Transcriptional regulation of proteoglycans and glycosaminoglycan chain-synthesizing glycosyltransferases by UV irradiation in cultured human dermal fibroblasts.

Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm(2) of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, ß1,3-glucuronyltransferase-1, ß1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of ß1,3-galactosyltransferase-6, ß1,4-galactosyltransferase-3, -7, ß-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

Molecular inactivation of testicular hyaluronidase in solid state after proton irradiation: a study based on target size, substrate binding and thermodynamic analysis of heat denaturation.

Dose response activity curve of testicular hyaluronidase (HDase) following proton irradiation in dry state follows complicated mechanisms which may involve multiple hits and multiple targets of variable sizes giving a constant G value of 1.66. Target analysis appears to be modified by slow recovery of activity when irradiated enzyme is brought to aqueous phase. However, pattern of irradiation at a dose of 1 x 10(5) to 8 x 10(5) Gy reveals that though binding affinity of enzyme to the substrate (hyaluronic acid) increases as shown by declining Km from 500 mg/l to 300-70 mg/l, the reaction rate of catalysis by irradiated HDase is decreased due to decrease in reaction velocity (Vmax: 266 versus 76 units at 8 x 10(5) Gy). Activation analysis of heat denaturation of nonirradiated HDase suggested the involvement of 78 kcal/mole of energy of activation (E*a) which declined to 63-52 k cal/mole after irradiation at 1 x 10(5) to 8 x 10(5) Gy for residual enzyme. The corresponding change in entropy of activation (delta S*) increased from a control value of -291 eu to -236 eu at 8 x 10(5) Gy. From thermodynamic analysis in association with recovery in aqueous phase, it is concluded that HDase is inactivated due to dissipation of proton energy among weak forces including H bonds associated with secondary/tertiary structure of molecules.

A reversed-phase ion-pair high-performance liquid chromatography method for bovine testicular hyaluronidase digests using postcolumn derivatization with 2-cyanoacetamide and ultraviolet detection.

A reversed-phase ion-pair HPLC method for separating hyaluronic acid oligomers, using a polymeric C18 column at alkaline pH, is described. As the concentration of the ion-pairing agent tetrabutylammonium hydroxide increased, over the range of 0.01 to 0.06M, the capacity factors (k') of tetra- to dodecasaccharide decreased. The change in k', for each increment in pairing agent, increased with oligomer molecular weight. When changing mobile phase pH from 7 to 8, k' dramatically decreased and remained unchanged from pH 8 to 11. The isocratic separation was optimized to resolve tetrato dodecasaccharide at pH 9.0 in under 19 min. The postcolumn derivatizing agent 2-cyanoacetamide reacted with the reducing N-acetylglucosamine end groups of hyaluronic acid oligomers to yield reaction products that were monitored at 27 nm. In a series of control experiments using decasaccharide and N-acetylglucosamine, it was found that maximum product formation took place at pH 9 and was greatly influenced by borate buffer concentration. The optimum concentration for 2-cyanoacetamide was 0.33% and a temperature of 100 degrees C gave the best signal to noise ratio for the postcolumn reaction. The method is linear and reproducible, and has a lower limit of detection for tetrasaccharide of 20 ng (25 pmol). This system is suitable for studying the degradation kinetics of purified hyaluronic acid oligomers by bovine testicular hyaluronidase. Extension of the method to fluorescent and electrochemical detection and its applicability to other glycosaminoglycans is discussed.

Catalytic properties of testicular hyaluronidase after gamma-irradiation.

The effect of gamma-irradiation on ovine testicular hyaluronidase was studied in aqueous solution. Following irradiation, hyaluronidase is inhibited, and the kinetics of inhibition follow a pattern in which Km and Vmax decline as radiation dose is increased. It was indicated that the binding affinity of the residual activity of hyaluronidase with substrate is enhanced and depends upon radiation damage. Effects of various agents such as pH, salts, PCMB and glutathione on irradiated hyaluronidase have been compared with non-irradiated enzyme. The irradiated hyaluronidase was more sensitive to inhibition by CuSO4 than the non-irradiated enzyme. The residual activity after irradiation is less refractory to FeCl3 inhibition and less sensitive to NaCl stimulation compared to non-irradiated hyaluronidase. pH response curves of ovine testicular hyaluronidase show two maxima which become more evident after irradiation.

Photoactivation of hyaluronidase by chlorpromazine.

The effects of UVA and chlorpromazine on the activity of hyaluronidase were investigated. Hyaluronidase was slightly activated by UVA alone. It was also activated by chlorpromazine, but the activation was enhanced by concomitant UVA irradiation. By pre-irradiation of chlorpromazine, it was found that a stable photoproduct of chlorpromazine was formed that activated hyaluronidase. As activation of hyaluronidase has been described in relation to tissue inflammation, the onset of chlorpromazine-induced photosensitivity may, in part, be related to the hyaluronidase activating effect of chlorpromazine plus UVA.

The influence of low temperature on the radiation sensitivity of enzymes.

When enzymes are exposed to ionizing radiation at low temperatures there is a progressive decrease in radiation sensitivity: considerably more enzymatic activity remains after the same dose of radiation at low temperature compared to room temperature. Detailed studies of five enzymes reveals the quantitative relationship between radiation sensitivity and temperature during exposure. Although 25 enzymes are shown to display this same relationship, recent reports have denied this effect in three enzymes. In this paper, we investigate two possible artifacts that could cause these discrepancies: 1) inaccurate determination of the temperature of the sample during irradiation, and 2) use of temperature-sensitive dosimeters to measure radiation dose. Procedures are described that carefully control these parameters. Thermoluminescent dosimeters are shown to be independent of temperature effects. These methods are used to investigate one of the enzymes, malate dehydrogenase, that has been reported to have a temperature-insensitive radiation inactivation. The radiation sensitivity of this enzyme is found to show the same temperature dependence as 24 other enzymes.