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The shortage of physicians has become a serious problem in Japan. It has been pointed out that an increase in the number of female doctors may contribute to the aggravation of this shortage because it is known that women work fewer hours than male doctors. Here, we investigated how many female doctors had ever resigned from a full-time position, and elucidated the reasons why female doctors find it difficult to stay in full-time employment. An alumnae survey of 2 private medical schools was conducted in 2007. A self-administered questionnaire was sent to 1423 graduates and 711 responded with informed consent (response rate, 50%; mean age, 39 years). Overall, 55% of the respondents had previously resigned from full-time employment, of which 90% resigned within 10 years of graduating from medical school. The difficulty in balancing work, childbirth and child rearing (45%) were the top 2 reasons for resignation, followed by physical problems (12%) and long working hours (8%). Among those who resigned, only 33% returned to full-time employment. Women who had at least 1 child were only 30% of those who had never resigned and 84% of those who had previously resigned. The majority of study subjects, regardless of experience of resignation (88%), agreed that women should continue to work even after childbirth. In conclusion, the results of this study suggested that many female doctors resigned from a full-time position within 10 years of graduating from medical school, largely because of the gender role stereotype and poor working conditions.

Ultraviolet radiation is the major environmental cause of skin damage. Although only 0.5% of ultraviolet B (UVB) radiation reaches the earth, it is the main cause of sunburn and inflammation and the most carcinogenic constituent of sunlight. We investigated whether the topical application of a novel, water-soluble gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG), could protect against UV-induced skin damage. Topical pre- or postapplication of gamma-TDMG solution significantly prevented sunburn cell formation, lipid peroxidation, and edema/inflammation that were induced by exposure to a single dose of UV irradiation. Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E(2) (PGE(2)) levels seen after UV exposure were significantly suppressed by pre- or posttreatment with gamma-TDMG. The increase in COX-2 activity was significantly inhibited by gamma-TDMG, suggesting that the reduction in PGE(2) concentration was due to the direct inhibition of COX-2 activity by gamma-TDMG. The derivative strongly inhibited inducible nitric oxide synthase mRNA expression and nitric oxide production. With the application of gamma-TDMG, the pigmentation in melanocytes was lightened and the increase melanin concentration was suppressed. Gamma-TDMG is converted to gamma-Toc in the skin and has higher bioavailability than gamma-Toc itself. These results suggest that gamma-TDMG-derived gamma-Toc acts as an antioxidant, antiinflammatory and antipigmentation agent. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing and reducing UV-induced skin damage in humans.

We previously demonstrated that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG) converts to gamma-Toc in the mouse skin and has a higher bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce photo-inflammation in mouse skin, and compared its effectiveness to that of alpha-Toc acetate (alpha-TA). Topical pre- or post-application of 5% gamma-TDMG significantly reduced the formation of edema and tempered the increase in cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E2 (PGE2) that were induced by a single dose of UV irradiation of 2 kJ/m2 (290-380 nm, maximum 312 nm). The pre-treatment of mouse skin with 10% alpha-TA had the same anti-inflammatory effect as did gamma-TDMG. In spite of same having the ability to reduce PGE2 levels, the effect of gamma-TDMG pre-treatment on the inhibition of COX-2 mRNA/protein expression was less than that seen with 10% alpha-TA. In contrast, the increase in COX-2 activity seen after UV exposure was reduced more by gamma-TDMG than by alpha-TA, suggesting that the reduction in PGE2 levels might have been due to the direct inhibition of COX-2 activity by gamma-TDMG-derived gamma-Toc. Both Toc derivatives strongly suppressed inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production, both of which play important roles in UV-induced inflammation. Both derivatives also significantly reduced lipid peroxidation in response to UV exposure, though gamma-TDMG's ability in this regard was less than that seen with alpha-TA, which correlated with their abilities to suppress COX-2 expression. Thus, the gamma-TDMG-derived gamma-Toc acts as an antioxidant, suppresses iNOS expression and directly inhibits COX-2 activity, all of which likely play a role in mediating its suppressive effects on photo-inflammation. Our data further suggest that the topical application of gamma-TDMG, a novel hydrophilic gamma-Toc derivative, may be efficacious in preventing and reducing UV-induced inflammation in humans.

The Shumiya cataract rat (SCR) is a hereditary cataract model in which lens opacity appears spontaneously in both the nuclear and perinuclear regions of 10‐11 week old animals. The expression of drug metabolizing enzymes, including cytochrome P450 (CYP) and conjugation enzymes, was investigated in the ocular tissues of SCRs during cataractogenesis. Significantly high levels of gene expression were detected for CYP1A1, CYP2B2, CYP2C11 and CYP2E1 in normal lenses, whereas CYP1A1 and CYP2C11 were at lower levels in the cataractous lenses. The gene expression profiles of conjugation enzymes were also compared between normal and cataractous ocular tissues in which a moderate reduction in ST1A1 and ST1B1 expression, and an extensive loss of UGT1A1 expression, were observed in the SCR animals that had developed cataracts. In contrast, the elevated expression of the GSTA family of genes was evident in cataractous lenses. The reduced expression of several drug metabolizing enzymes in the cataractous ocular tissues might therefore be related to cataractogenesis via the accumulation of endogenous or exogenous toxic substances.

畜水産物に使用される動物用医薬品は，生産性の向上や疾病予防や治療のため使用量が増大し，その残留の可能性が懸念されている．一方，食品の安全性確保のため公布された「ポジティブリスト制」施行に向け，動物用医薬品の一斉分析法の開発が急務となっている．そこで家畜用寄生虫駆除剤として広く使用されているベンズイミダゾール系薬剤について，高感度測定と構造解析能を併せ持つイオントラップ－高速液体クロマトグラフ/タンデム質量分析計（HPLC/MS/MS）に，固定相にペンタフルオロフェニルプロピル基が導入されたHPLCカラムを用いた分析法を検討した．移動相をアセトニトリル : 0.2％ ギ酸（50 : 50）（v/v）とし，ポジティブモードのエレクトロスプレーイオン化（ESI）でmultiple reaction monitoring（MRM）及びenhanced product ion scan（EPI）により測定を行った．オクタデシルシリカ（ODS）カラムとは選択性の異なる分析カラム用いることにより，アイソクラテックな移動相での9種のベンズイミダゾール系薬剤の一斉分析が可能となった．また，イオントラップ－HPLC/MS/MSのEPI分析では定性分析の指標として有効な化合物の構造情報が，MRM分析では高感度測定が得られた．HPLC/MS/MS法の活用は残留動物用医薬品分析の有効な手段となると思われる．

The quenching abilities of sodium L-ascorbyl-2-phosphate (APS) and ascorbic acid 2-glucose (AG) against UVB/A-generated free radicals in cultured mouse skin were investigated using electron spin resonance (ESR). The relation between their quenching ability and protective effects against photodamage were also compared to those of ascorbic acid (AsA) pretreatment. Both APS and AG were able to scavenge UVB/A-generated hydroxyl radicals under aqueous conditions (pH 7.2) in a manner similar to that seen with AsA; however, APS was a more effective scavenger than AG. Similar results were obtained ex vivo. Both derivatives could protect skin from UVB/A-induced photodamage, as determined by a reduction in the presence of sunburn cells and DNA fragmentation. However, AsA pretreatment had the weakest protective effect, even though cutaneous, its level was the highest among the three agents tested before irradiation. These results indicated that the superior protective effect of APS is related to its direct free radical scavenging ability, rather than to its conversion to AsA.

Accumulated evidence suggests that Parp-1 is involved in DNA repair processes, including base excision repair, single-strand and double-strand break repairs. To understand the precise role of Parp-1 in genomic stability in vivo, we carried out mutation analysis using Parp-1 knockout (Parp-1-/-) mice harboring two marker genes, gpt and red/gam genes. Spontaneous mutant frequencies of both genes in the bone marrows and livers did not differ significantly between Parp-1-/- and Parp-1+/+ mice (P>0.05). After treatment with an alkylating agent, N-nitrosobis(2-hydroxypropyl)amine (BHP), the mutant frequency of the red/gam genes in the liver in Parp-1-/- mice was 1.6-fold higher than that in Parp-1+/+ mice (P

We studied the intraocular pharmacokinetics of dorzolamide hydrochloride eye drops and the effect of dorzolamide on carbonic anhydrase activity and localization in ocular tissues. Carbonic anhydrase activity was detected in normal ocular tissues. The activity was inhibited in corneal endothelial cells, the ciliary body, lens epithelial cells, or the retina 1 to 8 hours after instillation of dorzolamide eye drops. In lens epithelial cells and the retina, the enzyme activity had not recovered even 10 hours after instillation of the drug. Immunostaining did not reveal any differences between the group administered dorzolamide eye drops and the control group administered a physiologically balanced solution. Time-related changes in dorzolamide concentrations in ocular tissues were measured by high-performance liquid chromatography (HPLC). In the cornea, anterior aqueous, iris, ciliary body and retina, drug concentrations increased 15 minutes after the instillation and peaked within 1 hour. These results suggest that dorzolamide immediately suppresses carbonic anhydrase activity in ocular tissues, and is rapidly distributed among the tissues of the eye when administered as eye drops.

Drosophila Crumbs and the mammalian homologues encoded by the Crb genes are transmembrane proteins required for determination of retinal cell polarity. We cloned a novel variant of mouse Crb1 and termed it Crb1s. Since the 3′-end of exon 6 remained unspliced, Crb1s coded for a short secretory protein lacking the transmembrane and cytoplasmic domains required for the function of Crb1. The Crb1 expression was confined to brain and eye, whereas Crb1s was detectable in various tissues including skin, lung, and kidney in adult mice. Active expression of Crb1s, but not Crb1, was observed during the skin development, in which localization of the Crb1s protein was altered from the basal layer to the upper layers. Cultured mouse keratinocytes synthesized the Crb1s protein and secreted a 80 kDa processed form to the supernatant. After Ca2+-induced differentiation, Crb1s became associated with focal adhesions and cell–cell contacts. Crb1s may play a role distinct from that of Crb1 in epidermal tissue morphogenesis.

Purpose: To evaluate the relationship of λ-crystallin to reduced nicotinamide adenine dinucleotide (NADH)-dependent dehydroascorbate (DHA) reductase found specifically in the rabbit lens. Methods: DHA reductase Fractions I–IV were separated from the λ/βL1-crystallin fraction of rabbit lens soluble protein by diethylaminoethyl (DEAE)-cellulose ion-exchange column chromatography, and then the enzyme was partially purified from Fraction II by rechromatography on the same ion-exchange column. The isolated DHA reductase fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, native isoelectric focusing and two-dimensional gel electrophoresis. Results: Using Western blot and a probe of antiserum to recombinant λ-crystallin, the main 33-kDa protein band was strongly stained in all the rabbit lens DHA reductase fractions, and most of the additional protein bands of approximately 25–30 kDa were also detectable. In the partially purified enzyme, the 33-kDa subunit alone was identified as a distinct protein band by SDS-PAGE, and a main basic protein was found at pI 7.6 by native isoelectric focusing. In addition, many bands of more acidic proteins were separated from other enzyme fractions, and protein spots corresponding to the 33 and/or approximately 25–30-kDa subunits were detected in each of the more acidic proteins by two-dimensional gel electrophoresis. Conclusion: These results suggest that λ-crystallin is closely related to the DHA reductase in the rabbit lens. The above heterogeneity of the enzyme-crystallin may arise from posttranslational modifications.

Urinary proteins from 14 patients with tubulointerstitial nephritis were analyzed by cellulose acetate membrane electrophoresis. Urinary total protein concentrations were measured, and urinary 15 proteins (prealbumin, albumin, α(1)‐microglobulin, α(1)‐antitrypsin, α(2)‐macroglobulin, haptoglobin, retinol binding protein, transferrin, β(2)‐microglobulin, IgA, IgG, κ‐ and λ‐light chains, cystatin C, and lysozyme) were identified by the use of a rapid and highly sensitive colloidal silver staining reagent suited for use with cellulose acetate membranes, as reported previously by Matsuda et al. (J Clin Lab Anal 15:171–174, 2001; Clin Chem47:763–766, 2001) and Hiratsuka et al. (J Clin Lab Anal 10:403–406, 1996). We also analyzed urinary total protein concentration and urinary protein fractions according to the presence of acute or nonacute interstitial nephritis. In addition, the relationship between urinary protein fraction and complications of interstitial nephritis was analyzed. The goal of this work was to find a useful index for the diagnosis of tubulointerstitial nephritis. J. Clin. Lab. Anal. 17:44–51, 2003. © 2003 Wiley‐Liss, Inc.

We have improved the agarose gel electrophoresis method reported previously for size-dependent separation of proteins. Good resolution in protein separation was achieved using a novel agarose gel, containing both sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate (LDS). The gel was divided into stacking and separating gels. We determined the optimal conditions for horizontal gel electrophoresis to be a voltage of 100V for the separating gel and 50V for the stacking gel, in an electrophoretic buffer solution of 25mM Tris-190mM glycine containing 0.03% LDS and 0.02% SDS (pH 8.3), which was also used to make the agarose gel. Separation of proteins in the molecular weight range 14.2-205K and of the proteins in human serum showed good resolution under these conditions. This method should be useful for clinical laboratories because it is simple and requires little bench space.

We have previously reported that connective tissue cells in the superficial dermis preferentially express α 1 (XVI) collagen rather than those in the lower dermis. Double immunofluorescence labeling using the antibodies for α 1 (XVI) collagen and factor XIIIa (plasma transglutaminase), which is a marker of dermal dendrocytes, demonstrated that both antibodies reacted with the same cells in the superficial dermis of normal skin as well as the lesional skins of dermal dendrocyte-related disorders, dermatofibroma, and psoriasis. Dermal dendrocytes are considered to be established by a culture of peripheral blood monocytes in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4. Reverse transcription–polymerase chain reaction, metabolic labeling, and immunofluorescence studies demonstrated that treatment of CD14 + peripheral blood monocytes with granulocyte macrophage-colony stimulating factor/interleukin-4 over a period of 8 d resulted in the induction of α 1 (XVI) collagen as well as factor XIIIa. The physiologic significance of colocalization of α 1 (XVI) collagen and factor XIIIa in the tissue and their coordinate induction in CD14 + monocyte-derived dendritic cells in vitro was studied. Considerable incorporation of [ 3 H]putrescine by factor XIIIa into recombinant noncollagenous domain (NC) 11 but not into collagenous domain (COL) 1·NC1 domain of the α 1 (XVI) polypeptide was found. Incubation of recombinant NC11 of α 1 (XVI) polypeptide with factor XIIIa in vitro produced a covalent cross-linking complex on sodium dodecylsulfate–polyacrylamide gel electrophoresis. The results indicate that α 1 (XVI) collagen is constitutively expressed by most dermal dendrocytes in the skin and dendritic cells differentiated from peripheral blood monocytes in vitro . Type XVI collagen is expressed in factor XIIIa + dermal dendrocytes and may form an intermolecular cross-linking through NC11 domain by the reaction catalyzed by factor XIIIa contributing to the structural integrity of factor XIIIa + dendritic cell-rich tissues.

腫瘍壊死因子α (TNF-α) は, 単球・マクロファージ由来の向炎症性サイトカインとして, 今日さまざまな分野で注目されている。免疫機能検査の新規診断薬として, 低用量のLipopolysaccharide (LPS) 刺激により数時間内に産生されるTNF-αを測定する系, SEK-5001システムを積水化学工業と共同で開発するに至った。このSEK-5001システム系に関する基礎的検討を行い, 検査法として十分使用できることが分かった。そこで本研究では運動による影響を調べ, その他の血中成分の検査値とも併せて比較し, 生体内における低用量LPS刺激によるTNF-α産生量の変動因子について検討した。低用量LPS刺激のTNF-α産生量は, 1か月間では時間の影響はみられなかったが, 年齢や軽度の運動による影響は反映した。特に軽度運動による本法のTNF-α変動は, 高用量LPSを用いたときよりも感度良く表れることが分かった。SEK-5001システムを用いてさらに多くの諸疾患患者のTNF-α産生量を測定し, 臨床現場でのより実用的な新規免疫機能検査法として確立されることが期待される。

Methods for separating proteins according to molecular weight are usually based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the toxicity of acrylamide and its handling during gel preparation constitute a significant problem. Therefore, we determined the molecular weights of proteins using agarose gel instead of polyacrylamide. We used the Mupid electrophoretic apparatus, because it is small, easy to use, and time-saving. We evaluated different types of agarose and buffer solutions, and parameters such as gel concentration, buffer SDS concentration, and quantity of gel (central thickness). The optimal gel was made from 5ml (central gel thickness 2mm) 4% NuSieve 3:1 agarose. An electrophoresis buffer containing 25mM Tris, 190mM glycine (pH8.3), and 0.1% SDS is optimal for protein separation. With this method, the standard curve was linear for reference proteins in the range of molecular weights from 14.4×103 to 97×103.

Composition and inducibility of cytochrome P450 (CYP) in ocular tissues were investigated using reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot techniques. Composition of ocular CYPs is more restricted than that of hepatic forms. High levels of CYP2B1/2 and reduced levels of CYP2C11 expression were detected in rat ocular tissues. Phenobarbital induced CYP2B1/2 expression in the lens but not in the rest of the eye, whereas CYP2C11 induction was observed in both regions. In addition, CYP2B1/2 proteins were found to be prominent in the retina. Our data indicate eye-specific regulation of CYP expression.

Tumor necrosis factor α (TNF‐α) has come into recent focus as a proinflammatory cytokine derived from monocytes/macrophages. We developed a novel system (the SEK‐5001 system) for measurement of ex vivo production of TNF‐α stimulated by lipopolysaccharide (LPS) as a marker of immune function. In the present study, we evaluated the performance of this system as a diagnostic tool. Furthermore, we compared TNF‐α levels with data from other immune function tests, including lymphocyte blast formation test and differential leukocyte counts. Incubation of whole blood with a stimulation of low‐dose LPS (100 EU/mL blood) for 4hr at 37°C gave acceptable results. After incubation, plasma TNF‐α levels were determined by enzyme‐linked immunosorbent assay (ELISA). The specificity, reproducibility, and recovery of the SEK‐5001 system were excellent. No correlation between TNF‐α levels and total leukocyte counts was found. Lymphocyte blast formation test and monocyte counts, however, were correlated with TNF‐α levels in blood from patients with hematological malignancy and aplastic anemia before treatment. This assay system may potentially be clinically applicable to assess in vivo immune function. J. Clin. Lab. Anal. 16:273–278, 2002. © 2002 Wiley‐Liss, Inc.

The protective effect of sodium-L-ascorbyl-2 phosphate (As-2P), a stable form of ascorbic acid (AsA), against photodamage induced by a single dose of UVB exposure (290-320 nm, Max 312 nm) was investigated using cultured mouse skin. When the cultured skin was treated with various As-2P concentrations, the cutaneous AsA level increased in proportion to the As-2P concentration. After 3 h of incubation, the AsA level in the cultured skin treated with 2, 20 and 100 mM As-2P increased 1.03-, 2.17- and 6.27-fold, respectively, compared with that of the control skin. These results suggest that As-2P was transported into the cultured mouse skin where it was converted to AsA. After 3 h, the cutaneous AsA level in irradiated (20 kJ/m2) skin was depleted to a half of that in the control skin. However, the level in skin pretreated with 20 mM As-2P was maintained within normal limits, even after 24 h. Pretreatment with 20 mM As-2P significantly prevented such photodamage as sunburn cell formation, DNA fragmentation and lipid peroxidation, which were caused by a single dose of UVB irradiation. These results suggest that the protective effect of 20 mM As-2P on UVB-induced cutaneous damage is due to the maintenance of a normal As level by conversion of As-2P to As in skin tissue.

The effects of stable vitamin C, magnesium-L-ascorbyl-2-phosphate (MAP), administered after acute and chronic exposure to UVB irradiation were investigated using hairless mice. Intraperitoneal administration of 100 mg/kg of MAP immediately after acute exposure to 15 kJ/m2 of UVB significantly prevented increases of UVB-induced lipid peroxidation in skin and sialic acid in serum, an inflammation marker. Administration of 50 mg/kg of MAP immediately after each exposure significantly delayed skin tumor formation and hyperplasia induced by chronic exposure to 2 kJ/m2 of UVB. Intraperitoneal administration of 200 mg/kg of MAP produced an increase in ascorbic acid (As) levels in the serum, liver and skin within 15 min. Serum As levels quickly returned to normal, but hepatic and cutaneous levels remained elevated before returning to normal after 24 h, suggesting that MAP was converted to As in the serum and in those tissues. Ultraviolet B-induced hydroxyl radical generation in murine skin homogenates was scavenged by As-Na addition, which was directly detected by electron spin resonance (ESR). These results suggest that postadministration of MAP delays progression of skin damage induced by UVB irradiation. It is presumed that MAP, once converted to As, exhibits such inhibitory effects by scavenging hydroxyl and lipid radicals generated as a direct or indirect result of UVB exposure.

Monkey and human lenses contain essentially the same glycosphingolipids, and Lewisxand sialylated Lewisxepitopes are expressed on the terminal structure of neolactotetraosylceramide. However, monolayer cultures of lens epithelial cells from rhesus monkey expressed gangliosides GM3, GD3 and a small amount of GM1, but not sialylated Lewisxepitopes. Eight-week-old cultures on various extracellular matrices resulted in morphological changes in lens epithelial cells. Monolayer of cells cultured on vitronectin or polylysine assembled into aggregates after 4 weeks of culture. Cells cultured on vitronectin expressed sialyl-Lewisxgangliosides and did not exhibit GD3. On collagens, fibronectin and laminin elongated cells were observed in cells cultured for 8 weeks. Thus, the interaction between cells and extracellular matrices influenced morphology and glycosphingolipid composition in lens epithelial cells.

Previously we showed a protective role of endogenous glutathione (GSH) in ultraviolet B (UVB) injury. Moderate UVE exposure to hairless mice receiving oral treatment with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, resulted in a greater number of SBCs in the epidermis. The evidence led to the hypothesis that increasing the level of endogenous GSH in the skin may reduce the skin damage caused by a high dose of UVB irradiation. Since systemic administration of a reduced form of GSH (reduced GSH) is understood to have poor permeability into the cells, in the current study we investigated transportability of esterified GSH and photoprotective effect of reduced GSH and the esterified derivative against UVB injury in vivo . Oral administration of esterified GSH revealed increased cutaneous GSH level more effectively than did reduced GSH. The number of sunburn cells (SBC) formed was significantly depressed in the skin exposed to UVIB in mice treated with esterified GSH as compared with non-GSH- or reduced GSH-treated mice. The suppressive effect of esterified GSH was prominent in BSO-treated animals.

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.

Urinary proteins of patients with diabetes mellitus (DM) were analyzed using cellulose acetate membrane electrophoresis, to determine the clinical usefulness of fraction patterns of the proteins in detecting the group at high risk for diabetic nephropathy. We divided the protein patterns into 5 groups. Four groups (I, II, III, IV) were found in the healthy group and a newly classified group was termed group 0 and was characterized by a prominent albumin peak with a negligible or small globulin peak. The incidence of groups 0, I, II, III, and IV, was 36.6%, 13.3%, 18.7%, 10.7% and 22.7%, respectively. This distribution was clearly different from that of healthy subjects and the most characteristic feature of diabetics was that group 0 accounted for 36.6% of the total cases. Characteristic features of each group were examined from the aspect of laboratory and clinical findings. Urinary protein patterns were concluded to be useful not only to predict the high risk group for diabetic nephropathy in the preclinical stage but also to discriminate nephropathic types of glomerular or tubular origin. It is useful for clinicians to know the risk stage and prognosis for diabetic nephropathy.

The protective effect of magnesium-L-ascorbyl-2-phosphate (MAP) on cutaneous photodamage such as lipid peroxidation and inflammation induced by ultraviolet B (UVB) exposure (290-320 nm, max. 312 nm) was investigated using hairless mice. When MAP was administered intraperitoneally to mice at a dose of 100 mg of ascorbic acid (AS) per kg body weight base immediately before irradiation (15 kj/m2), the expected increases in thiobarbituric acid reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, were significantly reduced. However, the expected decrease in the level of cutaneous AS was unchanged. Similar results were observed for animals given 100 mg of AS-Na per kg body weight before UVB irradiation. When MAP was administered intracutaneously immediately before irradiation, the expected UVB-induced increases in TBARS and sialic acid were again significantly prevented. Ascorbic acid-Na had a less protective effect than intracutaneous MAP administration. The cutaneous AS level was significantly higher in the MAP-treated mice than in the controls, and the UVB-induced decrease in tissue AS was prevented by intracutaneous MAP administration. These results suggest that MAP protects against UVB irradiation-induced lipid peroxidation and inflammation in cutaneous tissue, regardless of the drug administration route. We found, in an in vitro experiment, that MAP was converted to AS as it crossed the epidermis, but that AS-Na did not pass through the epidermis. Furthermore, MAP was also converted to AS in serum. These results suggest that the protective effect of MAP on UVB-induced cutaneous damage is due to conversion of MAP to AS.

The effect of glutathione (GSH) isopropyl ester on cellular sensitivity to UVB irradiation was investigated in HeLaS3 cells. Pretreatment with 0.1-0.5 mM GSH isopropyl ester for 4 h significantly inhibited the decrease of thymidine (TdR) incorporation caused by UVB irradiation at a dose of 500 J/m2, whereas pretreatment with a high dose (1 mM) had no effect. The colony formation ability of the pretreated cells (0.3 mM) was significantly better than that of cells that received irradiation only. When the cells were treated with GSH isopropyl ester, their intracellular GSH level increased dose-dependently over a 4 h period, suggesting that GSH isopropyl ester was transported into the cells and there converted to GSH. Within 2 min of exposure, the intracellular GSH level depleted rapidly to about 75% of that in non-irradiated normal cells. In contrast, the GSH level in cells pretreated with 0.3 mM GSH isopropyl ester was maintained at the same level as that in normal cells, indicating that the maintenance of intracellular GSH level is due to converted GSH from GSH isopropyl ester. These results clearly show that intracellular GSH is involved in cell protection against photodamage, and that GSH isopropyl ester is a useful antioxidant for protection against photooxidative injury.

As part of a trial course on interprofessional education (IPE) at Keio University our Faculty of Pharmacy initiated a joint seminar with the university’s medical and nursing departments in 2008. We had two joint seminars: one in June and another in October. In the seminars, students and participants actively discussed several issues with regard to the national and private health care systems and medical malpractice. They also listened to feedback lectures held by experts from the medical and social system. At Kyoritsu University of Pharmacy, we had already held similar joint seminars three times since 2006. The participants were not only from our school but from various universities around Japan, given that our school was then only a small college for pharmaceutical students. Our considerable experience in the field of small group learning (SGL) helped make these seminars a success. By making full use of the SGL method we have successfully lead the discussion sessions at the IPE seminars attended by students from various medical fields. After the merging of our small pharmaceutical college with Keio University, we continued holding joint seminars for the three medical faculties at Keio University. It has not been without tribulations, however. The newly created interprofessional seminars faced several problems. The seeming lack of interest shown by the medical school decreased the level of participation of medical students, while too many facilitators are required for such seminars. To resolve these issues, we have made some changes to future activities in this area. To start with, we plan to change the course from an elective to a required subject for the students of the three medical faculties in Keio University.

An increase in polydrug abuse is a major problem worldwide. The coadministration of methamphetamine and morphine increased subacute toxicity or lethality in rodents. However, the underlying mechanisms by which lethality is increased by the coadministration of methamphetamine and morphine are not yet fully understood. Coadministered methamphetamine and morphine induced lethality by more than 80% in BALB/c mice, accompanied by the rupture of cells in the kidney and liver, and an increase in poly (ADP-ribose) polymerase (PARP)-immunoreactive cells in the heart, kidney and liver. The lethal effect and the increase in the incidence of rupture or PARP-immunoreactive cells induced by the coadministration of methamphetamine and morphine was significantly attenuated by pretreatment with mepacrine (phospholipase A(2) inhibitor) or fullerene (a radical scavenger), or by cooling from 30 to 90 min after drug administration. Furthermore, based on the results of the electron spin resonance spin-trapping technique, hydroxyl radicals were increased by the administration of methamphetamine and morphine, and these increased hydroxyl radicals were potently attenuated by fullerene and cooling. These results suggest that hydroxyl radicals plays an important role in the increased lethality induced by the coadministration of methamphetamine plus morphine. The potency of cooling or drugs for decreasing the subacute toxicity or lethality induced by the coadministration of methamphetamine and morphine was in the order fullerene=cooling>mepacrine. These results indicate that fullerene and cooling are beneficial for preventing death that is induced by the coadministration of methamphetamine and morphine.

Ultraviolet radiation is the major environmental cause of skin damage. Although only 0.5% of ultraviolet B (UVB) radiation reaches the earth, it is the main cause of sunburn and inflammation and the most carcinogenic constituent of sunlight. We investigated whether the topical application of a novel, water-soluble gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG), could protect against UV-induced skin damage. Topical pre- or postapplication of gamma-TDMG solution significantly prevented sunburn cell formation, lipid peroxidation, and edema/inflammation that were induced by exposure to a single dose of UV irradiation. Cyclooxygenase-2 (COX-2)-catalyzed synthesis of prostaglandin E(2) (PGE(2)) levels seen after UV exposure were significantly suppressed by pre- or posttreatment with gamma-TDMG. The increase in COX-2 activity was significantly inhibited by gamma-TDMG, suggesting that the reduction in PGE(2) concentration was due to the direct inhibition of COX-2 activity by gamma-TDMG. The derivative strongly inhibited inducible nitric oxide synthase mRNA expression and nitric oxide production. With the application of gamma-TDMG, the pigmentation in melanocytes was lightened and the increase melanin concentration was suppressed. Gamma-TDMG is converted to gamma-Toc in the skin and has higher bioavailability than gamma-Toc itself. These results suggest that gamma-TDMG-derived gamma-Toc acts as an antioxidant, antiinflammatory and antipigmentation agent. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing and reducing UV-induced skin damage in humans.

The present investigation aims to evaluate the NADH binding ability of lambda-crystallin, a taxon-specific enzyme-crystallin, in the rabbit lens.A lambda/betaL1-crystallin fraction was separated from the rabbit lens soluble fraction by gel filtration and the enzyme-crystallin was partially purified by subsequent affinity column chromatography. Analysis of NADH bound to the lambda-crystallin preparation was performed using spectrophotometric and enzymological methods. Binding of added NADH to the enzyme-crystallin preparation was also analyzed using a simple ultrafiltration method, which was theoretically equivalent to equilibrium dialysis, to study additional NADH binding to the protein.The prepared lambda-crystallin samples clearly exhibited an absorption maximum at 340 nm, even though they were thoroughly dialyzed. This was due to the presence of nondialyzable NADH bound tightly to the protein. The bound NADH was removed by charcoal treatment, and extracted by 0.1% SDS or 70 degrees C heat treatment. A dissociation constant (Kd) of less than 5 nM indicated tight binding of NADH. The quantity of bound NADH in the 88% purified 33 kDa enzyme-crystallin was estimated to be 20.5 nmol/mg protein, suggesting a stoichiometry of 0.7 mol of the nucleotide/mol of the 33 kDa protein. Additional looser binding of added NADH to lambda-crystallin was observed in both the lambda/betaL1-crystallin fraction (including the full-length 33 kDa protein: 34%; 25-30 kDa proteins, most of which might be generated by cleavage of the 33 kDa protein: 64%) and the partially purified enzyme-crystallin. It was assumed from the analysis of binding titration that some (about 30%) of the 33 kDa protein and most of the lower molecular weight proteins still possessed the ability to loosely bind NADH. Kd values of their lower affinity binding were determined to be 2 or 6 microM.From the present study, we conclude that lambda-crystallin plays a sufficiently important role as a NADH binding protein to maintain high levels of this nucleotide in the rabbit lens.

We previously reported that a novel hydrophilic gamma-tocopherol (gamma-Toc) derivative, gamma-tocopheryl-N,N-dimethylglycinate hydrochloride (gamma-TDMG) gets converted to the antioxidant gamma-Toc in skin. We also found that this derivative displayed greater bioavailability than gamma-Toc itself. In the present study, we determined whether gamma-TDMG could reduce UV-induced skin pigmentation in brownish guinea pigs. gamma-TDMG (0.1 or 0.5%) was topically applied to the skin before and after it was exposed to UVB plus UVA (3 times/week for 1 week), and then 10 times/week for 4 weeks thereafter. Treatment with 0.5% gamma-TDMG resulted in significant skin lightening (70% of the pigmentation of irradiated controls). We also found that melanin synthesis was dose-dependently inhibited by gamma-TDMG in murine B16 melanoma cells. When gamma-TDMG or kojic acid (250 microM) were added to homogenates of B16 melanoma cells, their tyrosinase activity was significantly inhibited by approximately 40% and 75%, respectively. Mushroom tyrosinase activity was significantly inhibited by 200 microM gamma-Toc and kojic acid, but not gamma-TDMG. When B16 cells were incubated with 250 microM gamma-TDMG for 24 or 48 h, their intracellular gamma-Toc concentrations rose over 100 fold to 10.5 and 11.2 nmol/10(6) cells, respectively, suggesting that gamma-TDMG was rapidly converted to gamma-Toc in these cells and that their reduced melanin synthesis may have been due to the activity of gamma-Toc. Our data further suggest that the topical application of gamma-TDMG may be efficacious in preventing photo-induced skin pigmentation in humans.

Mouse milk fat globule-EGF factor 8, MFG-E8, is the ortholog to the human mammary tumor marker, lactadherin, and comprises two spliced variants, the L and S forms. Recent studies have suggested that MFG-E8-L produced by macrophages and Langerhans cells in the skin serves as a linker between phagocytic cells and apoptotic cells, and that MFG-E8-S, also termed SED1, facilitates sperm-egg interaction for fertilization. However, Mfge8 gene expression occurs in various tissues apparently unrelated to these critical events. Our in situ hybridization study has revealed that Mfge8 is expressed in the periderm (the premature epidermis) on embryonic day-14, well before Langerhans cells begin to grow in the prenatal phase. Mfge8 transcript is detectable in the basal and spinous layers throughout skin development, whereas immunostaining has revealed MFG-E8 protein accumulation in the spinous layer. Cultured keratinocyte stem cells consistently express Mfge8-L and -S mRNAs and produce the L protein, which is primarily detectable in the culture supernatant, and the S protein, which is mostly associated with the cells. Upon Ca(2+)-stimulated differentiation, which is detected by a decrease in keratinocyte stem cell marker p63(p51) and the induction of keratin1, we have observed suppression of Mfge8, and the protein becomes localized to the cell-cell borders. Papillomas and carcinomas caused by chronic UV-B irradiation produce MFG-E8 as determined by immunostaining. Thus, undifferentiated and poorly differentiated keratinocytes produce the L and S forms of MFG-E8 during normal and pathological tissue development, probably to support an as yet unidentified membrane function.

In the clinical field of nephrology, a noninvasive approach employing the analysis of electrophoretic patterns in urinary protein has been established. In this study a total of 52 urine samples with IgA nephropathy (IgAN), anti‐neutrophil cytoplasmic antigen‐associated crescentic glomerulonephritis (GN), and other types of GN were analyzed. Patients with high α(1) globulin (α(1)G) fractions, which contained α(1)AT in cellulose acetate membrane electrophoresis (CAE), tended to have α(1) antitrypsin (α(1)AT) of normal molecular weight (57 kDa and 49 kDa), while patients with a deficit α(1)G fraction tended to have α(1)AT of low molecular weight (

We investigated whether the topical application of a novel, water-soluble gamma-tocopherol (gamma-Toc) derivative, gamma-tocopherol-N,N-dimethylglycinate hydrochloride (gamma-TDMG), could protect against UV-induced skin damage in hairless mice. Topical pre- or post-application of a 5% (93 mM) gamma-TDMG solution in water/propylene glycol/ethanol (2:1:2) significantly prevented sunburn cell formation, lipid peroxidation and edema/inflammation that were induced by exposure to a single dose of UV irradiation of 5 kJ/m2 (290-380 nm, maximum 312 nm). This effect was greater than that seen with two alpha-Toc derivatives, alpha-tocopherol acetate (alpha-TA) and alpha-tocopherol-N,N-dimethylglycinate (alpha-TDMG). When a 5% solution of gamma-TDMG was applied to mouse skin for 1 h, cutaneous gamma-Toc increased by 25-fold after 24 h; levels of cutaneous alpha-Toc increased by only two- and eight-fold in alpha-TDMG and alpha-TA treated skins, respectively. These findings indicated that gamma-TDMG immediately converted to gamma-Toc in the skin and suggest that ability of gamma-TDMG to protect the skin from the damaging effects of irradiation was due to its conversion to gamma-Toc. When a 5% solution of gamma-Toc was applied to mouse skin for 1 h, cutaneous gamma-Toc rapidly increased by 25-fold, but fell to baseline levels by 24 h. In contrast, the concentration of gamma-Toc in skin that was treated with gamma-TDMG similarly increased, but these high levels were maintained after 24 h. These results suggest that gamma-TDMG may be a more effective source of gamma-Toc in skin. Thus, the topical application of gamma-TDMG may be efficacious for the prevention of UV-B-induced skin damage.

Ascorbate free radical (AFR) reductase with diaphorase activity was isolated from the rabbit lens soluble fraction to characterise some molecular properties of the enzyme. The isolation was accomplished using gel filtration (Sephadex G-75 superfine or Sephacryl S-200 HR), affinity chromatography (Affi-Gel Blue), native isoelectric focusing and two-dimensional gel electrophoresis. A major soluble AFR reductase was found at an isoelectric point of 8·4 and a molecular weight of 31 kDa, and a few minor enzymes were also detected in the range of pI 7·0–8·6. An unknown N-terminal partial amino acid sequence was determined in one peptide fragment prepared from the major enzyme fraction. From the sequence analysis, it is discussed that the lens soluble AFR reductase may differ from NADH-cytochrome b5 reductase reported to be involved in the membrane-bound AFR reductase activity of mitochondria, microsomes and plasma membrane.

We have developed a new method for measuring total urinary protein using acid violet 6B (AV6B) pigment. Nine purified components of human urinary proteins and urine samples collected randomly from 123 diabetic outpatients were used. There were 62, 36, and 25 cases of prenephropathy, early nephropathy, and overt nephropathy, respectively. All samples were measured by Coomassie brilliant blue G 250 (CBB), pyrogallol red‐molybdate (PRM), and AV6B methods using an optical photometer. In healthy subjects, the major components of urinary proteins, such as γ‐globulins, IgG, IgA Tamm‐Horsfall protein, and transferrin, the reactivity values of the AV6B and PRM methods were similar. The CBB method was the least sensitive of the three methods. In the urine samples from diabetic patients, the urinary protein values measured by the AV6B method were higher than those measured by the CBB method in the prenephropathy stage. The values obtained by the AV6B method (y) correlated well with those from the CBB method (x) (y = 1.243x+ 3.61, r = 0.904). When the values from the AV6B method (y) were compared to those from the PRM method (x), correlation was low (y = 1.406x−29.15, r = 0.786). In conclusion, the AV6B method was more useful than the CBB and PRM methods for low levels of urinary protein. J. Clin. Lab. Anal. 17: 147–154, 2003. © 2003 Wiley‐Liss, Inc.

The ability of the novel water-soluble provitamin E, alpha-tocopherol-6-O-phosphate, to protect against ultraviolet B-induced damage in cultured mouse skin was investigated and compared with the protectiveness of alpha-tocopherol acetate in cultured mouse skin. Pretreatment of skin with 0.5% (9.4 mM) alpha-tocopherol-6-O-phosphate in medium for 3 h significantly prevented such photodamage as sunburn cell formation, DNA degradation, and lipid peroxidation, which were induced in control cultured skin by a single dose of ultraviolet B irradiation at 0 to 40 kJ per m2 (290-380 nm, maximum 312 nm). This protection was greater than that seen with alpha-tocopherol acetate, the most common provitamin E that is used in commercial human skin care products. The concentration of alpha-tocopherol in cultured skin pretreated with 0.5% alpha-tocopherol-6-O-phosphate rose to approximately two to three times that found in the control skin and the reduction in cutaneous alpha-tocopherol that was induced by ultraviolet irradiation was significantly inhibited. In the group pretreated with 0.5% alpha-tocopherol acetate, however, conversion of alpha-tocopherol acetate to alpha-tocopherol was not observed, although the level of provitamin incorporated into the cultured skin was the same as that for alpha-tocopherol-6-O-phosphate. These findings indicated that the enhanced ability of alpha-tocopherol-6-O-phosphate to protect against ultraviolet B-induced skin damage compared with alpha-tocopherol acetate may have been due to alpha-tocopherol-6-O-phosphate's conversion to alpha-tocopherol. Moreover, following pretreatment with a 0.5% alpha-tocopherol-6-O-phosphate, alpha-tocopherol-6-O-phosphate was incorporated into the human skin in a three-dimensional model and 5% of the incorporated alpha-tocopherol-6-O-phosphate was converted to alpha-tocopherol. These results suggest that treatment with the novel provitamin E, alpha-tocopherol-6-O-phosphate may be useful in preventing ultraviolet-induced human skin damage.

To clarify the physiological role(s) of metallothionein (MT) in carcinogenesis, we studied the susceptibility of MT-null mice to chemically mediated carcinogenesis in the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA)-induced two-stage carcinogenesis model. The MT-null mice were subjected to a single topical application of DMBA (50 or 100 micro g/mouse) and, 1 week later, to promotion with TPA (10 micro g/mouse) twice a week for 20 weeks. At week 21, nearly all of the MT-null mice developed tumors in the skin, in contrast to only 10-40% of wild-type mice. No tumors were observed in MT-null or wild-type mice that were administered TPA alone. By using the PCR-restriction fragment length polymorphism and PCR-single strand conformation polymorphism methods, we found a transversion of A182 to T in codon 61 of c-Ha-ras in the papilloma tissue of MT-null mice and wild-type mice but failed to find any mutations in the c-Ki-ras and c-N-ras genes. In two-stage skin carcinogenesis induction by DMBA/TPA, p53 and p21(WAF1/Cip1) expression levels were found to be increased in MT-null mice compared with wild-type mice. As to an earlier change at the promotion stage triggered by TPA application, MT-null mice were found to have both hyperplasia of the epithelium and a marked degree of inflammation in the basal layer, indicating that the induced as well as endogenous MT acted as a protective factor against tumorigenesis. In conclusion, the present study has demonstrated that MT has antitumorigenic potential in both the initiation and promotion stages of the two-stage chemical skin carcinogenesis model.

P-glycoprotein (P-gp), the product of the multiple drug resistance (mdr) gene, can actively pump toxic drugs out of cells, but its pathophysiologic role is not yet fully understood. In this study, we examined the expression of P-gp in dextran sodium sulfate (DSS)-induced colitis in mice. Eight-week-old Balb/c female mice were given drinking water containing 7% DSS ad libitum for 7 days. Mice receiving DSS were sacrificed on days 3, 5, and 7 for histopathologic study. Tissue samples were examined by hematoxylin and eosin (HE) staining, and immunostained against mdr, CD4+, CD8+, and B220+. RNA was isolated from the large intestine and the expression of mdr1a was determined by RT-PCR. The function of P-gp was evaluated by rhodamine123 efflux using the everted sac method. The induction of colitis in mice was confirmed by body weight changes, HE staining and immunohistological grading of the large intestine with reference to CD4+, CD8+, and B220+ after 7 days of treatment. Severe inflammation was observed in the large, but not the small, intestine on day 7. The expression of mdr1a in the large intestine was reduced on days 3, 5, and 7. In addition, the P-gp function and the expression of PXR were also reduced in the large intestine of DSS-treated mice on day 3. This reduction was consistent with the immunohistologic observations. The expression of the mdr1a gene was reduced before severe symptoms appeared. These results suggest that P-gp expression may be related to the pathology of colitis.

We fractionated normal urinary proteins obtained from 40 healthy subjects using cellulose acetate membrane electrophoresis and stained them with Acid Violet 17. The electrophoretic patterns were classified into four groups. Each of groups I, II, III, IV had an albumin peak and 1, 2, 3, and 4 additional globulin peaks, respectively. Within-day variation study showed that the pattern was fundamentally specific to the individual, although some intermediate cases were observed. We were unable to determine which type was standard for normal subjects. However, the concentration of Tamm-Horsfall protein was speculated to be an important factor in determining the patterns. Group III showed significantly higher values than group I in urine albumin, total protein, and beta-N-acetyl-D-glucosaminidase and this group was believed to include subjects in the subclinical stage of a glomerular disease. All specimens belonging to group IV showed an obvious fraction of alpha 1 globulin which is often found in urine specimens of patients with renal diseases of tubular origin or other pathological conditions.

We investigated the expression of heat shock protein 27 (HSP27) at intermediate stages of a cutaneous tumor induced by UVB-irradiation stress (290-380 nm, max. 312 nm) using an immunostaining method. After 15-20 weeks of chronic exposure to UVB irradiation at a dose of 2 kJ/m2, HSP27 was found in the upper cell layers of bowenoid multilayers of epidermis, in areas of the lesions where normal stratification seems to be conserved. After 25 weeks, HSP27 was weakly expressed in squamous cell carcinoma (SCC). The HSP27 distribution patterns during cutaneous tumor progression resemble that of cytokeratin 10, a differentiation marker in keratinocytes. In SCC, a low degree of HSP27 expression was detected in the well-differentiated carcinomatous areas, but not in the poorly differentiated areas. These results indicate that the level of HSP27 decreases significantly as epithelial carcinoma growth progresses upon UVB-exposure. The expression of HSP27 may be associated with the onset of skin keratinocyte differentiation, but not with progression of SCC.

We investigated the changes in urinary albumin and urinary transferrin as glomerular proteins, and in urinary N-acetyl-β-D-glucosaminidase and urinary β2-microglobulin as tubular proteins, in patients with impaired glucose tolerance. We attempted to compare the proteins of normal subjects to those of diabetics with pre-nephropathy. Transferrin and N-acetyl-β-D-glucosaminidase levels were significantly increased in patients with impaired glucose tolerance, while albumin and β2-microglobulin levels were only slightly increased. In addition, there was no significant difference in transferrin levels between patients with impaired glucose tolerance and type 2 diabetics with pre-nephropathy. In our observation, although albumin levels were only slightly increased in patients with impaired glucose tolerance, a sharp increase in transferrin levels was reflected in patients with glomerular disorders. In addition, since N-acetyl-β-D-glucosaminidase levels varied markedly, tubular disorders were suspected. It should be stressed that increased parameters for both glomerular and tubular disorders in group C—patients who showed abnormal levels in three proteins—had already been observed in some patients with impaired glucose tolerance. Therefore, the evaluation of the mutual relationships between various urinary protein components in patients with impaired glucose tolerance will become a more important assessment tool than that of single urinary protein components. J. Clin. Lab. Anal. 12:351–355, 1998. © 1998 Wiley-Liss, Inc.

— We investigated the induction, cellular localization and phosphorylation of a low-molecular weight stress protein (heat shock protein 27, HSP27) by UVB (290-320 nm, max. 312 nm) irradiation stress using immunoblot and indirect immunofluorescence analysis in in vivo and in vitro experiments. The HSP27 was constitutively expressed and distributed in the cytoplasmic fraction of Pam 212 cells (mouse keratinocyte line) or dorsal skin. The increase in the cytoplasm HSP27 level induced by UVB irradiation was less than two-fold that in nonirra-diated controls. On the other hand, the translocation of HSP27 from cytoplasm to the nucleus or perinuclear area was time- and dose-dependently induced by UVB irradiation. After UVB irradiation, three isoforms having different isoelectric points were detected in nucleic HSP27 by two-dimensional immunoblotting. The most basic isoform was the unphosphorylated type and the two acidic isoforms were phosphorylated, suggesting that HSP27 is phosphorylated in response to UVB irradiation and accumulates in or around the nucleus as a phosphorylated isoform. These results suggest that the translocation and phosphorylation of HSP27 are induced in response to UVB-irradiation stress.

— The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290–400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kj/m2) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kj/m2, three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photo-damage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.

–The role of metallothionein (MT) in protecting skin cells against UVB irradiation was investigated. Fibroblast strains from normal adult (HS-K) and neonatal (NB1RGB) human skins as well as keratinocyte strains from human skin (SV40-HSK) and newborn Balb/c mouse skin (Pam 212) were exposed to UVB irradiation. The sensitivity of HS-K and NB1RGB cells to UVB irradiation was similar; those of SV40-HSK and Pam 212 cells were two- and six-fold as sensitive to UVB irradiation as HS-K cells, respectively. The HS-K cells contained the greatest cellular reduced form of glutathione (GSH) levels compared to the three other skin cells: the levels were 13-, 7- and 6-fold of those in NB1RGB, SV40-HSK and Pam 212 cells, respectively. These results indicated that the sensitivity of skin cells to UVB irradiation was not always associated with their endogenous GSH levels. In particular, despite the fact that NB1 RGB cells contained a relatively small amount of GSH, they were less sensitive to UVB irradiation. NB1RGB cells contained 4–30 times more MT than those in other skin cells examined. The sulfhydryl residues of MT molecules in the NB1RGB cells were estimated to be mostly unoccupied by metals, suggesting they act in a similar way to those of GSH. Moreover, NB1RGB cells in which the MT content was elevated by dexamethasone (1 μg/mL) or Zn2+ (7 μg/mL) treatment were more resistant to UVB irradiation than nontreated ones. These results suggest that, at least in neonatal human skin fibroblasts, MT may play a role in protection against UVB irradiation.