Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry.

OBJECTIVES: To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. METHODS: Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis. RESULTS: 2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. CONCLUSION: Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.

OBJECTIFS: évaluer l'amélioration de l'extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d'expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l'abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d'un gel 2DE JRH ont été clivés par enzyme pour l'analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l'analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d'abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d'un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées. CONCLUSION: l'extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu'il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

Electron microscopy and tomography reveal that sodium 2-naphthalene sulfonate incorporated into perming solutions swells and tilts trichocyte intermediate filaments causing straightening of curly Japanese human hair.

OBJECTIVE: A new hair-care process has been specifically developed for the straightening of curved Japanese woman's hair . The process included sodium 2-naphthalene sulfonate (SNS) in the reduction and oxidation steps of a conventional perming process. Our objective was to develop an understanding of how this process caused hair straightening by measuring the changes to morphology and ultrastructure between untreated, conventionally permed and SNS permed hair. Untreated and SNS permed Merino wool fibres were used to confirm structural changes. METHODS: Japanese hair samples were measured for single-fibre curvature before and after perming treatments. A silver staining method was developed to stain hair fibres without changing fibre curvature so that transmission electron microscopy could be used to measure changes in the lateral dimensions of all structural components from the cellular to protein filament level. Electron tomography determined intermediate filament slopes and slope changes after SNS perming relative to the central longitudinal axis of the fibre. RESULTS: SNS perming was found to cause greater lateral swelling than conventional perming of: the paracortical cells of wool; the cuticle, the cuticular cell membrane complex and the macrofibrillar centre-to-centre distance of hair; and of the intermediate filaments in wool and hair. In curved hair, SNS perming caused the intermediate filaments of the helical macrofibrils to simultaneously swell and to tilt further, resulting in the slight longitudinal contraction of the macrofibrils. The overall swelling and tilting was greatest in the helical macrofibrils of Type B cortical cells predominately located in the convex fibre half. The presence of a higher percentage of helical macrofibrils in the convex fibre half than in the concave fibre half caused a contraction differential between the two halves leading to straighten of the curved fibre. A mechanical model was proposed to explain how SNS perming straightened curly hair. CONCLUSION: The effects of conventional and SNS perming on the morphological and ultrastructural components of curved Japanese hair and high-curl Merino wool fibres have given clear insights into understanding the mechanism of fibre curvature change.

OBJECTIF: Un nouveau procédé de soin des cheveux a été spécialement conçu pour lisser les cheveux ondulés des Japonaises[1]. Le procédé utilise le sulfonate de naphthalène-2 sodium (SNS) dans les étapes de réduction et d'oxydation du procédé conventionnel de permanente. Notre objectif était de comprendre la façon dont ce procédé induisait le lissage des cheveux en mesurant les différences de changement morphologique et ultrastructural entre les cheveux non traités et ceux soumis à une permanente conventionnelle et une permanente à base de SNS. Des fibres de laine de mérinos non traitées et soumises à une permanente à base de SNS ont été utilisées pour confirmer les changements structurels. MÉTHODES: Des échantillons de cheveux japonais ont été utilisés pour mesurer la courbure d'une fibre isolée avant et après le traitement de permanente. Une méthode de coloration argent a été mise au point pour colorer les fibres de cheveux sans changer la courbure des fibres afin de pouvoir utiliser la microscopie électronique en transmission pour mesurer les modifications des dimensions en largeur de tous les composants structurels du filament, de la cellule aux protéines. Une tomographie électronique a déterminé les pentes intermédiaires et les changements de pente des filaments après permanente à base de SNS par rapport à l'axe longitudinal central de la fibre. RÉSULTATS: On a constaté que la permanente à base de SNS induisait un gonflement en largeur plus important que la permanente classique des cellules paracorticales de la laine; de la cuticule, du complexe de la membrane cellulaire cuticulaire et de la distance centre à centre des macrofibrilles du cheveu; et des filaments intermédiaires dans la laine et les cheveux. Dans les cheveux ondulés, la permanente à base de SNS a provoqué à la fois un gonflement et une inclinaison des filaments intermédiaires des macrofibrilles hélicoïdales, entraînant une légère contraction longitudinale des macrofibrilles. Au total, le gonflement et l'inclinaison étaient plus importants dans les macrofibrilles hélicoïdales des cellules corticales de type B situées principalement dans la moitié convexe de la fibre. La présence d'un pourcentage plus élevé de macrofibrilles hélicoïdales dans la moitié convexe par rapport à la moitié concave de la fibre a entraîné une contraction différentielle entre les deux moitiés qui a entraîné le redressement de la fibre courbée. Un modèle mécanique a été proposé pour expliquer comment la permanente à base de SNS lissait les cheveux bouclés. CONCLUSION: Les effets de la permanente conventionnelle et à base de SNS sur les composants morphologiques et ultrastructuraux des cheveux japonais ondulés et des fibres de laine très frisés de mérinos ont permis de mieux comprendre le mécanisme du changement de courbure des fibres.

Randomized, double-blind, phase III trial of palonosetron versus granisetron in the triplet regimen for preventing chemotherapy-induced nausea and vomiting after highly emetogenic chemotherapy: TRIPLE study.

BACKGROUND: There has been no phase III study of comparing the efficacy of first- and second-generation 5-HT3 receptor antagonists in the triplet regimen with dexamethasone and aprepitant for preventing chemotherapy-induced nausea and vomiting after highly emetogenic chemotherapy (HEC). PATIENTS AND METHODS: Patients with a malignant solid tumor who would receive HEC containing 50 mg/m(2) or more cisplatin were randomly assigned to either palonosetron (0.75 mg) arm (Arm P) or granisetron (1 mg) arm (Arm G), on day 1, both arms with dexamethasone (12 mg on day 1 and 8 mg on days 2-4) and aprepitant (125 mg on day 1 and 80 mg on days 2-3). The primary end point was complete response (CR; no vomiting/retching and no rescue medication) at the 0-120 h period and secondary end points included complete control (CC; no vomiting/retching, no rescue medication, and no more than mild nausea) and total control (TC; no vomiting/retching, no rescue medication, and no nausea). RESULTS: Between July 2011 and June 2012, 842 patients were enrolled. Of 827 evaluable, 272 of 414 patients (65.7%) in Arm P had a CR at the 0-120 h period when compared with 244 of 413 (59.1%) in Arm G (P = 0.0539). Both arms had the same CR rate of 91.8% at the acute (0-24 h) period, while at the delayed (24-120 h) period, Arm P had a significantly higher CR rate than Arm G (67.2% versus 59.1%; P = 0.0142). In secondary end points, Arm P had significantly higher rates than Arm G at the 0-120 h period (CC rate: 63.8% versus 55.9%, P = 0.0234; TC rate: 47.6% versus 40.7%, P = 0.0369) and delayed periods (CC rate: 65.2% versus 55.9%, P = 0.0053; TC rate: 48.6% versus 41.4%, P = 0.0369). CONCLUSION: The present study did not show the superiority of palonosetron when compared with granisetron in the triplet regimen regarding the primary end point. CLINICAL TRIAL REGISTRY IDENTIFIER: UMIN000004863.

Ceramide limits phosphatidylinositol-3-kinase C2ß-controlled cell motility in ovarian cancer: potential of ceramide as a metastasis-suppressor lipid.

Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2ß-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2ß and affect its compartmentalization, thereby suppressing PI3KC2ß activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2ß knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2ß in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2ß-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.

Diverse coordination modes in tin analogues of a cyclopentadienyl anion depending on the substituents on the tin atom.

Reactions of an anionic heavy ruthenocene with CCl4, MeI, EtBr and Me3SiCl afforded the first stannole monoanion complexes. Surprisingly, coordination modes of the stannole rings are highly dependent on the substituents on the tin atom. The chloro derivative exhibits a η(4)-fashion-like coordination mode with a bent stannole ring, whereas the trimethylsilyl derivative adopts the conventional η(5)-coordination mode. Coordination modes of the alkyl derivatives are in between the two types. Cyclic voltammograms for these complexes reveal that the electron-donating character of the stannole ligand becomes stronger as the stannole ring becomes planar. Theoretical calculations elucidate that the different coordination modes originate from both electronegativity of an adjacent atom to the tin atom and bulkiness of a substituent on the tin atom.

Comparison of pregnancy outcomes between women with gestational diabetes and overt diabetes first diagnosed in pregnancy: a retrospective multi-institutional study in Japan.

AIMS: To determine differences in pregnancy outcomes including diabetic complications, maternal and perinatal complications between gestational diabetes mellitus and overt diabetes in pregnancy in Japan. METHODS: A multi-institutional retrospective study compared pregnancy outcomes between gestational diabetes mellitus and overt diabetes in pregnancy. We examined pregnant women who met the former criteria for gestational diabetes mellitus and received dietary intervention with self-monitoring of blood glucose with or without insulin. Overt diabetes in pregnancy was defined as ≥2 abnormal values on 75-g oral glucose tolerance test, fasting glucose ≥126 mg/dl (7.0 mmol/l) and 2-h postprandial glucose ≥200 mg/dl (11.1 mmol/l), or glycated hemoglobin levels ≥6.5% (48 mmol/mol). RESULTS: Data were collected on 1267 women with gestational diabetes and 348 with overt diabetes in pregnancy. Pregestational body mass index was higher (26.2 ± 6.1 vs. 24.9 ± 5.7 kg, P<0.05) and gestational age at delivery was earlier (37.8 ± 2.5 weeks vs. 38.1 ± 2.1 weeks, P<0.05) in overt diabetes than in gestational diabetes. Glycated hemoglobin (6.8 ± 1.1% [51 mmol/mol] vs. 5.8 ± 0.5% [40 mmol/mol], P<0.05) and glucose on 75-g oral glucose tolerance test and prevalence of retinopathy (1.2% vs. 0%, P<0.05) and pregnancy-induced hypertension (10.1% vs. 6.1%, P<0.05) were higher in overt diabetes than in gestational diabetes. Pregnancy-induced hypertension was associated with pregestational body mass index, gestational weight gain, chronic hypertension, and nulliparity but not with 75-g oral glucose tolerance test. CONCLUSIONS: Overt diabetes in pregnancy is significantly associated with maternal complications such as retinopathy and pregnancy-induced hypertension.

Randomised, phase III trial of epoetin-ß to treat chemotherapy-induced anaemia according to the EU regulation.

BACKGROUND: Erythropoietin-stimulating agents (ESAs) effectively decrease the transfusion requirements of patients with chemotherapy-induced anaemia (CIA). Recent studies indicate that ESAs increase mortality and accelerate tumour progression. The studies also identify a 1.6-fold increased risk of venous thromboembolism. The ESA labelling was thus revised in Europe and the United States in 2008. This is the first randomised, phase III trial evaluating the efficacy and safety of epoetin-ß (EPO), an ESA, dosed in accordance with the revised labelling, which specifies that ESAs should be administered to CIA patients with a haemoglobin level of ≤ 10 g dl⁻¹ and that a sustained haemoglobin level of > 12 g dl⁻¹ should be avoided. METHODS: A total of 186 CIA patients (8.0 g dl⁻¹ ≤ haemoglobin ≤ 10.0 g dl⁻¹) with lung or gynaecological cancer were randomised to receive EPO 36,000 IU or placebo weekly for 12 weeks. RESULTS: The proportion of patients receiving transfusions or with haemoglobin < 8.0 g dl⁻¹ between week 5 and the end of the treatment period as the primary end point was significantly lower in the EPO group (n=89) than in the placebo group (n=92; 10.0% vs 56.4%, P < 0.001). The proportion receiving transfusions was significantly lower in the EPO group (4.5% vs 19.6%, P=0.002). Changes in quality of life were not different. No significant differences in adverse events - for example, the incidence of thromboembolic events was 1.1% for each group - or the 1-year overall survival were observed between groups. CONCLUSION: Weekly EPO administered according to the revised labelling approved by the European Medicines Agency is effective and well tolerated for CIA treatment. Further investigations are needed on the effect of ESAs on mortality.

Chemotherapeutic choice of ranimustine or nimustine on the basis of regional polyamine levels in rat brain.

The aim of the present study was to improve the chemotherapeutic efficacy of anticancer drugs by choosing on the basis of the polyamine level induced by the drug in each host cancer-bearing tissue. We propose an "organ-specific therapy" in the article. The polyamines, putrescine, spermidine and spermine are strongly associated with tumor cell growth. The effects of ranimustine (MCNU) and nimustine (ACNU) on body weight, regional brain weights and concentrations of putrescine, spermidine and spermine in the cerebellum, hippocampus, corpus striatum, cortex, combined thalamus and hypothalamus and diencephalon of the brain were examined in rats. MCNU and ACNU reduced spermidine and spermine in the corpus striatum, and spermine in the diencephalon, but increased putrescine in the corpus striatum and combined thalamus and hypothalamus. These results indicate that both MCNU and ACNU are suitable for the treatment of cancers of the corpus striatum, but ACNU is not suitable for cancers of the corpus striatum, thalamus and hypothalamus.

PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer.

Carcinoma is an altered state of tissue differentiation in which epithelial cells no longer respond to cues that keep them in their proper position. A break down in these cues has disastrous consequences not only in cancer but also in embryonic development when cells of various lineages must organize into discrete entities to form a body plan. Paraxial protocadherin (PAPC) is an adhesion protein with six cadherin repeats that organizes the formation and polarity of developing cellular structures in frog, fish and mouse embryos. Here we show that protocadherin-8 (PCDH8), the human ortholog of PAPC, is inactivated through either mutation or epigenetic silencing in a high fraction of breast carcinomas. Loss of PCDH8 expression is associated with loss of heterozygosity, partial promoter methylation, and increased proliferation. Complementation of mutant tumor cell line HCC2218 with wild-type PCDH8 inhibited its growth. Two tumor mutants, E146K and R343H, were defective for inhibition of cell growth and migration. Surprisingly, the E146K mutant transformed the human mammary epithelial cell line MCF10A and sustained the expression of cyclin D1 and MYC without epidermal growth factor. We propose that loss of PCDH8 promotes oncogenesis in epithelial human cancers by disrupting cell-cell communication dedicated to tissue organization and repression of mitogenic signaling.

Vesicouterine ligament contains abundant autonomic nerve ganglion cells: the distribution in histology concerning nerve-sparing radical hysterectomy.

The aim of this study is to describe the histologic architecture of the tissues corresponding to the surgically developed connective tissue bundle commonly referred to as the posterior leaf of the vesico-uterine ligament (VUL), and to examine distribution of ganglion cells. Serial macroscopic slices, each 15-20 mm in thickness, were made from eight specimens (obtained from six female elderly cadavers). In these macroslices, the location of the deep uterine vein was used to identify the deep leaf of the VUL. The specimens were trimmed and semi-serial histologic sections in thickness were prepared at 1 mm intervals. Vesical veins and the associated nerve elements were enclosed by fascia and formed a common pedicle. The base of the pedicle contained the deep uterine vein trunk. The fascia encircling the pedicle varied in thickness and connective intensity between specimens. This vesical neurovascular bundle contained abundant ganglion cells. On average, 48.0% of the ganglion cells along the vesical tributaries of the deep uterine vein were located on the medial or vaginal side of the veins, 19.2% were located between veins, 13.0% on the lateral side of the veins, and 19.8% on the dorsal side. The interindividual variability was greatest on the dorsal side of vesical veins and ranged 11-202 cells. We conclude that in order to achieve maximal preservation of the ganglion cells during the surgical dissection of the posterior leaf of the VUL, care must be taken when the medial or vesical aspect of the ligament is separated. The standard nerve-sparing radical hysterectomy should be modified to reflect differences in the distribution of ganglion cells and in connective intensity between ganglions and veins.

Vaginal tumors with histologic and immunocytochemical feature of gastrointestinal stromal tumor: two cases and review of the literature.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the gastrointestinal tract (GI). Although positive immunohistochemistry for c-kit protein (CD117) and CD34 is critical in establishing diagnosis, the clinicopathologic features of CD117-positive mesenchymal tumors without obvious connection to the GI tract are not well documented. We describe the clinicopathologic features of two cases of extra-GIST. Case 1 was a 42-year-old woman who presented with a submucosal tumor located in the posterior vaginal wall. Case 2 was a 66-year-old woman who presented with a mass that bulged from the right side of the middle vaginal wall. Both tumors were excised locally. The results of microscopic examination and immunohistochemistry for both cases indicated the diagnosis of GIST. Case 2, in addition, showed oncogenic mutation in KIT exon 11. Case 1 is healthy without evidence of recurrence 4 years after surgery. Case 2 started imatinib therapy after resection of a recurrent mass. Gynecologists as well as diagnostic pathologists should be aware of GIST manifesting as a vaginal mass. Recognition of microscopic patterns and characteristic immunohistochemical phenotype, plus genetic study, is mandatory for establishing the correct diagnosis of GIST.

Effect of spontaneous exercise on antioxidant capacity in rat muscles determined by electron spin resonance.

AIMS: The effect of physical activity on antioxidant capacity in muscle remains unknown. This study investigated the effect of spontaneous exercise on antioxidant capacity in rat muscles determined by electron spin resonance (ESR), which is a technique for the direct detection of free radicals. METHODS: Ten-week-old male Wistar rats were housed individually in cages with a running wheel. Rats were classified as high activity (HA), middle activity (MA) or low activity group (LA), based on an assessment of running distance covered over a 23-week period. After 23 weeks of housing, soleus (Sol), plantaris (Pl), gastrocnemius [deep/surface portions (GasD/GasS)] and heart (Hrt) muscles were isolated, and scavenging activity against superoxide anions (O(2)(*-)) and hydroxyl radicals (HO(*)) was determined by ESR using a spin-trap chemical. The citrate synthase (CS) activity was used as a marker of mitochondrial oxidative phosphorylation capacity. RESULTS: Among the parameters measured, only O(2)(*-) scavenging activity in GasD significantly correlated with the running distance. The highest scavenging activity was observed in Hrt of HA rats. The O(2)(*-) scavenging activity in Pl of MA rats was significantly higher than that of LA rats. The O(2)(*-) scavenging activity of Sol and GasS was not significantly different between the three groups. Furthermore, the HO(*) scavenging activity of any muscle specimens was similar among the three groups and did not correlate with running distance at all. CS activity did not significantly differ between the three groups. CONCLUSION: These data suggest that O(2)(*-) scavenging activity in specific types of muscle tissues would increase especially in spontaneously active animals. However, HO(*) scavenging activity would not.

Crescentic glomerulonephritis associated with renal cell carcinoma after cancer immunotherapy.

A 59 year-old woman showed rapidly progressive glomerulonephritis during immunotherapy for metastatic renal cell carcinoma. She received unilateral nephrectomy and cytotoxic T lymphocyte (CTL) therapy for the treatment of retroperitoneal lymph node metastasis of renal cell carcinoma. With CTL therapy, her retroperitoneal lymph node mass decreased in size. One year after the third round of CTL therapy, her serum creatinine was increased and massive proteinuria occurred. Her renal biopsy specimen revealed necrotizing and crescentic glomerulonephritis with immune complex deposition. Her retroperitoneal lymph node mass continued to decrease in size. Consequently, for the purpose of avoiding interfering with the CTL therapy, we performed double filtration plasmapheresis (DFPP) monotherapy for removal of immune complexes without using immunosuppressive drugs or prednisolone. After 24 sessions of DFPP, her serum IgG was reduced from 3,942 mg/dL to 2,400 mg/dL, and proteinuria (from 9.0 g/day to 0.9 g/day) and renal function (serum creatinine; from 5.6 mg/dL to 2.2 mg/dL) also improved. However, 3 months after the final DFPP, she expired due to perforation of the colon. The autopsy sample of the kidney showed that most of the glomeruli were obsolescent, but immunoglobulin depositions were reduced and necrotizing lesions were diminished. In the patients with RPGN associated with renal cell carcinoma, renal functional recovery has not been observed upon immunosuppressive treatment. Consequently, plasmapheresis is considered to be one of the effective and safe methods for patients with this association. We also discuss previous reports of RPGN associated with renal cell carcinoma, or RPGN after cancer immunotherapy.

Nutcracker syndrome associated with severe anemia and mild proteinuria.

A 70-year-old man was referred to our hospital with the chief complaint of gross hematuria. Urinalysis revealed gross hematuria (3+, RBC 100/HPF or more) and mild proteinuria (3+, 1.8 g/day) with no urinary casts. Computed tomography of the abdomen showed compression of the left renal vein between the superior mesenteric artery and the aorta. Ultrasonography showed an increased flow velocity at the stenotic portion of the left renal vein. An aortography and selective left renal arteriography showed that there was no evidence of tumor vessels or arterial abnormalities in the arterial phase. However, the venous phase revealed a stenosis of the left renal vein just lateral to the aorta as well as a reflux of contrast material toward the left gonadal vein which was dilated. In addition, cystoscopy revealed left ureteral bleeding. Based on these findings, we made the diagnosis of gross hematuria caused by nutcracker syndrome (NCS). We concluded that the main cause of the anemia and proteinuria in our patient was leakage of blood and this is confirmed by the relationship of red blood cells to protein in the urine because we proved whole blood and plasma protein loss in the urine by calculation. Fourteen months after discharge, both the gross hematuria and proteinuria spontaneously disappeared. This case strongly suggested that the first therapy for hematuria and proteinuria with NCS should be observation.

Glomerular crescents predominantly express cadherin-catenin complex in pauci-immune-type crescentic glomerulonephritis.

AIMS: To investigate the expression of the cadherin complex in human crescentic glomerulonephritis to elucidate the role of intercellular adherens junction molecules in crescent formation. METHODS AND RESULTS: Immunostaining revealed cadherin complexes localized in Bowman's epithelial cells, but not in podocytes, of normal human glomeruli. Eight adult cases with myeloperoxidase anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA)-related (pauci-immune type) crescentic glomerulonephritis were examined on immunofluorescence microscopy with anti-pan cadherin, p120 catenin, and beta-catenin antibodies. The specimens provided six cellular crescents, 12 fibrocellular crescents, and four fibrotic crescents. Immunofluorescence was semiquantitatively estimated by the rate of the field of localization within the whole area of the crescent, according to the four-grade system [(-) - (++)]. All the tested molecules consisting of the cadherin complex were abundantly observed in cytokeratin-positive epithelial components in crescents, each with an equivalent area of localization. The expression of the cadherin complex was closely associated with that of cytokeratin and both diminished as the crescents developed from cellular to fibrotic. CONCLUSIONS: The cadherin-catenin complex is a specific marker of Bowman's epithelial cells in human glomeruli. The cellular crescents in pauci-immune-type crescentic glomerulonephritis possess adherens junction molecules, indicating a principle parietal epithelial cell phenotype.

The antioxidant EPC-K1 ameliorates brain injury by inhibiting lipid peroxidation in a rat model of transient focal cerebral ischaemia.

BACKGROUND: Cerebral ischaemia-reperfusion injury is associated with the generation of reactive oxygen species during the early phases of reoxygenation. EPC-K1, a phosphate diester of vitamins C and E, has been reported to possess potent hydroxyl radical scavenging activity. This study was performed to investigate the effectiveness of EPC-K1 in attenuating cerebral ischaemia-reperfusion injury in a rat model of transient focal cerebral ischaemia. METHOD: We evaluated the efficacy of EPC-K1 by measuring the concentration of cerebral thiobarbituric acid reactive substances (TBARS), an indicator of the extent of lipid peroxidation by free radicals, and infarct size in rats subjected to one hour of cerebral ischaemia and 4, 24, or 72 hours of reperfusion. FINDINGS: EPC-K1 significantly reduced both the cerebral TBARS level and the infarct size in a rat model of transient focal cerebral ischaemia. These results indicate that EPC-K1 administration during the early stages of reperfusion ameliorates ischaemic brain injury by inhibiting lipid peroxidation. INTERPRETATION: This report is the first to describe the protective mechanism of EPC-K1 by measuring both the TBARS level and infarct size in a rat model of transient focal cerebral ischaemia, and may suggest a potential clinical approach for the treatment of ischaemic cerebrovascular disease.

A nonspectroscopic method to determine the photolytic decomposition pathways of 3-chloro-3-alkyldiazirine: carbene, diazo and rearrangement in excited state.

C(60) acts as a mechanistic probe for the formation of carbene, diazo compound, and for the rearranged product via the excited state in the photolysis of 3-chloro-3-isopropyldiazirine and 3-chloro-3-chloromethyldiazirine. The carbene adds to C(60) to form methanofullerene, whereas the diazo compound adds to C(60) to form fulleroid. The olefin product arises as a result of the rearrangement in the excited state.

Synthesis, structure, electrochemistry, and spectroelectrochemistry of hypervalent phosphorus(V) octaethylporphyrins and theoretical analysis of the nature of the PO bond in P(OEP)(CH(2)CH(3))(O).

A variety of phosphorus(V) octaethylporphyrin derivatives of the type [P(OEP)(X)(Y)](+)Z(-) (OEP: octaethylporphyrin) (X = CH(3), CH(2)CH(3), C(6)H(5), F; Y = CH(3), CH(2)CH(3), OH, OCH(3), OCH(2)CH(3), On-Pr, Oi-Pr, Osec-Bu, NHBu, NEt(2), Cl, F, O(-); Z = ClO(4), PF(6)) were prepared. X-ray crystallographic analysis of eleven compounds reveals that the degree of ruffling of the porphyrin core becomes greater and the average P-N bond distance becomes shorter as the axial ligands become more electronegative. Therefore, the electronic effect of the axial substituents plays a major role in determining the degree of ruffling although the steric effect of the substituents plays some role. A comparison of the (1)H NMR chemical shifts for the series of [P(OEP)(CH(2)CH(3))(Y)](+)Z(-) complexes with those of the corresponding arsenic porphyrins, which possess a planar core, indicates a much smaller ring current effect of the porphyrin core in the severely ruffled phosphorus porphyrins. The electrochemistry, spectroelectrochemistry and ESR spectroscopy of the singly reduced compounds are also discussed. The OH protons of [P(OEP)(X)(OH)](+) are acidic enough to generate P(OEP)(X)(O) by treatment with aq dilute NaOH. X-ray analysis of P(OEP)(CH(2)CH(3))(O) reveals that the PO bond length is very short (1.475(7) A) and is comparable to that in triphenylphosphine oxide (1.483 A). The features of the quite unique hexacoordinate hypervalent compounds are investigated by density functional calculation of a model (Por)P(CH(2)CH(3))(O) and (Por)P(F)(O) (Por: unsubstituted porphyrin).

Mg@C72 MNDO/d evaluation of the isomeric composition.

Temperature development of the relative stabilities of isomers of Mg@C72 (which has not yet been isolated) is computed using the recently introduced MNDO/d method. Four isomers originally considered for the Ca@C72 case are treated: one isolated-pentagon-rule (IPR) structure, two structures with a pair of adjacent pentagons, and one cage with a heptagon. The IPR structure comes as the lowest in MNDO/d potential energy, being rather closely followed by the two structures with a pentagon-pentagon pair. On the other hand, the structure with a heptagon is located too high in potential energy to be of any experimental significance. The entropy contributions are evaluated by the MNDO/d-based partition functions so that the relative concentrations can be treated accordingly. The computations suggest that if Mg@C72 is isolated, it should be a mixture of either two or three isomers. The prediction depends on temperature prehistory. If preparation takes place at temperatures of approximately 1000 K, two isomers should be produced. If temperatures are increased to approximately 2000 K, there will already be three isomers with significant relative concentrations. The study supplies a further interesting example of the profound role of enthalpy-entropy interplay in stabilities of isomeric fullerenic structures.

Antimicrobial susceptibility of Staphylococcus intermedius isolated from healthy and diseased dogs.

A total of 90 strains of Staphylococcus intermedius isolated from dogs were examined for antimicrobial susceptibility. There were no significant differences in the distribution patterns of MICs between strains from 1982 to 1985 and those from 1999, and between strains from healthy dogs and those from diseased dogs. All of the strains were susceptible to ABPC, DMPPC, CEX, TDM, ERFX, BFLX, and FF at concentrations of 0.05 to 6.25 microg/ml. The MICs of OTC, KM, EM, AIV-TS, and LCM were distributed in a broad range of 0.1 to >100 microg/ml, indicating the existence of resistant as well as susceptible populations of S. intermedius. Thirty-three strains (36.7%) were resistant to one or more anitmicrobial agents such as OTC (n=32), KM (n=9), EM (n=7), AIV-TS (n=7), and LCM (n=7).