Crucial role of plasmacytoid dendritic cells in the development of acute colitis through the regulation of intestinal inflammation.

Disruption of intestinal homeostasis can lead to inflammatory bowel diseases endowed susceptibility genes and environmental factors affecting intestinal accumulation and activation of colitogenic phagocytes. Plasmacytoid dendritic cells (pDCs) are immune cells that had been proposed to control innate and adaptive immunity through the massive secretion of type I interferon (IFN-I). However, the contribution of pDCs to the progression of intestinal inflammation remains unclear. Here we show a critical role of pDCs in the initiation of acute colonic inflammation using T-cell-independent acute colitis model with a selective ablation of pDCs. Although pDCs accumulated in the inflamed colon upon mucosal injury, deficiency of pDCs attenuated the development of acute colitis independent of IFN-I signaling, accompanied by the diminished colonic production of proinflammatory cytokines. Furthermore, deficiency of pDCs impaired the mobilization of colitogenic phagocytes into the inflamed colon possibly mediated by the abrogated mucosal production of C-C chemokine receptor 2 ligand. Thus, our findings highlight a critical role of pDCs in the induction of the colonic inflammation that regulates the colonic accumulation of inflammatory phagocytes leading to the initiation and exacerbation of acute colitis, and they may serve a key role in controlling gut mucosal immune homeostasis.

Mouse monoclonal antibody (FKH1) detecting human melanoma-associated antigens.

A mouse monoclonal antibody, FKH1, was produced to detect cytoplasmic melanoma-associated antigen. FKH1 was raised using cultured human melanoma cell line KHm-6 as an immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against cell lines and normal and neoplastic tissues. Positive reactions were seen against 5 human melanoma cell lines. It stained cytoplasm of melanoma cells in a diffuse and granular pattern in indirect immunofluorescence. Immunoelectron microscopy showed diffuse distribution of immunoreactant in the cytoplasm of KHm-1 cells excluding melanosomes and other organelles. Reactivity against frozen and alcohol-fixed, paraffin-embedded melanocytic tumors was also tested with IIF or indirect or avidin biotinylated horseradish peroxidase complex immunoperoxidase techniques. All cases of frozen sections from benign and malignant melanocytic tumors showed positive staining with FKH1. In fixed tissues, however, reactivity was 11 of 14 (79%) in malignant melanoma and 28 of 42 (67%) in other melanocytic tumors. FKH1 did not react against normal melanocytes and nonmelanocytic tumors except APUDoma and 2 glioblastoma cell lines. It failed to stain the B-16 mouse melanoma cell line, neuroblastoma cell line, breast carcinoma cell line, and T-cell lymphoma cell line. Normal human peripheral nerves were nonreactive with FKH1. In immunoelectroblot study, FKH1 bound with proteins having molecular weight of 71,000 and 55,000 extracted from KHm-6 cells. It was suggested that FKH1 is a useful monoclonal antibody in diagnostic study of human malignant melanoma specimens.

Effect of medroxyprogesterone acetate on endothelium-dependent vasodilation in postmenopausal women receiving estrogen.

BACKGROUND: Estrogen increases endothelium-dependent vasodilation in postmenopausal women. However, use of progestins in combination with estrogen may counter beneficial effects of estrogen on endothelium. We investigated the effect of medroxyprogesterone acetate (MPA) on estrogen-induced increase in endothelium-dependent vasodilation in postmenopausal women. METHODS AND RESULTS: Postmenopausal women were treated daily with conjugated equine estrogen (CEE) 0.625 mg (n=14), CEE 0.625 mg and MPA 2.5 mg (n=15) or CEE 0.625 mg and MPA 5.0 mg (n=16) for 3 months. Plasma lipids and hormones were measured before and after treatment. Vasodilatory responses of the brachial artery were evaluated by measuring flow-mediated vasodilation (FMD) and nitroglycerin-induced vasodilation by use of high-resolution ultrasonography. Susceptibility of LDL to oxidation was analyzed by incubation with CuSO(4) while kinetics of conjugated diene formation was monitored. Plasma total and LDL cholesterol concentrations were decreased significantly in all groups. CEE increased FMD significantly, from 4.5+/-1.7% to 8.5+/-2.8% (P<0.001). Addition of MPA reversed this effect in a concentration-dependent manner (for MPA 2.5 mg, from 5.0+/-3.2% to 6.2+/-3.1%; for MPA 5.0 mg, from 4.9+/-3.4% to 3.6+/-3.7%; P=NS for each). No treatment significantly altered nitroglycerin-induced dilation. Lag time for conjugated diene formation was prolonged significantly in all groups, and the oxidation rate was significantly reduced. CONCLUSIONS: Concurrent MPA administration may offset favorable effects of estrogen on endothelial function in postmenopausal women. Because MPA did not diminish LDL-lowering and antioxidant effects of estrogen, MPA-induced inhibition of endothelium-dependent vasodilation may be independent of changes in oxidative susceptibility and plasma concentration of LDL.

Estrogen-induced small low density lipoprotein particles may be atherogenic in postmenopausal women.

OBJECTIVES: The purpose of this study was to investigate the susceptibility of estrogen-induced small low density lipoprotein (LDL) particles to oxidation. BACKGROUND: Estrogen replacement therapy in postmenopausal women has an antioxidant effect that opposes oxidation of LDL particles. Estrogen-induced increases in plasma triglyceride concentrations, however, decrease LDL particle size, which may act counter to this antioxdant effect. It has not been evaluated whether estrogen-induced small LDL particles are atherogenic. METHODS: In 24 lean and healthy postmenopausal women treated with conjugated equine estrogen (0.625 mg daily) for three months, plasma lipid concentrations and diameter of LDL particles were measured before and after therapy. Susceptibility of LDL to oxidation was determined by measuring the concentration of thiobarbituric acid-reactive substances (TBARS) after incubation with CuSO4. RESULTS: Estrogen significantly decreased plasma concentrations of total cholesterol, LDL-cholesterol and apolipoprotein B, while increasing concentrations of triglyceride, high-density lipoprotein cholesterol and apolipoprotein A-I. Estrogen-induced changes in LDL particle diameter correlated negatively with changes in plasma triglyceride concentrations (r = -0.55, p < 0.005) and with changes in concentrations of LDL-derived TBARS (r = -0.49, p < 0.005). In subjects with substantial estrogen-induced plasma triglyceride increases, estrogen significantly reduced the diameter of LDL particles (p < 0.05) and significantly increased the concentration of LDL-derived TBARS (p < 0.05). In contrast, estrogen significantly reduced the concentration of LDL-derived TBARS (p < 0.05) and caused no significant change in LDL particle diameter in subjects whose plasma triglyceride concentration was unchanged with estrogen therapy. CONCLUSIONS: Because estrogen-induced plasma triglyceride increases may produce small LDL particles that are more susceptible to oxidation, antioxidant effects of estrogen might be offset in patients showing such a triglyceride increase.

A novel synthetic vitamin A-like compound (a polyprenoic acid derivative, E-5166) inhibits the release of arachidonic acid stimulated by epidermal growth factor.

Little is known about the mechanisms of anti-inflammatory activity of retinoids. A new synthetic vitamin A-like compound (polyprenoic acid derivative, E-5166) has a strong in vitro binding affinity to intracellular binding proteins for acidic retinoids. In order to elucidate the anti-inflammatory activity of E-5166, we studied the effect of E-5166 on the epidermal growth factor (EGF)-stimulated arachidonic acid (AA) release of pig epidermis. E-5166 significantly inhibited the EGF-stimulated AA release and this inhibitory effect of E-5166 required a longer incubation than hydrocortisone did. Furthermore, E-5166 inhibited the EGF-stimulated phosphatidylinositol (PI) turnover of pig epidermis. These results indicate that E-5166 inhibited the EGF-stimulated AA release through the inhibition of the EGF-stimulated PI turnover.

Beta-adrenergic stimulation induces intracellular Ca++ increase in human epidermal keratinocytes.

Intracellular Ca++ ([Ca++]i) is one of the most important second messengers of extracellular signals that induce cellular responses. In epidermal keratinocytes, both extracellular and intracellular Ca++ are reported to be important to cell differentiation and proliferation. Several mechanisms that increase [Ca++]i have been elicited in various tissues; however, in epidermal keratinocytes they remain unknown. Thus, we investigated the [Ca++]i modulation in cultured human epidermal keratinocytes and the stimulation that increases the concentration. The [Ca++]i concentration of keratinocytes was increased immediately and transiently by epinephrine. Methoxamine hydrochloride and clonidine (alpha-1- and 2-adrenergic agonists) did not induce an increase in [Ca++]i. The beta-antagonist, propranolol, inhibited the [Ca++]i increase induced by epinephrine and salbutamol (a beta-2-agonist). These results reveal that the beta-adrenergic stimulation induces an immediate and transient [Ca++]i increase in human keratinocytes. Beta-adrenergic stimulation is known to induce adenylate cyclase activation, which results in cyclic AMP accumulation through stimulatory guanosine 5-triphosphate (GTP) binding proteins in the keratinocytes. Also, epinephrine is reported to inhibit cultured epidermal cell proliferation. The effect of epinephrine has been demonstrated by cyclic AMP accumulation; however, beta-adrenergic stimulation revealed a [Ca++]i increase in keratinocytes in our study. One of epinephrine's regulatory effects on epidermal cell proliferation is assumed to occur through the [Ca++]i increase as well.

Monoclonal antibody (AFH1) immunoreactive on morphologically abnormal basal melanocytes within dysplastic nevi, nevocellular nevus nests, and melanoma.

The mouse monoclonal antibody AFH1 was produced using formalin-fixed, sham paraffin-embedded human melanoma cell culture line A375 as immunogen. Reactivity of this antibody was assessed by immunohistochemical techniques against formalin- or acid alcohol-fixed paraffin-embedded tissue as well as formalin- or acid alcohol-fixed unembedded lesions. Ninety-seven nevomelanocytic lesions, neurofibromas, epithelial lesions, and a plasmacellular infiltrate were evaluated. AFH1 was immunoreactive on 54 of 55 nevocytic lesions (98.2%), 15 of 16 primary melanomas (93.7%), a lentigo maligna, and nests in 21 of 21 dysplastic nevi (100%). Of 100 consecutive basal melanocytes of intraepidermal melanoma cells counted in each lesion, mean AFH1 immunoreactivity for nonnested basal melanocytes in nevocellular nevi was 3.8%; for dysplastic nevi, 13.8%; and for intraepidermal melanoma cells, 78.0%. When nonnested basal melanocytes were subdivided into cytologically normal and abnormal cell groups, AFH1 immunoreactivity was 9.4% and 72.6%, respectively. AFH1 recognition of the lentiginous portion of dysplastic nevi corresponds statistically to the appearance of abnormal melanocyte cytology, nest formation, or both. Using 50% immunoreactive nonnested melanocytes as the criterion, AFH1 seems to distinguish primary melanoma from dysplastic nevi with a sensitivity of 93.8% and a specificity of 95.8%.

Epidermal growth factor stimulates phosphorylation of pig epidermal keratin protein.

Endogenous protein phosphorylation of pig epidermis by epidermal growth factor (EGF) was studied to elucidate biologic roles of EGF on epidermal cells. EGF stimulated phosphorylation of keratin proteins (Mr: 65,000, 60,000, 56,000, and 51,000) identified by the Ouchterlony immunodiffusion analysis, a low Mr protein (16,000 dalton) of the urea-SDS-mercaptoethanol soluble fraction, and a 30,000 dalton Tris-HCl soluble protein. The phosphorylated epidermal proteins such as keratin proteins and a 30,000 dalton protein of the Tris-HCl soluble fraction were slightly dephosphorylated following the addition of unlabeled phosphate. Anti-EGF serum eliminated the EGF-stimulated phosphorylation of keratin proteins, a low Mr protein, and a 30,000 dalton Tris-HCl soluble protein. The overall results indicate that EGF specifically stimulated phosphorylation of several epidermal proteins, one of which was keratin protein.

Ad4BP in the human adrenal cortex and its disorders.

Ad4BP, a zinc finger DNA-binding protein, is a transcription factor that regulates the expression of the steroidogenic P450 genes. We performed immunoblotting and immunohistochemistry of Ad4BP in 34 human adrenal cortex specimens, which included adrenocortical adenomas and carcinomas. Immunoblotting revealed a single band of 53K, corresponding to the mol wt of Ad4BP. The immunohistochemical studies demonstrated that Ad4BP immunoreactivity was present exclusively in the nuclei of nearly all of the adrenocortical parenchymal cells in both the normal and the pathological human adrenal specimens. Ad4BP was immunostained with equal intensity and frequency among the different cell types. Ad4BP immunoreactivity was also observed in areas of marked degenerative changes, such as lipomyelomatous lesions, and in poorly differentiated carcinoma cells. These results suggest a close association of Ad4BP expression with the biological phenotype of adrenocortical parenchymal cells. Ad4BP therefore seems to play important roles in the induction and maintenance of the transcription of all steroidogenic P450 genes in human adrenocortical cells, even after malignant transformation.

Changes of serum gonadotropin levels and sex differences in premature and mature infant during neonatal life.

We measured FSH and LH concentrations by RIA in 130 cord sera and 213 peripheral sera obtained as serially as possible from 67 infants who were 5-75 days old and were born between the 28th and 42nd gestational weeks. Cord serum FSH and LH (+hCG) levels were 3.9-13.6 mIU/ml and 43.3-88.6 mIU/ml, respectively; they decreased with advancing gestational age. Postnatal FSH levels in male infants maintained low levels (3.7-8.7 mIU/ml). However, those in female infants increased with peak levels (51.8-270.3 mIU/ml) between 11 and 30 days after delivery, and then decreased; the surge was more marked and prolonged in preterm infants than in term infants. Postnatal LH levels in both sexes decreased rapidly after birth, which may be due to a decrease of placental hCG, and thereafter displayed patterns similar to FSH levels. We found a significant sex difference of serum gonadotropin levels in newborn infants and differences between term and preterm infants. Our results suggest that the sex difference of pituitary gonadal function exists and that the function matures during the fetal and neonatal life.

Messenger ribonucleic acid in situ hybridization analysis of estrogen receptors alpha and beta in human breast carcinoma.

We examined the expression of a recently characterized novel estrogen receptor (ER) beta in 25 cases of invasive ductal carcinoma of the breast, using messenger RNA (mRNA) in situ hybridization, and compared the findings with those of ERalpha, to study its localization and its possible biological significance in human breast cancer. ERalpha and ERbeta hybridization signals were both detected, predominantly in carcinoma cells and in some stromal cells, in 18 of 25 (72%) and 11 of 25 (44%) cases, respectively. The cases in which more than 25% of carcinoma cells demonstrated mRNA hybridization signals were 13 of 25 (52%) and 2 of 25 (8%) cases for ERalpha and ERbeta, respectively. Among the cases expressing ERbeta, 10 of 11 (91%) also expressed ERalpha mRNA; and in these 10 cases, coexpressing both ERalpha and beta, the number of carcinoma cells expressing ERalpha was greater than that expressing ERbeta in 9 cases. Eight cases demonstrated only ERalpha mRNA hybridization signals in carcinoma cells. These results indicate that ERbeta is coexpressed with ERalpha in most ERbeta-positive breast carcinoma cells, which suggests that the expression of ERbeta depends on the presence of ERalpha in the great majority of human breast cancer. In addition, the number of carcinoma cases and/or the ratio of carcinoma cells expressing ERalpha was much greater than those expressing ERbeta. The relative ratio of ERalpha and ERbeta expression in carcinoma cells may be related to various estrogen-dependent biological features of human breast cancer.

Cell turnover in normal cycling human ovary.

We investigated cell proliferation and apoptosis in 37 normal cycling human ovaries to determine cell turnover in the various stages of follicular and luteal development. We examined cell proliferation by immunostaining for both Ki67 and proliferative cell nuclear antigen (PCNA) and by silver staining of nucleolar organizer regions (AgNORs). Apoptosis was examined by 3'-hydroxy nick-end labeling and by immunostaining of an apoptosis-related antigen, Ley. The labeling indexes of Ki67, PCNA, and AgNORs were significantly increased in antral follicles. However, there were no significant differences in the labeling index of Ki67, PCNA, and AgNORs between dominant and nondominant follicles, including nonovulated follicles in the luteal phase. These results indicate that the transformation of granulosa cells from quiescence to active growth is important in early folliculogenesis. Immunoreactivity for Ki67 and PCNA were observed predominantly in the functioning corpus luteum, but not in the degenerating corpus luteum, indicating proliferation only during the luteal phase. Immunoreactivity for Ley and nick end-labeling reactive cells were not observed in the follicular and luteal phases, except for scattered cells in the degenerating corpus luteum. This may be because of the relatively long process of human follicular growth and atresia.

Immunohistochemical localization of Ad4-binding protein with correlation to steroidogenic enzyme expression in cycling human ovaries and sex cord stromal tumors.

Ad4-binding protein (Ad4BP) has been demonstrated recently as a transcription factor that serves as a general regulator of all steroidogenic P450 genes. We examined the expression of Ad4BP in 32 normal cycling human ovaries and 22 human ovarian sex cord stromal tumors by immunoblotting and immunohistochemistry. Immunoblotting of normal cycling human ovaries revealed a single band of 53 kilodaltons, corresponding to the mol wt of Ad4BP. We also correlated Ad4BP expression with the immunolocalization of the steroidogenic enzymes (side-chain cleavage cytochrome P450, cytochrome P450 17 alpha-hydroxylase, and cytochrome P450 aromatase). Ad4BP immunoreactivity, which was present only in the nuclei, was observed sporadically in the granulosa cells and adjacent stromal cells in the preantral follicles. In the dominant antral follicles, Ad4BP was detected in both granulosa and theca interna cells. However, in the nondominant antral follicles, Ad4BP was observed only in theca interna cells. In the corpus luteum, Ad4BP was present in both luteinized granulosa and thecal cells. Ad4BP was also expressed in some atretic follicles and degenerating corpora lutea. The spatial and temporal localization of Ad4BP in the normal cycling human ovary generally correlated well with that of steroidogenic enzymes. However, expression of the steroidogenic enzymes followed that of Ad4BP during the developing stages of the preantral follicle and vice versa during the process of follicular atresia. In ovarian sex cord stromal tumors, Ad4BP expression was observed in tumor cells that were positive for steroidogenic enzymes, but not in nonsteroidogenic tumor cells. These results, especially the in situ colocalization of Ad4BP and the steroidogenic enzymes, suggest that Ad4BP has the potential to control steroidogenic P450 expression in both normal and pathological human ovaries.

Mutation of cytochrome P-45017 alpha gene (CYP17) in a Japanese patient previously reported as having glucocorticoid-responsive hyperaldosteronism: with a review of Japanese patients with mutations of CYP17.

A 17-yr-old female Japanese patient, who was reported in 1968 as having glucocorticoid-responsive hyperaldosteronism but was presumed to have a defect of 17 alpha-hydroxylation mainly in the adrenal glands as the etiology of her illness, was followed. The relationship between clinical manifestations and molecular abnormalities in cytochrome P-45017 alpha gene (CYP17) was also reviewed based on the literature on Japanese patients with 17 alpha-hydroxylase deficiency. She has been treated with dexamethasone, resulting in normal blood pressure and normokalemia for 28 yr. She had almost normal gonadal function with regular menstruation on her first admission. Because of sustained genital bleeding, however, she underwent total hysterectomy with an ovarian biopsy at the age of 42 yr. No follicles or corpus luteum were detected in the ovarian specimen. At the age of 45 yr, the basal levels of sex steroids were decreased, while those of gonadotropins were increased. A genetic study on CYP17 revealed a homozygous deletion of phenylalanine (Phe) codon (TTC) at either amino acid position 53 or 54 in exon 1. A review of the literature revealed 4 patients with this type of CYP17 mutation, including the present patient, out of a total of 11 young adult Japanese patients. The clinical manifestations caused by congenitally deficient gonadal function were not marked in any of these 4 patients, but were marked in 5 of the 7 patients with different mutations of CYP17. The remaining 2 female patients had irregular menstruation. The pretreatment urine/plasma values of aldosterone were variable, normal to high, in individual patients, regardless of the structural abnormalities of CYP17. The following conclusions were suggested: 1) this type of CYP17 mutation is associated with well preserved gonadal function in young adult patients, but it likely causes early reduction of gonadal function with increasing age in these patients; 2) the prevalence of this type of CYP17 mutation is quite high in Japanese patients; and 3) the pretreatment hyperaldosteronism observed in the present patient seems not to be related to the mutation of CYP17.

Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: relationship between the enzyme activity and immunointensity.

To understand changes associated with the menstrual cycle in the human ovary, it is very important to examine chronological changes in P450 aromatase (P450arom) enzymatic activity in the normal cycling ovary. Therefore, we initially examined the correlation between intensity of P450arom immunoreactivity and its biochemical enzymatic activity in five estrogen-producing human cancer cell lines (HHUA, Ishikawa, HEC-59, OMC-2, and MCF-7). P450arom immunointensity per cell was evaluated by the CAS 200 computed image analysis system, and its catalytic activity per 10(6) culture cells was analyzed by the tritiated water method. A significant correlation (r = 0.959) was demonstrated between P450arom immunoreactivity and enzymatic activity under optimal conditions of tissue fixation and immunohistochemical procedures. We then investigated P450arom immunointensity in 31 specimens of normal cycling human ovaries to examine chronological changes in P450arom activity per cell throughout the menstrual cycle. In the follicular phase, P450arom was observed in the granulosa cells of one selected antral follicle per case during the mid- to late proliferative period, and its immunointensity per granulosa cell in the follicle was not significantly different between mid- and late proliferative periods, although serum estradiol level was markedly elevated in the late proliferative period. In the luteal phase, both P450arom immunointensity per luteinized granulosa cell in a corpus luteum and serum estradiol level reached a peak in the mid-secretory period. These findings indicate that different factors may influence ovarian P450arom activity during the follicular and luteal phases, i.e., an increased number of granulosa cells in the selected follicle during the follicular phase but changes in P450arom activity per luteinized granulosa cell in the corpus luteum during the luteal phase.

Temporal and spatial localization of steroidogenic enzymes in premenopausal human ovaries: in situ hybridization and immunohistochemical study.

In situ hybridization and immunohistochemical localization of cytochrome P450 cholesterol side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), cytochrome P450 17 alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) was performed in 50 morphologically normal human premenopausal ovaries, and correlated these findings with their endometrial phase. In general, mRNA expression of these enzymes examined by in situ hybridization were in good agreement with immunolocalization examined by immunohistochemistry. Expression of P450scc, 3 beta HSD and P450c17 was observed in large-sized preantral follicles, consisting of more than five layers of granulosa cells, preovulatory follicles, corpora lutea, and some degenerating corpora lutea and atretic follicles in all endometrial phases. Several follicles and/or corpora lutea positive for these enzymes were observed in the same ovary. Expression of P450arom was generally observed in only one follicle (antral or preovulatory follicle) or corpus luteum per case in mid proliferative to premenstrual phase, and was not observed in menstrual to early proliferative phase. These findings indicated that (1) expression of steroidogenic enzymes was associated with the continual human ovarian process including follicular development and atresia, and (2) especially, P450arom expression may occur only in a selected antral follicle and may have an important role in dominant follicular development.

Thermal-induced optical properties of a PdOx mask layer in an optical data storage system with a superresolution near-field structure.

Optical-thermal and thermal-optical properties of a PdOx mask layer in a system with a superresolution near-field structure are investigated with a Z-scan technique and a heating experiment. The high photothermal stability of the PdOx mask is shown, and the reversible limit of the PdOx mask layer and a weak switch effect are revealed. The PdOx decomposition, which results in a bubble with Pd particles, is confirmed, and the laser-induced physical and chemical mechanisms in the PdOx mask layer are clarified and discussed. Our microscopic studies and heating analysis are consistent with the Z-scan results. The PdOx mask sample is also compared briefly with a PtO2 mask layer that has the same structure.

Somatic alterations of the DPC4 and Madr2 genes in colorectal cancers and relationship to metastasis.

The DPC4 and Madr2 genes are located at 18q21, and the LOH on chromosome 18q21 has been shown to occur frequently in colorectal cancers. To investigate the role of these genes in advanced colorectal cancers, we analyzed 29 colorectal specimens for alterations in the DPC4 and Madr2 genes. Twelve (63.2%) of 19 informative primary colorectal cancers showed allelic loss of chromosome 18q21.3 marker. An alteration of the DPC4 gene sequence was identified in 6 (20.7%) of 29 colorectal carcinomas, and the distinct Madr2 gene mobility shifts were present in 3 (10.3%) cancers. Somatic mutations were identified in these tumors by sequencing analysis. DPC4 gene alterations of 4 cases were detected in Mad homology 2 domains. There was no significant correlation between the somatic alteration of Madr2 and clinicopathological findings. However, the frequency of DPC4 mutation was significantly higher in tumors associated with liver metastasis than in those without such metastasis. Our findings suggest that somatic alteration of the DPC4 gene may play a role in tumorigenesis and liver metastasis of human colorectal cancers.

Transcription factor adrenal 4 binding protein as a marker of adrenocortical malignancy.

Adrenal 4 binding protein (Ad4BP) is a transcription factor that regulates the expression of the steroidogenic enzymes and is expressed primarily in steroidogenic cells. We immunolocalized Ad4BP in adrenocortical carcinoma (eight cases) and various malignancies that histologically simulate an adrenocortical carcinoma to evaluate the value of Ad4BP as an immunohistochemical marker of adrenocortical carcinoma. These malignancies examined were renal cell carcinoma (20 cases), hepatocellular carcinoma (10 cases), malignant melanoma (eight cases), ovarian (six cases) and uterine (three cases) clear cell carcinoma, large cell carcinoma of the lung (five cases), and pheochromocytoma (three cases). Nuclear Ad4BP immunoreactivity was observed only in adrenocortical carcinoma cases but not in other tumors examined. Almost all of the adrenocortical carcinoma cells were immunohistochemically positive for Ad4BP including cells associated with bizarre nuclei. These results show that application of Ad4BP immunostain can contribute greatly to the differential diagnosis of adrenocortical carcinoma.

Translocation of protein kinase C from cytosol to membrane fractions in human epidermal keratinocytes by recombinant human interferon-gamma.

The activation of protein kinase C involves its translocation from a cytosol fraction to a membrane fraction. Effects of interferon-gamma (IFN-gamma) on the epidermal protein kinase C were investigated. The treatment of recombinant human IFN-gamma on intact human epidermis resulted in the translocation of protein kinase C from a cytosol to a membrane fraction. The human IFN-gamma had no translocation effect on pig epidermal protein kinase C. Tumor promoter, 12-o-tetradecanoylphorbol-13-acetate (TPA), and a membrane-permeable diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), both of which are well-known activators of protein kinase C, translocated the epidermal protein kinase C. The IFN-gamma had no direct effect on the epidermal protein kinase C; the addition of the IFN-gamma to partially-purified pig epidermal protein kinase C had no effect on its activity. The effect of the IFN-gamma on human epidermal protein kinase C appears to be through the species specific IFN-gamma receptors. It has been reported that the epidermal beta-adrenergic adenylate cyclase response is decreased following the TPA- (and OAG-) induced activation of protein kinase C. Human recombinant IFN-gamma, however, had no effect on the beta-adrenergic response of the human epidermis. Our results indicate that IFN-gamma affects intact keratinocytes in vitro, resulting in the activation of protein kinase C, which might be related to the physiological effect of IFN-gamma on keratinocyte.