Coexpression of an unusual form of the EWS-WT1 fusion transcript and interleukin 2/15 receptor betamRNA in a desmoplastic small round cell tumour.

BACKGROUND: The beta chain of the interleukin 2/15 receptor (IL-2/15Rbeta) is induced by the expression of the EWS-WT1. A case of desmoplastic small round cell tumour (DSRCT) expressing only an unusual EWS-WT1 treated by us is reported here. AIM: To characterise an unusual form of EWS-WT1. METHODS: Frozen tissue sections of the axillary tumour were examined using a laser-assisted microdissection technique and reverse transcriptase polymerase chain reaction. RESULTS: The novel fusion of exon 8 of EWS and the defective exon 10 of WT1 (-KTS) was detected. Although it was an unusual form, the coexpression of the present EWS-WT1, IL-2/15Rbeta and Janus kinase (JAK1) mRNA was detected in the tumour cells. IL-2 and signal transducers and activators of transcription (STAT5) mRNA were detected in both tumour and stromal cells. CONCLUSION: The induction of the IL-2/15 receptor signalling pathway may contribute to tumorigenesis in DSRCT through a paracrine or an autocrine system, even though the EWS-WT1 was an unusual form.

Vitamin D and intact PTH status in patients with hip fracture.

INTRODUCTION: The prevalence of hypovitaminosis D in patients with acute hip fracture was examined in a population on Sado Island in Japan. There were 85 cases of hip fracture among this population in 2004, giving an overall incidence of hip fracture of 121.4 per 100,000 population per year. This study included 50 of the 85 cases, and these cases were defined as the hip fracture group. Patients older than 70 years without established osteoporosis who were admitted to the hospital on the island during almost the same period for treatment of an orthopedic condition other than a hip fracture were defined as the control group. MATERIALS AND METHODS: The levels of serum 25-hydroxyvitamin D (25-OHD), intact parathyroid hormone (intact PTH), alkaline phosphatase (ALP), albumin, and the number of remaining teeth were examined in each group. In the hip fracture group, serum calcium, serum phosphorus, urine N-terminal cross-linking telopeptide of type I collagen (NTx), bone mineral density (BMD) of the nonfractured hip, the presence of a vertebral fracture on X-ray, severity of dementia, and physical activity level were also examined. RESULTS: Both the serum 25-OHD and serum albumin levels were significantly lower in patients with hip fracture than in controls, and the intact PTH level was significantly higher in patients with hip fracture. The number of remaining teeth was correlated with age, and was also significantly correlated with 25-OHD. In the hip fracture group, 62% of the subjects had hypovitaminosis D (25-OHD <20 ng/ml) and one-fifth of cases with hypovitaminosis D showed elevated PTH levels (>65 pg/ml). On the other hand, in the control group, hypovitaminosis D occurred in 18.9% of the subjects, and only one case showed elevated PTH. The serum 25-OHD level showed a decrease as the severity of dementia progressed and the activity level decreased. CONCLUSION: Our results indicate that about two-thirds (62%) of hip fracture patients had vitamin D insufficiency, suggesting that this condition may be closely associated with hip fracture in elderly people. Therefore, the serum 25-OHD level may be a useful index for the risk of hip fracture in elderly people.

Overexpression of anti-apoptotic Mcl-1 in testicular germ cell tumours.

AIMS: To determine the expression of Mcl-1 in testicular germ cell tumours in order to clarify the role of this anti-apoptotic factor in these tumours. Various members of the Bcl-2 family have been implicated in the apoptotic mechanisms regulating germ cell apoptosis. Mcl-1 is an anti-apoptotic Bcl-2 family member and has recently been reported to be related to the progression of malignancy; however, the involvement of Mcl-1 in the development of germ cell tumours is still unknown. METHODS AND RESULTS: Mcl-1 expression in testicular germ cell tumours was investigated by immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR). By immunohistochemistry, overexpression of Mcl-1 was present in all germ cell tumours that were studied, including embryonal carcinoma and yolk sac tumour, as well as choriocarcinoma and teratoma. In teratomas, Mcl-1 was widely distributed in the epithelial, myogenic, neural and mesenchymal components. RT-PCR analysis after microdissection revealed high levels of Mcl-1 mRNA in all tumour variants compared with non-neoplastic germ cells. CONCLUSION: Overexpression of anti-apoptotic Mcl-1 may function to enhance the viability of testicular germ cells, thereby leading to tumorigenesis.

A monoclonal antibody against insect CALNUC recognizes the prooncoprotein EWS specifically in mammalian cells. Immunohistochemical and biochemical studies of the antigen in rat tissues.

A monoclonal antibody against insect CALNUC was shown to recognize an 85-kDa nuclear protein specifically in mammalian cells. Amino acid sequencing of the protein purified from rat liver revealed it to be EWS, a prooncoprotein for Ewing sarcomas and related tumors. Using the antibody, distribution of EWS was studied in rat tissues fixed with 4% paraformaldehyde by immunohistochemical methods. On thaw-fixed cryosections or those of perfusion-fixed tissues, almost all cell nuclei showed the specific staining. In immersion-fixed tissues, the staining unexpectedly disappeared in particular tissues (kidney cortex, liver, etc.), although it was recovered by autoclaving the cryosections. Western blotting also demonstrated the ubiquitous expression of EWS in the tissues. In extracts from the liver, the 85-kDa band rapidly disappeared in a Ca(2+)-dependent manner, but never in the testis. The antigen was very labile in kidney homogenates even without Ca2+. Biochemical studies with digoxigenin-labeled EWS showed that the Ca(2+)-dependent disappearance was associated with upward mobility shifts of EWS. These suggested that EWS was ubiquitously expressed in rat tissues, and that the antigen was masked in particular tissues during the immersion fixation.

A simple contrast enhancement by potassium permanganate oxidation for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution.

A simple contrast enhancement method is presented for Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution. The sections were treated with an acidified potassium permanganate oxidizing solution followed by uranyl acetate and lead citrate staining. The method, designated KMnO4-UA/Pb staining, provided a much greater contrast in electron microscopy than conventional UA/Pb staining. In detail, the visibility of plasma membrane was especially improved and the nuclear heterochromatin, mitochondria and cytoplasmic ribosomes showed an adequate increase in electron density. In the mucous cells of rat Brunner's glands, the Golgi cisternae were well defined with the KMnO4-UA/Pb staining. Interestingly, the membranes of the intermediate compartments were moderately reactive to the KMnO4-UA/Pb staining, whereas the cis and the trans compartments were only faintly stained. It should be emphasized that the KMnO4 oxidation following colloidal gold labelling did not cause a remarkable reduction of immunogold labelling and the enhanced contrast helped us to examine the gold particles with high accuracy. This contrast enhancement method is highly promising, with the potential to become a useful tool for histochemical investigation, including immunocytochemistry with the Lowicryl K4M ultrathin sections prepared by high pressure freezing/freeze substitution techniques.

Expression of peroxisome proliferator-activated receptor subtypes in human atherosclerosis.

To clarify the involvement of peroxisome proliferator-activated receptors (PPARs) in atherosclerotic plaque formation, we investigated the expression patterns of mRNA and protein of PPARalpha and PPARgamma in human aorta. Atheromatous plaque, fatty streak, and diffuse intimal thickening (DIT) were separated macroscopically, and each sample was divided into halves. Half of them were used for analysis of mRNA expression with reverse transcription-polymerase chain reaction and the others were used for histologic analysis. Both PPARalpha and PPARgamma mRNA were detected in all atheromatous plaques, all fatty streaks, and in some DIT. However, expressions of PPARalpha and PPARgamma were obviously less frequently found in DIT than in atheromatous plaques, and the intensity of these expressions was stronger in the atheromatous plaques than in the DIT. Compared with PPARalpha, PPARgamma mRNA was expressed more frequently in atheromatous plaques. In atheromatous plaques, PPARgamma mRNA was expressed independently, whereas PPARalpha mRNA was coexpressed with PPARgamma. PPARgamma protein was obviously found in the nuclei of endothelial cells, macrophages, mononuclear cells, and smooth muscle cells in the aortic intima. These results suggest that expressions of PPARalpha and PPARgamma in human aortic wall are involved in atherogenesis from the early stages.

CALNUC (nucleobindin) is localized in the Golgi apparatus in insect cells.

A mouse monoclonal antibody 12B1 was raised against Golgi fractions from Sf21 insect cells and selected as Golgi-specific by immunostaining of the cells. The antigen was purified from the cells by immunoaffinity chromatography with the monoclonal antibody, and its N-terminal and internal amino acid sequences were determined. Based on the partial amino acid sequences, cDNA encoding the antigen protein was cloned and sequenced. The amino acid sequence deduced from the cDNA nucleotide sequence showed a homology to those of CALNUC family proteins, CALNUC (or nucleobindin, a calcium-binding Golgi protein with DNA-binding activity) and protein NEFA (a cell surface protein with DNA-binding, EF-hand, and acidic domains). The insect protein had two EF-hand loops at the same sites as the mammalian CALNUC family proteins, but had no leucine zipper which the mammalian homologues commonly have. An electron microscopic immunoperoxidase study demonstrated that the insect protein was localized in the cis-Golgi cisternae and cis-Golgi networks. Since this localization is identical to that of mammalian CALNUC, the insect protein was considered to be a homologue of CALNUC rather than that of NEFA. Assays involving proteinase K digestion, sodium carbonate extraction and Triton X-114 extraction revealed that the insect CALNUC-like protein was a soluble protein tightly associated with the luminal surface of Golgi membranes as reported for mammalian CALNUC. The insect protein was also shown to have calcium-binding activity as does mammalian CALNUC. These data verify that the insect protein is CALNUC. The existence of CALNUC in insect cells suggests that CALNUC is an essential calcium-binding Golgi protein in a wide range of the animal kingdom. A phylogenetic tree analysis, however, suggested that NEFA was derived from CALNUC long after the segregation of a mammalian ancestor from an insect ancestor.

A comparative study of cultured smooth muscle cell proliferation and injury, utilizing glycated low density lipoproteins with slight oxidation, auto-oxidation, or extensive oxidation.

We investigated the influence of glycated low density lipoprotein (LDL) for vascular smooth muscle cell (SMC) proliferation or injury. We utilized glycated, slightly oxidized LDL (GLDL-LOX), glycated, auto-oxidized LDL (GLDL) and glycated, metal-induced extensively oxidized LDL (GLDL-OX) to examine the effect of glycation itself or combined glycation and oxidation on SMC. GLDL-LOX induced SMC proliferation and migration, and increased the number of platelet-derived growth factor receptor, beta subunits, (PDGF-R) positive SMC. Also, GLDL-LOX promoted protease activity, compared with the other groups including native LDL (control). GLDL and GLDL-OX demonstrated SMC injury with apoptosis and Bax protein expression, compared with native LDL and GLDL-LOX. These results suggested that LDL glycation contributed to the progression of atherosclerosis by promoting SMC migration and proliferation, with little dependence on oxidative modification. Secondary auto-oxidation adding to glycation induced SMC apoptosis, and SMC injury occurred in the state of strong oxidation with glycation. We concluded that LDL glycation might play a key role in the progression of atherosclerosis in diabetes, and glycated LDL promoted atherosclerosis, even with little assistance from oxidation.

[A pathomorphological study on atherosclerotic regression with references between apoptosis and atherosclerotic plaque with central depression in rabbit aorta].

Atherosclerotic plaque with central depression (depressed lesions) have been proposed as one morphological feature of atherosclerotic regression in the elderly. We have also revealed that depressed lesions have been found not only in the aorta of elderly human but also in rabbit aorta. In this study a relationship between apoptosis and atherosclerotic regression was immunohistochemically studied to clarify the possibility of the pathogenesis of depressed lesion in rabbit aorta. Twenty male New Zealand White rabbit (NZW) were fed with a 1.0% cholesterol diet during a 12-week atherogenic induction period. Then they were fed only with a basic diet for 36-week (n = 6) and 48-week (n = 6, experimental group) regression periods. A control group was fed with 1.0% cholesterol diet for 12-week (n = 2) and 60-week (n = 6). They were killed and aortas were fixed with formalin. Sections obtained from aortas were processed for histological and immunohistochemical studies including the TUNEL method, staining with Ki-67 and others. Several depressed lesions were found in the aortas of NZW animal, but none were noted in control aortas. Results of surface involvement in the experimental groups were significantly lower than in the control, and the aortas of the experimental group had atherosclerotic regression. Apoptosis was found in the depressed lesions, which had more apoptotic cells than non-regressed atherosclerotic plaques. Furthermore, the apoptotic cells were significantly greater in the center of depressed lesions than in marginal areas. Ki-67 staining was positive in the atherosclerotic plaque, but negative in the depressed lesions. It appears that the NZW aortic atherosclerotic plaques may transform to depressed lesions with apoptosis. Atherosclerotic regression has been associated with apoptosis.

Multistratified expression of polysialic acid and its relationship to VAChT-containing neurons in the inner plexiform layer of adult rat retina.

We investigated the localization of polysialic acid (PSA), neural cell adhesion molecule (NCAM), and vesicular acetylcholine transporter (VAChT) in adult rat retina by using immunofluorescence with a confocal laser scanning microscope. Western blot analysis showed a typical broad smear of PSA and isoforms of NCAM (120, 140, and 180 kD). PSA immunofluorescence revealed multistratification in the inner plexiform layer (IPL). Dual immunostaining for PSA and NCAM exhibited the selective co-expression of PSA and NCAM on Müller cells. Moreover, dual immunolabeling for PSA and VAChT completely separated the five strata in the IPL. Strata 1, 3, and 5 were immunoreactive for PSA and Strata 2 and 4 for VAChT. These results suggest the possibility that PSA molecules on Müller cells are spatially related to ON and OFF retinal channels in the IPL.

Polaprezinc protects gastric mucosal cells from noxious agents through antioxidant properties in vitro.

BACKGROUND: Polaprezinc has been shown to exert an anti-oxidant property in a tube experiment, protect gastric mucosa from experimental ulcerations in vivo, and accelerate the healing of gastric ulcer in humans. AIM: To examine a possible protective effect of polaprezinc on oxidant-mediated injury in primary monolayer cultures of rat gastric fundic mucosa. METHODS: Cytotoxicity was quantified by measuring 51Cr release. Whether or not polaprezinc exerts an antioxidant property was investigated by determining the effect of this agent on hydrogen peroxide (H2O2)-induced injury. The effects of polaprezinc on superoxide (O2-. ) generation as well as on ethanol (EtOH)-induced injury were also examined. Generation of O2-. was assessed by the reduction in cytochrome c. RESULTS: H2O2 caused a time- and dose-dependent increase in 51Cr release. The dose-response curve of 51Cr release by H2O2 shifted to the right in the presence of polaprezinc. Polaprezinc, at submillimolar concentrations, prevented H2O2-induced 51Cr release. EtOH also caused a dose-dependent increase in 51Cr release, which was prevented by the addition of polaprezinc. The incubation of cells with EtOH caused an increase in cytochrome c reduction, as the concentrations of EtOH increased. Polaprezinc inhibited EtOH-induced cytochrome c reduction. Protection by polaprezinc was microscopically associated with the prevention of monolayer disruption. CONCLUSIONS: Polaprezinc is antioxidative and directly protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro. This finding may provide the theoretical basis for the usage of an antiulcer drug with antioxidant properties for the treatment of gastric inflammation, such as that induced by ethanol.

Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of rat gastric chief cells.

The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Lowicryl K4M embedding allows us histochemical studies of dynamic cellular processes with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To address this problem, we examined the effects of KMnO4 oxidation applied to Lowicryl K4M embedded rat gastric glands processed by high pressure freezing. The KMnO4 oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential staining of the zymogen granules of rat gastric chief cells. This technique could be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO4 oxidation-toluidine blue staining provided sufficient contrast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO4 oxidation plays an essential role in the differential staining of the zymogen granules. Combined use of the KMnO4 oxidation with phospholipase A2-immunostaining demonstrated that gold labeling was localized to the zymogen granules without the loss of immunolabeling. Energy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO4 oxidation will become a useful tool for histochemical investigations combined with cryofixation/freeze substitution and low temperature embedding techniques.

[Regression of atherosclerosis and centrally depressed lesions in rabbit aortas].

Atherosclerotic plaque with central depression (depressed lesion) has been hypothesized to be a morphological feature of atherosclerosis regression. We tested this hypothesis in New Zealand white rabbits. After the animals were fed a diet containing 1% cholesterol for three months, they were changed to a normal diet for 6 to 9 months. Several aortas had centrally depressed lesions similar to those found in elderly people, and the animals had low serum cholesterol levels. Immunohistochemical study showed that the depressed lesions contained more smooth muscle cells and collagen type IV, and fewer macrophage-derived foam cells than did common atherosclerotic elevated lesions found in rabbits. We know of no other report of depressed lesions in rabbits with atherosclerosis. Thus we believe that the centrally depressed lesion is a histological characteristic of regression of atherosclerosis.

Effects of copper-zinc type superoxide dismutase on the proliferation and migration of cultured vascular smooth muscle cells induced by oxidized low density lipoprotein.

We have investigated the effects of copper-zinc type superoxide dismutase (Cu, Zn-SOD) on the function of oxidized low density lipoprotein, utilizing cultured smooth muscle cells (SMC), obtained from rabbit aorta. We added native LDL (nLDL), minimally oxidized LDL (MmLDL) and copper ion-induced oxidized LDL (OxLDL) to the culture media. No remarkable change was found out by adding nLDL. The numbers of SMC, including migrated SMC, were increased by the addition of MmLDL. Cu, Zn-SOD significantly inhibited the reactions induced by MmLDL. The SMC numbers were markedly decreased by OxLDL addition without recovery by adding Cu, Zn-SOD. Thus, MmLDL significantly promoted the SMC proliferation and migration. OxLDL revealed strong cytotoxicity against SMC. Cu, Zn-SOD inhibited both the migration and the proliferation of SMC induced by MmLDL, and did not alter the effect of OxLDL. In conclusion, Cu, Zn-SOD inhibited some functions of MmLDL, and may play an important role in protecting against the atherosclerotic processes evoked by MmLDL.

[Atherosclerosis regression in the elderly--correlation between centrally depressed lesions and risk factors].

Atherosclerotic plaque with central depression (depressed lesion) may indicate regression of atherosclerosis in the aorta. Aortic depressed lesions have a solitary elevated area of plaque with a sharply-bordered roung depression in its center and no area ulceration. This may be interpretable as a sign of regression of atherosclerosis. To clarify the pathogenesis of depressed lesion, we studied clinical risk factors such as hypercholesterolemia in patients with depressed lesions that were confirmed at autopsy. The patients were divided into 3 groups according to their total cholesterol level at autopsy: a high-risk group (> or = 220 mg/dl), a moderate-risk group (180-220 mg/dl), and a low-risk group (< or = 180 mg/dl). Depressed lesions were found in 16.4% of those in the high-risk group, in 14.6% of those in the moderate-risk group and in 69.0% of those in the low-risk group. Severe aortic atherosclerosis was found in 69.8% of the patients; 50.9% of those with severe disease were in the-low risk group. Depressed lesions were also found in those with low levels of low-density lipoprotein cholesterol (< or = 140 mg/dl), 58.8% of whom were found to have severe atherosclerosis. There was no relationship between total cholesterol level and the presence of depressed lesions. However, a clinical prevention trial may result in sufficient control of ahterosclerosis among those in the high-risk group and may also lead to regression of aortic lesions.

Correlation of two argyrophilic nucleolar organizer region counting methods with the Ki-67 labeling index in uterine smooth muscle cell tumors.

The argyrophilic nucleolar organizer regions (AgNOR) are silver stained granules that are thought to correlate with cell proliferation activity. Two AgNOR counting methods: the mean AgNOR count (mAgNOR, the mean number of AgNOR granules in 100 cells) and the AgNOR proliferative index (pAgNOR, the percentage of cells exhibiting five or more AgNOR granules per nuclei) have been proposed. In this study, the two counting methods were applied to 58 cases of normal uterine corpus and uterine corpus tumors and were compared with the Ki-67 labeling index (Ki-67 LI) using MIB-1 monoclonal antibody and other histopathological criteria. Notable differences in the number of AgNOR and the Ki-67 LI were observed between benign and malignant smooth muscle tissue. Histopathologic features are well correlated to the proliferative activity of tumors. Although the most reliable method of predicting malignant potential cannot be determined, the methods outlined by this study are thought to be highly useful in assessing proliferative activities.

[Study of atherosclerosis regression in the elderly--central depression and its correlation to ulcerated plaques].

Atherosclerotic plaque with central depression (depressed lesion) was firstly proposed in our previous report as one of the morphological features of regressed lesions, which was characterized by the presence of isolated, well defined lesions with a centrally depressed area and smooth surface. They were obviously different from atherosclerotic plaques with ulceration (ulcerated plaques) in elderly autopsy cases. In this study, 30 ulcerated plaques obtained from specimens of the elderly aortas were histologically and immunohistochemically investigated to clarify the morphogenesis of the depressed lesion and its correlation to the ulcerated plaque. These depressed lesions were divided into 4 groups according to their derivation; (a) fused lesion of multiple fibrous plaques, (b) regressing lesion of plaques, (c) healed ulcerated plaques, and (d) mixed type of these lesions. Regeneration of endothelial cell was noted in the peripheral zone of ulcerated plaques, and collagen type IV was also increased in the stroma of these ulcerated plaques. These were healed ulcerated plaques. The ulcerated plaques with complete restoration of endothelial cells on the ulcerated surface may become atherosclerotic plaques with central depression. These lesions are one of the histological features of regression in advanced atherosclerosis.