Biochemical and ultrastructural analyses of IgLON cell adhesion molecules, Kilon and OBCAM in the rat brain.

Kilon (kindred of IgLON) and opioid-binding cell adhesion molecule belong to the IgLON subgroup of immunoglobulin superfamily together with the limbic system-associated membrane protein and neurotrimin. In the present study, we have analyzed biochemical and ultrastructural characterization of Kilon and opioid-binding cell adhesion molecule such as regional and developmental expression patterns, light and electron microscopic localization, and intermolecular interactions. Western blotting revealed a widespread distribution pattern of Kilon with high expression levels in the olfactory bulb, cerebral cortex, diencephalon, hippocampus, and cerebellum and low expression levels in the medulla oblongata and spinal cord. In contrast, opioid-binding cell adhesion molecule showed a regionally restricted expression pattern with high levels only in the cerebral cortex and hippocampus. Expression of Kilon and opioid-binding cell adhesion molecule was increased gradually during postnatal development and maintained until adulthood. Light microscopic immunohistochemistry demonstrated that the localization of opioid-binding cell adhesion molecule and Kilon coincided well with that of vesicle-associated membrane protein 2, a synaptic marker protein, in the cerebral cortex and hippocampus of adult brain. In the cerebellum, Kilon-immunoreactive puncta were observed to colocalize well with that of vesicle-associated membrane protein 2, while opioid-binding cell adhesion molecule immunoreactivity was observed only at part of synaptic glomeruli in the granular layer and rare in the molecular layer. Electron microscopic analysis revealed that Kilon and opioid-binding cell adhesion molecule immunoreactivity was observed mainly at postsynaptic sites of dendritic and somatic synapses in adult cerebral cortex and hippocampus. Only trace levels of Kilon and opioid-binding cell adhesion molecule were detected in the soluble fraction of a cortical homogenate, although a substantial amount of F3 was present in the soluble fraction. A binding analysis using a cross-linker and the immunoprecipitation technique demonstrated that Kilon and opioid-binding cell adhesion molecule interacted heterophilically and homophilically. These findings show that Kilon and opioid-binding cell adhesion molecule are clearly distinguishable from each other in regional expression and localization, and binding patterns. These differences possibly represent diverse functions of each IgLON molecule.

Expression of the 67-kDa elastin receptor in perforating skin disorders.

BACKGROUND: Perforating skin dermatoses include elastosis perforans serpiginosa (EPS), reactive perforating collagenosis, Kyrle's disease and perforating folliculitis. In addition to these four diseases, an acquired form of perforating dermatosis associated with diabetes mellitus and/or chronic renal failure has been reported for which the term acquired perforating dermatosis (APD) was proposed. The molecular mechanism of transepidermal elimination of dermal components in perforating skin dermatoses remains unclear. We recently demonstrated that the 67-kDa elastin receptor can be detected in the epidermis eliminating altered elastic fibres in EPS, suggesting that the elastin-keratinocyte interaction may play a role in transepidermal elimination in EPS. OBJECTIVES: To determine whether the 67-kDa elastin receptor is involved in other perforating diseases. METHODS: Paraffin-embedded skin specimens from new cases of EPS (n = 2), APD (n = 15) and perforating granuloma annulare (PGA; n = 2) were studied immunohistochemically using a specific antibody to the 67-kDa elastin receptor. In one case of EPS, two different sites from a single lesion, a central atrophic area and a peripheral keratotic area, were studied. RESULTS: Expression of the elastin receptor was detected in the epidermis surrounding the elastic materials in both cases of EPS. The elastin receptor was not detected in the central inactive area, whereas it was expressed strongly in the peripheral keratotic active area. The elastin receptor was also detected in three of 15 cases of APD in which a few elastic fibres were found in the eliminated dermal materials. In one case of APD, the elastin receptor was not detected in spite of the presence of a few elastic fibres in the eliminated materials. The elastin receptor was not detected in either case of PGA. CONCLUSIONS: Expression of the elastin receptor in EPS was seen in both cases studied and was dependent on the stage of the lesion. Expression of the elastin receptor in APD appeared to be related to the amount of elastic fibres in the eliminated materials. Thus, expression of the elastin receptor in perforating skin disorders may depend on the stage of the lesion and/or the content of elastic fibres in the dermal materials being eliminated.

EST analysis of genes that are expressed in the neural complex of Ciona intestinalis adults.

A subtractive cDNA library was made corresponding to mRNAs expressed in the neural complex relative to those expressed in the pharynx of adults of the ascidian Ciona intestinalis. Determination and comparison of expressed sequence tags (ESTs) of a set of 1,527 randomly selected clones demonstrated that they represent 832 independent sequences. Five hundred seventy-two of the clones contained amino-acid-encoding sequences. BLASTX analyses showed that 342 of the 572 clones were strong matches (P < 10(-7)) to previously identified proteins, while the remaining 230 fell into the "no match" category. Among the clones matching previously identified proteins, about 80 clones represented proteins that are involved in the formation, maintenance of the structure, and function of the nervous system: 22 proteins are associated with signal transduction, five proteins are related to the synapse, 11 to transcription factors, nine to transporters, five to enzymes, and 13 to extracellular matrix and cytoskeletal components, and six to apoptosis. In addition, sequence information for genes associated with the immune system and for genes encoding proteins with interesting functions were obtained. These data provide cues for further studies on genes that are expressed in and function in the ascidian nervous system.

Expression of type XVI collagen in cultured skin fibroblasts is related to cell growth arrest.

The expression of type XVI collagen in various phases of cell growth in cultured skin fibroblasts was studied. A marked increase in type XVI collagen mRNA level was found in stationary phases of cell growth (non-adherent and confluent phases), whereas the expression of type I and III collagens was undetectable in the non-adherent phase but became greater in the confluent phase. When suspended cells were further cultured over 72 h (suspension arrest), mRNA level and gene transcription of type XVI collagen were time-dependently increased whereas those of type I collagen remained undetectable. When the confluent cells were further cultured for 72 h under the condition of serum deprivation (serum deprivation arrest), mRNA levels of both type XVI collagen and type I collagen were elevated. The level of type XVI collagen polypeptide in the culture media of suspension-arrested and serum deprivation-arrested cells paralleled the mRNA level of type XVI collagen. The results indicate that expression of type XVI collagen (a member of the fibril-associated collagens with interrupted triple helices), unlike interstitial collagens (type I collagen), is related to cell growth arrest brought about by two different growth inhibiting systems, suspension arrest and serum deprivation arrest.

Expression of type XVI collagen in human skin fibroblasts: enhanced expression in fibrotic skin diseases.

Abundance of type XVI collagen mRNA in normal human dermal fibroblasts explanted from different horizontal layers was determined using RNase protection assays. Type XVI collagen mRNA level in the fibroblasts explanted from the upper dermis was greater than those of the middle and lower dermis. The antibody raised against the synthetic N-terminal noncollagenous region reacted with approximately 210 kDa collagenous polypeptide in the culture medium of fibroblasts. Immunohistochemical study of normal human skin demonstrated that the antibody reacted preferentially with the fibroblasts and the extracellular matrix in the upper dermis rather than those in the middle and lower dermis. Type XVI collagen mRNA level was elevated 2.3-fold in localized scleroderma and 3.6-fold in systemic scleroderma compared with keloid and normal controls. Immunofluorescent study revealed that an intense immunoreactivity with the antibody was observed in the upper to lower dermal matrix and fibroblasts in the skin of systemic scleroderma as compared with normal skin. The results suggest that expression of type XVI collagen, a member of fibril-associated collagens with interrupted triple helices, in human skin fibroblasts can be heterogeneous in the dermal layers and can be modulated by some fibrotic diseases.

Role of copper accumulation in spontaneous renal carcinogenesis in Long-Evans Cinnamon rats.

Spontaneous renal cell tumors in totals of 223 male and female Long-Evans Cinnamon (LEC) rats of 51-120 weeks old, 157 male F344 rats of 51-120 weeks old, and 14 male Long-Evans Agouti (LEA) rats of 51-70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long-term treatment of LEC rats with D-penicillamine, a copper-chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.

Non-carcinogenicity of capsaicinoids in B6C3F1 mice.

The carcinogenicity of a mixture of capsaicinoids (64.5% capsaicin and 32.6% dihydrocapsaicin) was examined in B6C3F1 mice. In a 13-week toxicity study, renal toxicity was observed in 1% capsaicinoid-treated males. Next, groups of 50 mice of each sex were given 0, 0.025, 0.083 or 0.25% capsaicinoids in powdered diet for 79 weeks and killed in week 83. Food intake was reduced in mice of all capsaicinoid-treated groups, especially females, because of the pungency of capsaicinoids, and inhibition of body weight gain was apparent in females. The numbers of tumour-bearing females in the high-dose groups were significantly lower than that in the controls, and the incidences of hepatocellular neoplasms in both sexes were negatively correlated with the dose of capsaicinoids (Cochran-Armitage trend test). Renal cell adenomas developed in one mouse each of 0.025 and 0.25% capsaicinoid-treated males. The incidences of other tumours were similar in the treated and control groups. Thus, the present study indicated that a mixture of capsaicinoids is not carcinogenic in B6C3F1 mice.

AutoCyte SCREEN interactive automated primary cytology screening system. A preliminary evaluation.

OBJECTIVE: To perform a preliminary evaluation of the relative sensitivity of a new interactive automated cytology screening system (AutoCyte SCREEN, AutoCyte, Inc., Elon College, North Carolina, U.S.A.) designed as a primary screening system for AutoCyte PREP monolayer slides (AutoCyte). STUDY DESIGN: Monolayer slides were prepared and evaluated in a masked review both manually and using the automated system. Human and machine performance was monitored and measured. RESULTS: Five hundred eighty-three monolayer preparations, including 56 clearly abnormal cases, were evaluated. The dual and combined computer and cytologist image evaluation resulted in a relatively low false positive rate, 20.8%, and 1.8% false negative rate. Use of the system was efficient, significantly reducing cytotechnologist screening time. CONCLUSION: The integrated AutoCyte cell preparation and automated screening approach seems to hold significant promise for primary cytology screening applications.

Effects of partial nephrectomy on the expression of osmolyte transporters.

Na+/myo-inositol cotransporter (SMIT) and Na+/Cl-/betaine-gamma-amino-n-butyric acid transporter (BGT-1) are the major osmolyte transporters that are regulated by extracellular osmolarity. We have recently shown localization and rapid regulation of the mRNAs for these transporters in rat kidney. In the present study, we examined the expression of SMIT and BGT-1 in partial nephrectomized rats in order to assess the change in local osmolarity following reduction of renal mass. Four weeks after 5/6 nephrectomy (NX), the rats were compared to sham-operated control animals (CONT). Northern analysis using RNA of whole kidney indicated that there were little differences in the levels of SMIT and BGT-1 mRNAs between the two groups. In situ hybridization revealed that signals for both transporter mRNAs were markedly reduced in the inner medulla of the remnant kidney. In contrast, these signals in the outer medulla increased following nephrectomy. SMIT signals in the cortex increased as well. Grain density, determined by counting grain number per cell, revealed that the signals in the inner medullary collecting ducts were markedly reduced whereas those in the thick ascending limbs of Henle (TAL) as well as macula densa cells were significantly increased. The signals in the TAL and macula densa were reduced by furosemide administration. The increased expression in NX rats may reflect the increased NaCl transport and high local osmolarity in this segment.

Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomerular epithelial cells in vivo.

The aim of this study was to seek a promoter, transactivated selectively in renal cells in vivo by using transgenic (tg) mouse technology. We generated two kinds of tg mouse lines carrying a green fluorescence protein (GFP) cDNA driven either by cytomegalovirus enhancer and beta-actin/beta-globin promoter (CX-GFP) or by elongation factor 1alpha promoter (EF-GFP), and investigated the expression of GFP in the kidney. Microscopic examination of the renal tissues in CX-GFP-tg mice revealed that GFP was expressed only in glomeruli, mainly epithelial cells, but not in tubules, arteries and interstitium. Moreover, in situ hybridization demonstrated that GFP mRNA expression was localized in the glomerular cells. In contrast, GFP was not detectable in the kidney in any of the lines of EF-GFP-tg mouse. To exclude the possible involvement of the GFP cDNA as an enhancer, we constructed tg mice carrying the CX promoter driving a human CD4 cDNA. It was confirmed that the expression patterns of human CD4 in the kidney were quite similar to those of GFP in the kidney of CX-GFP-tg mice. These results strongly suggest that CX promoter could be transactivated in glomerular epithelial cells in vivo.

Effects of inhibition of myo-inositol transport on MDCK cells under hypertonic environment.

To investigate the role of myo-inositol under hypertonic conditions, we examined the effects of inhibition of myo-inositol transport in Madin-Darby canine kidney (MDCK) cells using an analog of myo-inositol, 2-O,C-methylene-myo-inositol (MMI). We first characterized the inhibitory effects of MMI on myo-inositol transport in MDCK cells. The Na+-dependent component of [3H] myo-inositol uptake was inhibited by MMI in a concentration-dependent manner, although MMI did not inhibit the activities of the betaine transporter and system A neutral amino acid transporter. We found decreased affinity for myo-inositol in the presence of MMI, whereas the maximal velocity (Vmax) of the transporter did not change. Thus MMI behaves as a competitive inhibitor of myo-inositol transport with a relatively high inhibition constant (K(i)) value (1.6 mM). Myo-inositol content in hypertonic MDCK cells was markedly reduced in the presence of 5 mM MMI, but MMI itself did not accumulate in these cells. The hypertonic cells began to detach in the presence of MMI 3 days after increasing medium osmolality, whereas MMI did not affect the cells in isotonic medium. We also examined the effects of MMI on colony-forming efficiency of MDCK cells. MMI decreased colony-forming efficiency in a concentration-dependent manner, and addition of myo-inositol returned the efficiency to the value without MMI. Addition of betaine also increased colony-forming efficiency in the presence of MMI. These results indicate that myo-inositol plays an important role in survival and growth under hypertonic environment.

Carcinosarcoma of the uterine body of mesonephric origin.

A case of carcinosarcoma of mesonephric origin in a 58 year old woman is reported. A cystic tumor with a solid area, measuring 14 cm in greatest diameter, was detected in the pelvic cavity by computerized tomography and ultrasound. Although it was diagnosed as an ovarian cancer for surgical removal, it was found to be entirely located in the myometrium of the left lateral wall of the uterine body and neither ovary was remarkable. Histologically the tumor was composed of epithelial and sarcomatous components. The former showed low papillary pattern, crowded solid nests and cords of cells, and focal tubular structures. The latter showed a solid growth pattern with differentiation to leiomyosarcoma. In the uterine cervix, a 1.2 cm mesonephric (Gartner's) cyst was found. Neither neoplastic lesions nor endometriosis were identified in the cervix, endometrium, fallopian tubes or ovaries. Based on the histologic features and the specific location of the tumor, the coexistence of Gartner's cyst, and the normal appearance of the endocervical mucosa as well as the endometrium, it was diagnosed as a mesonephric carcinosarcoma. The serum levels of carcinoembryonic antigen, CA125, CA19-9, and CA72-4 were within normal ranges in the clinical course. The patient died of disease 8 months after surgery.

Induction of soft tissue tumours in F344 rats by subcutaneous, intramuscular, intra-articular, and retroperitoneal injection of nickel sulphide (Ni3S2).

The carcinogenicity of nickel sulphide (Ni3S2) injected into subcutaneous (s.c.), intramuscular (i.m.), or retroperitoneal intrafat (i.f.) tissue, or the intra-articular space (i.a.) of male F344 rats was studied. Rats were given a single injection of 0.5 mg of Ni3S2 and were observed for 48 weeks. Malignant soft tissue tumours were induced in 18/19 rats (95 per cent) by s.c. injection, 19/20 rats (95 per cent) by i.m. injection, in 16/19 rats (84 per cent) by i.a. injection, and in 9/20 rats (45 per cent) by i.f. injection of Ni3S2. The i.f. injection of Ni3S2 resulted in a lower tumour incidence and the appearance of tumours 10 weeks later than its injection by other routes. The tumours were examined histologically, ultrastructurally, and immunohistochemically with antibodies against desmin, vimentin, and cytokeratin. The 62 tumours induced by injection of Ni3S2 by different routes were identified as rhabdomyosarcomas (RMS, 35), malignant fibrous histiocytomas (MFH, 18), fibrosarcomas (FS, 5), and unclassified sarcomas (4). All 19 tumours induced by i.m. injection of Ni3S2 were rhabdomyosarcomas; those induced by s.c. or i.f. injection were mainly MFHs. However, a number of RMSs were also found in groups that received i.a., s.c., and i.f. injections; five FSs also developed in these groups. Four sarcomas induced by s.c. and i.a. injections were not classified. No synovial sarcoma developed.

Nonspecific esterase reaction in hyperplastic urinary bladder epithelium induced by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine, freezing and formalin instillation in rats.

Chronological changes in nonspecific esterase (NSE) activity in hyperplasia of the bladder mucosa in Wistar rats induced by the administration of 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for up to 20 weeks and in reversible regenerative hyperplasia by freeze ulceration and 20% formalin instillation in the bladder were compared. In regenerative hyperplasia foci with strong NSE activity could not be proved throughout the experimental period, while the foci were detected in hyperplastic epithelium induced by BBN treatment for more than 3 weeks. The focus of NSE high activity persisted for 56 weeks after withdrawal of the carcinogen and the focus or area with the same NSE reaction appeared in papilloma and transitional cell carcinoma seen in weeks 7 to 20 of BBN treatment. The appearance of focal strong activity of NSE seemed to be a promising marker for the precursor lesions of bladder tumors. Short uniform, pleomorphic microvilli were observed on the cell surface of preneoplastic and carcinomatous lesions by BBN as well as on that of regenerative hyperplasia after freeze ulceration and formalin instillation.

Effects of 12-O-tetradecanoylphorbol-13-acetate on carcinogenesis in the heterotopically transplanted rat urinary bladder.

12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent promoter of mouse skin carcinogenesis, was tested for possible tumor-enhancing effects on urinary bladder carcinogenesis using the heterotopically transplanted bladder (HTB) model. Weekly administration of TPA at 1.0 microgram/week to N-methyl-N-nitrosourea-initiated HTBs did not increase tumor incidence, but instead, resulted in a significantly high incidence of nodulopapillary hyperplasia, an early neoplastic lesion, suggesting possible tumor enhancement by TPA. In addition, administration of a high dose of TPA with or without a carcinogen treatment led to the development of numerous finger-like epithelial projections on the luminal surface of the HTBs. Evidence indicates that epithelial projections are formed as a result of proliferation of intermediate cells. Whether these structures evolve into true neoplastic lesions is at present unknown.