stratum corneum of 4-month-old guinea pigs, and in 2-month-old rats, Gly, Ser and Arg show the highest degree of release. Much lower amino acid concentrations were observed in 20-month-old rats. This was also reflected by the high levels of fluorescamine-reactive substances released from young rat skin as compared to the old animals. The release of the neuropeptide substance P into the aqueous medium was increased 3.2 times upon heat stimulus as compared to control skin. Amines and other compounds released from the skin may serve as markers for skin aging or for certain skin disorders, leading to a new approach for their treatments.

Prevention of cutaneous phototoxicity in photodynamic therapy

Photodynamic therapy using hematoporphyrin derivative (Hpd), or its semipurified form, dihematoporphyrin ether (DHE), in combination with light, has proven effective in the treatment of several types of neoplasms including squamous cell carcinoma. A major disadvantage of this therapy is the long-term retention of these drugs in skin, which may result in cutaneous phototoxicity for weeks after treatment. Singlet oxygen has been identified as the primary cytotoxic product in the reaction of Hpd and light in vitro. This study demonstrates the protective effect in vivo of the singlet oxygen scavengers 1,3 diphenylisobenzofuran and l-tryptophan on the skin of young rats exposed to light after receiving DHE.

Isolation and identification of 24-dehydroprovitamin D3 and its photolysis to 24-dehydroprevitamin D3 in mammalian skin.

Until now it had been assumed that mammalian skin contains only one provitamin D, 7-dehydrocholesterol, that is eventually converted to vitamin D3 after the skin is exposed to sunlight. Examination by reverse phase high performance liquid chromatography of lipid extracts from young rat skin, however, led to the observation that 7-dehydrocholesterol is not the only provitamin D in rat skin. Another provitamin D, accounting for 22 +/- 3% of the total provitamin content of the skin, was resolved from 7-dehydrocholesterol, and, on the basis of ultraviolet spectrophotometry, mass spectrometry, and nuclear magnetic resonance spectrometry, was identified as 24-dehydroprovitamin D3 (cholesta-5,7,24-trien-3 beta-ol). This new cutaneous provitamin D is not unique to the rat because it was also detected in the skin of reptiles, amphibians, birds, aquatic mammals, and humans. To be certain that the cutaneous 24-dehydroprovitamin D3 was as susceptible as 7-dehydrocholesterol to ultraviolet photolysis, rat skin was exposed to ultraviolet radiation. A reverse phase high performance liquid chromatographic analysis of a lipid extract of rat skin previously exposed to ultraviolet radiation demonstrated the presence of both previtamin D3 and 24-dehydroprevitamin D3. Therefore, these observations demonstrate for the first time that mammalian skin has the capacity to produce not one but at least two different vitamin Ds.

Skin sulfhydryl oxidase purification and some properties

A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol or by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, d-penicillamine, and l-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 ± 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.

Effects of oxytetracycline on solubility and synthesis of collagen in young rats.

In a recent study we demonstrated that the antibiotic oxytetracycline reduces both growth and mechanical strength of bone and skin in young rats. The present study deals with the effects of 14 days of oxytetracycline medication on salt solubility of collagen (cross-linking) and conversion of 14C-proline to 14C-hydroxyproline in collagen (synthesis). At the end of the medication period, significantly higher solubility of collagen was found in the femurs and skin of rats receiving oxytetracycline than in controls. No effect of the antibiotic on the rate of collagen synthesis could be demonstrated. These findings may indicate that oxytetracycline in young rats causes reduced mechanical strength of bone and skin by interfering with the cross-linking of collagen.

Effects of cloxacillin, doxycycline, fusidic acid and lincomycin on mineralization and solubility of collagen in young rats.

The antibiotics doxycycline, fusidic acid and lincoymcin have recently been shown to reduce growth of bones and tensile strength of skin in young rats. Such adverse effects were not found in rats receiving cloxacillin. The present investigation deals with the influences of these four antibiotics on the mineralization of bones and on the solubility of collagen in skin. In rats receiving doxycycline the content of calcium and phosphorus in bones and the concentration of albumin in serum were reduced. None of the antibiotics had a detectable effect on the solubility of collagen.

Effects of cloxacillin, doxycycline, fusidic acid and lincomycin on the mechanical properties of bone and skin in young rats.

The influence of cloxacillin, doxycycline, fuside acid and lincomycin on the mechanical properties of bone and skin in young rats was examined. The concentrations of the antibiotics in plasma corresponded to therapeutic levels in man. After 14 days of medication the weights of the rats receiving cloxacillin or doxycycline were significantly less when compared with the controls. The doxycycline, the fusidic acid and the lincomycin treated rats had reduced longitudinal growth of femur and reduced tensile strength of intact skin. No differences between any of the antibiotic groups and the control group were found in the tensile strength of incisional skin wounds or in the mechanical properties of the femur and tibia.

Effects of oxytetracycline on the mechanical properties of bone and skin in young rats.

The influence of the tetracycline antibiotics on growing bones has been disputed. In the present study 58 young male rats were given intraperitoneal injections twice a day for 2 weeks; half of the rats received oxytetracycline and the other half placebo. The concentration of oxytetracycline in plasma was comparable with therapeutic levels in man. Compared with the control animals the oxytetracycline rats had, at the end of the medication period, a significantly lower weight (7 per cent), shorter bones (1--2 per cent), lower bending strength of both the tibia diaphysis (9 per cent) and the distal femur metaphysis (22 per cent) and even lower tensile strength of intact (17 per cent ) and sutured (27 per cent) skin. The effect oxytetracycline seems to be reversible as no differences between the two groups could be detected 1--3 weeks after the end of medication.

Studies on the incorporation of (U-14C)glucose and (35S)sulphate into the acid glycosaminoglycans of neonatal rat skin.

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.

Studies on the Effect of Methotrexate on Amino Acid Incorporation in the Epidermis of the Young Rat

Studies on the effect of methotrexate on the uptake and distribution of tritiated amino acids in young rat epidermis are described. The drug given up to 72 hours before the tritiated precursors had no demonstrable effect on either the quantitative uptake using a new solubilization technique for scintillation counting, or on the qualitative distribution using autoradiography. There was no apparent effect over 24 hours if given shortly after the tritiated amino acids using autoradiography. It is concluded that methotrexate has no gross effects on amino acid incorporation in young rat skin.

Relationship between body collagen and urinary hydroxyproline excretion in young rats fed on a low-protein or low-calorie diet.

1. Hydroxyproline was determined in urine, carcass, and skin of young rats fed on low-protein and low-calorie diets.2. Protein and calorie deprivation in young rats resulted in a marked fall in the soluble as well as insoluble collagen content of the skin.3. The collagen content of the carcass (excluding skin) was slightly greater in the deficient rats than in the weanling rats, but very much lower than in the well-fed rats.4. In rats subjected to protein and calorie deficiency, urinary hydroxyproline was markedly reduced. In these rats, most of the reduction in urinary hydroxyproline was observed within 1 week of feeding the deficient diet. Protein deficiency had affected urinary excretion of hydroxyproline more severely than calorie deficiency.5. The reduction of urinary hydroxyproline in the deficient animals can be attributed to decreased turnover rate of their body collagen. This apparent decrease in turnover rate is explained as being due to an increase in the proportion of collagen of slow turnover in these animals.6. Protein and calorie deficiency had also resulted in the depletion of non-collagen nitrogen of both carcass and skin.

The tissue content and turnover rates of intermediates in the biosynthesis of glycosaminoglycans in young rat skin.

1. The tissue contents of hexose monophosphate, N-acetylglucosamine 6-phosphate, UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and UDP-glucuronic acid were determined in the skin of young rats less than 1 day post partum. Tissue-space determinations were used to calculate their average cellular concentrations. 2. The incorporation of [U-(14)C]-glucose into the intermediates was recorded with time and their rates of turnover were calculated. The results demonstrated product-precursor relationships along the pathway of hexosamine synthesis and that of hexuronic acid synthesis. The rates of synthesis of UDP-N-acetylhexosamine and UDP-glucuronic acid were 1.5+/-0.3 and 0.24+/-0.03mmumoles/min./g. of tissue respectively. These results indicated the average turnover time of the total tissue glycosaminoglycans to be about 5 days.

Quantitative Histochemistry of the Primate Skin: VIII Enolase

Quantitative Histochemistry of the Primate Skin: VIII Enolase

Citric Acid Cycle in Young Rat Skin

Citric Acid Cycle in Young Rat Skin

Autoradiographic Studies of the Incorporation of Thymidine-H3into Deoxyribonucleic Acid in the Skin of Young Rats

The synthesis of deoxyribonucleic acid (DNA) takes place in the cell cycle sometime before cell division and the replicated DNA is transferred to the “daughter” nuclei during mitosis. If tritiated thymidine, a specific precursor of DNA, is present during the period of DNA production, radioactive DNA is synthesized and the daughter nuclei also become radioactive. By autoradiography, the epidermal cells of mice and rats have been observed to be labeled at lj (1), 3 and 8 hours (2, 3) after injection of thymi-dine-H3sp>. The labeled cells were seen in the deeper part of the epidermis where mitoses are usually seen and DNA duplication must occur. Six months later, however, none of the labeled cells were present in the epidermis (2). Since DNA is considered to be metabolically inert under ordinary conditions, loss of tritium must have occurred either by (a) dilution of the label resulting from many cell divisions, so that the level of radioactivity was too low for identification; or (b) disappearance by the same process as causes the loss of the nucleus at the surface of the epidermis. Cells formed in the deeper part of the epidermis slowly move out towards the surface and are lost by desquamation, although the precise role played by each cell in this action is not yet known.

Glucose metabolism in young rat skin

Glucose metabolism in young rat skin slices has been studied with the aid of glucose-1-C 14, glucose-6-C 14, and glucose-U-C 14. A balance study showed that after 3 hr. about 36% of the starting glucose was converted to carboxylic acids (lactic, acetic, and formic), about 2% was oxidized to CO 2, 5% appeared as amino acids. 5% as phosphorylated compounds, and 28% as residual hexose. Lactic acid accounted for nearly all of the C 14 in the acidic material. Lactate degradation results showed the operation of the Embden-Meyerhof pathway. Glucose-1-C 14 was somewhat better as a source of C 14O 2 than was glucose-6-C 14, indicating qualitative evidence, for the presence of a pentose cycle. This is the first report of the presence of formic acid in skin. As glucose carbon is a poor source of formic acid, most of the compound was produced from endogenous sources.

Glycolytic activity in extracts of young rat skin

1. 1. Data have been presented which indicate the presence of most of the glycolytic enzymes in the skin of the young rat. 2. 2. A cell-free extract of this tissue will stoichiometrically convert fructose diphosphate to equimolar quantities of phosphoglyceric acid and phosphoglycerol in the presence of fluoride and trace quantities of diphosphopyridine nucleotide and inorganic phosphate under anaerobic conditions. Phosphoglycerol is formed from dihydroxyactone phosphate by a “diphosphopyridine nucleotide”-requiring glycerophosphate dehydrogenase. If P 32-labeled inorganic phosphate is used, 60% of the total radioactivity is incorporated equally into phosphoglyceric acid and phosphoglycerol. This incorporation is inhibited by iodoactate.