Membrane localization of protein-tyrosine phosphatase 1B is essential for its activation of sterol regulatory element-binding protein-1 gene expression.

Sterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor in stimulating lipogenesis in the liver. Protein-tyrosine phosphatase 1B (PTP1B) induces SREBP-1 gene expression via protein phosphatase 2A (PP2A) activation. PTP1B is reported to be anchored on the endoplasmic reticulum (ER) via its C-terminal tail, and change in intracellular localization of PTP1B by C-terminal-truncation did not alter its inhibitory effects on insulin signaling. In this study, we investigated whether the change in intracellular localization of PTP1B could influence SREBP-1 gene expression. Overexpression of C-terminal truncated PTP1B (PTP1BdeltaCT) in rat Fao cells did not induce SREBP-1 gene expression. Furthermore, PTP1BdeltaCT failed to bind PP2A, resulting in impaired PP2A activation, whereas overexpression of wild-type PTP1B (PTP1BWT) associated with PP2A. Moreover, a membrane-targeted PTP1BDeltaCT activated PP2A with restored PP2A binding, despite the absence of its C-terminal region. Finally, overexpression of PTP1BdeltaCT into mouse primary cultured hepatocytes failed to enhance SREBP-1c mRNA, whereas membrane-targeted PTP1BdeltaCT led to enhanced SREBP-1c mRNA in hepatocytes as well as PTP1BWT. In conclusion, membrane localization of PTP1B is essential for PP2A activation, which is crucial for its enhancement of SREBP-1 gene expression.

Human papillomavirus-associated plantar epidermoid cyst related to epidermoid metaplasia of the eccrine duct epithelium: a combined histological, immunohistochemical, DNA-DNA in situ hybridization and three-dimensional reconstruction analysis.

BACKGROUND: We recently proposed that certain palmoplantar epidermoid cysts may be related to eccrine ducts and that human papillomavirus (HPV) 60 may play a role in their pathomechanism. However, the origin of palmoplantar epidermoid cysts is still controversial. OBJECTIVES: To examine the contribution of eccrine ducts and HPV 60 in the development of epidermoid cysts. METHODS: Five epidermoid cysts and four ridged warts that had developed on the soles of a patient were studied histologically, immunohistochemically and by DNA-DNA in situ hybridization. Using serial sections obtained from its entire body, a three-dimensional reconstruction (3DR) analysis was performed on the smallest cyst to analyse the relationship between the epidermoid cyst, eccrine duct and the overlying epidermis. RESULTS: Histological and DNA-DNA in situ hybridization analyses demonstrated both homogeneous intracytoplasmic inclusion bodies pathognomonic for HPV 60 infection and HPV 60 DNA sequences not only in all of the epidermoid cysts and ridged warts but also in the acrosyringeal portion of an eccrine duct, with the dermal portion of which the smallest cyst had been revealed to connect by 3DR analysis. However, immunohistochemical analyses using antibodies against human carcinoembryonic antigen (CEA), involucrin and several cytokeratins (CKs) revealed that the immunoreactivity of the cyst was not identical to that of the eccrine dermal duct but was identical to that of suprabasal layers of the epidermis. CONCLUSIONS: It was clearly demonstrated that an HPV 60-associated epidermoid cyst with immunoreactivities for CEA, involucrin and CKs which were identical to those of the epidermis connected with the eccrine dermal duct, supporting the idea that certain palmoplantar epidermoid cysts may develop following the epidermoid metaplasia of eccrine ducts with HPV 60 infection.

Eccrine-centred distribution of human papillomavirus 63 infection in the epidermis of the plantar skin.

BACKGROUND: The primary target cell of human papillomaviruses (HPVs) is an unsettled issue. Recent studies have suggested that the hair follicle is an important candidate as the reservoir of certain HPV types. However, little is known about the cells which serve as the target or the reservoir of HPVs in nonhairy palmoplantar skin. OBJECTIVES: To investigate whether the eccrine sweat gland, the only skin appendage in nonhairy palmoplantar skin, also serves as the target or the reservoir of HPVs. METHODS: HPV 63-induced warts were employed in this study, because the virus induces tiny warty lesions of a punctuate appearance in the plantar skin and shows peculiar intracytoplasmic inclusion bodies as a diagnostic histopathological marker of infection: this seemed to provide a useful model for the present study. Serial sections were obtained from the entire body of each biopsy specimen and were investigated histologically, immunohistochemically and using DNA-DNA in situ hybridization (ISH) for the histological localization of HPV 63 infection. RESULTS: On microscopy, HPV 63 histopathological changes were seen closely associated with eccrine ducts. Using ISH, HPV 63 DNA was detected not only in keratinocytes resident around acrosyringia but also in the uppermost portion of the eccrine dermal duct. A few keratinocytes harbouring HPV 63 DNA were also identified in acrosyringeal areas in the normal plantar skin adjacent to the wart lesions. CONCLUSIONS: On the basis of our results, it seems likely that HPV 63 targets keratinocytes resident in or around the eccrine ducts in the plantar skin. The results may also suggest that not only hair follicles but also eccrine ducts serve as reservoirs for certain HPV types, including HPV 63, especially in the nonhairy plantar skin.

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.

Insulin-induced c-Jun N-terminal kinase activation is negatively regulated by protein kinase C delta.

We investigated the role of protein kinase C (PKC) in insulin-induced c-Jun N-terminal kinase (JNK) activation in rat 1 fibroblasts expressing human insulin receptors. Insulin treatment led to increased SAPK/ERK kinase 1 (SEK1) phosphorylation, and then stimulated JNK activity in a dose- and time-dependent manner, as measured either by a solid-phase kinase assay using glutathione S-transferase (GST)-c-Jun fusion protein as a substrate, or by quantitation of the levels of phosphorylated JNK by Western blotting using anti-phospho-JNK antibody. Insulin-induced JNK activation was potentiated by either preincubating cells with 2 nM GF109203X (PKC inhibitor) or down-regulation of PKC by overnight treatment with 100 nM tetradecanoyl phorbol acetate. In contrast, brief preincubation with 100 nM tetradecanoyl phorbol acetate inhibited the insulin- induced JNK activation. Furthermore, we found that 5 microM rottlerin, a PKCdelta inhibitor, enhanced insulin-induced JNK activation, but a PKCbeta inhibitor, LY333531, had no effect. Consistent with these findings, overexpression of PKCdelta led to decreased insulin-induced JNK activation, whereas overexpression of PKCbeta had no effect. Although overexpression of wild-type PKCdelta attenuated insulin-induced JNK activation, a kinase-dead PKCdelta mutant did not cause such attenuation. Finally, we found that the magnitude of insulin-induced JNK activation was inversely correlated with the expression level of PKCdelta among different cell lines. In conclusion, the expression of PKCdelta may negatively regulate insulin-induced JNK activation.

Insulin production in a neuroectodermal tumor that expresses islet factor-1, but not pancreatic-duodenal homeobox 1.

We studied a 60-yr-old female with a brain tumor who showed severe symptoms of hypoglycemia (plasma glucose, 2.2 mmol/L) and hyperinsulinemia (1.28 nmol/L) after radiotherapy. The cystic brain tumor contained proinsulin and insulin at concentrations of 13.6 and 1.22 nmol/L, respectively. Immunohistochemical studies showed the tumor cells were ectodermal in origin but not endodermal, based on three diagnostic features of neuroectodermal tumors 1) pseudorosette formation noted under light microscopy, 2) finding of a small number of dense core neurosecretory granules on electron microscopy, and 3) positive immunostaining for both neuronal specific enolase and protein gene product 9.5. These cells also expressed the transcription factor, neurogenin-3, NeuroD/beta 2, and islet factor I, which are believed to be transcription factors in neuroectoderm as well as in pancreatic islet cells, but not pancreatic-duodenal homeobox 1, Pax4, or Nkx2.2. In addition, they did not express glucagon, somatostatin, or glucagon-like peptide-1. Our results show the presence of proinsulin in an ectoderm cell brain tumor that does not express the homeobox gene, pancreatic-duodenal homeobox 1, but expresses other transcription factors, i.e. neurogenin3, NeuroD/beta 2, and islet factor-1, which are related to insulin gene expression in the brain tumor.

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells.

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.

Deoxynybomycin is a selective anti-tumor agent inducing apoptosis and inhibiting topoisomerase I.

Deoxynybomycin was identified as an inducer of p21the/WAF1 gene following screening using a reporter, p21/luciferase. The present study examined its anti-proliferative effect on human tumor cell lines. Deoxynybomycin selectively inhibited growth of human osteoblastic sarcoma Saos-2, gastric cancer TMK-1, and monocytic leukemia THP-1 cells, but did not affect survival of normal human fibroblasts at doses up to 5 microg/ml. Results from an assay system using a panel of 39 human cancer cell lines indicated that deoxynybomycin has selective cytotoxic activity against lung carcinoma cell lines. Deoxynybomycin induced apoptosis in Saos-2, TMK-1, and THP-1 cells as revealed by DNA fragmentation and TUNEL assays. It inhibited topoisomerase I but not topoisomerase II. These results suggest that deoxynybomycin may be useful in cancer chemotherapy.

The carcinoembryonic antigen (CEA) family (CD66) expressed in melanocytic naevi is not expressed in blue naevuscell naevi in dendritic type.

BACKGROUND: Although sporadic reports have regarded the expression of the carcinoembryonic antigen (CEA) family in melanoma, there has been no information about the expression in precursor lesions of melanoma such as melanocytic naevi and blue naevi. METHODS: The expression was immunohistochemically studied in frozen biopsy specimens of 45 acquired and 16 congenital melanocytic naevi and 20 blue naevi, using a panel of monoclonal and polyclonal antibodies that recognize different epitopes of CEA and related molecules. RESULTS: Members of the CEA glycoprotein family were strongly expressed in all of the subtypes of melanocytic naevus. A reduced expression of the CEA glycoproteins with increased dermal depth or acquisition of a spindled morphology of naevus cells was apparent. The expression was not seen in the present blue naevi and normal epidermal melanocytes. CONCLUSIONS: Although the significance of the expression was not clarified, this report has clearly demonstrated that the CEA family is strongly expressed in melanocytic naevi and immunoreactivity is divergent between melanocytic naevi and blue naevi and between dermal naevus cell types, suggesting that the expression may be altered with architectural changes in the melanocyte-lineage cells.

Nail bed keratinocytes express an antigen of the carcinoembryonic antigen family.

Using a panel of monoclonal and polyclonal antibodies raised against human carcinoembryonic antigen (CEA) and CEA-related molecules, we detected strong expression of an antigen, with immunoreactivity consistent with non-specific cross-reacting antigen (NCA) (CD66c), in all of 26 normal human nail specimens obtained from various fingers and toes. In longitudinal sections, strong and constant expression of the NCA-like antigen was seen on keratinocytes distributed in the upper epithelial cell layers of the nail bed, while in transverse sections the expression was limited to the major central portions of the nail bed, but only where longitudinal epidermal ridges were observed. In the hyponychium and the ventral aspect of the proximal nail fold, the expression was weak or lacking. No expression was seen in the nail matrix, the nail plate, or the dorsal aspect of the proximal nail fold. The same results were obtained for all of the 26 nails studied. This report is the first to demonstrate that an antigen of the CEA family, with NCA-like immunoreactivity, is expressed in a specific subpopulation of keratinocytes in the nail bed. The specific expression pattern suggests that the antigen may play a part in adhesion of the nail plate to the nail bed.

A case of viral warts with particular fibrillar intracytoplasmic inclusion bodies.

A new type of skin wart was observed in a Japanese patient. It was characterized by intracytoplasmic inclusions with a 'fibrillar' structure which were distinct from previously described wart-associated inclusions. The papillomavirus (HPV)-group-specific antigen could be detected, but DNA hybridization and PCR amplification using probes or PCR primers specific for the main skin HPV genotypes (including HPV-63 which is also associated with 'filamentous' inclusions) were negative. We consider that this cytopathic effect could correspond to an HPV genotype which has not yet been characterized.

A rapamycin-sensitive pathway down-regulates insulin signaling via phosphorylation and proteasomal degradation of insulin receptor substrate-1.

Insulin receptor substrate-1 (IRS-1) is a major substrate of the insulin receptor and acts as a docking protein for Src homology 2 domain containing signaling molecules that mediate many of the pleiotropic actions of insulin. Insulin stimulation elicits serine/threonine phosphorylation of IRS-1, which produces a mobility shift on SDS-PAGE, followed by degradation of IRS-1 after prolonged stimulation. We investigated the molecular mechanisms and the functional consequences of these phenomena in 3T3-L1 adipocytes. PI 3-kinase inhibitors or rapamycin, but not the MEK inhibitor, blocked both the insulin-induced electrophoretic mobility shift and degradation of IRS-1. Adenovirus-mediated expression of a membrane-targeted form of the p110 subunit of phosphatidylinositol (PI) 3-kinase (p110CAAX) induced a mobility shift and degradation of IRS-1, both of which were inhibited by rapamycin. Lactacystin, a specific proteasome inhibitor, inhibited insulin-induced degradation of IRS-1 without any effect on its electrophoretic mobility. Inhibition of the mobility shift did not significantly affect tyrosine phosphorylation of IRS-1 or downstream insulin signaling. In contrast, blockade of IRS-1 degradation resulted in sustained activation of Akt, p70 S6 kinase, and mitogen-activated protein (MAP) kinase during prolonged insulin treatment. These results indicate that insulin-induced serine/threonine phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway, which is downstream of PI 3-kinase and independent of ras/MAP kinase. The pathway leads to degradation of IRS-1 by the proteasome, which plays a major role in down-regulation of certain insulin actions during prolonged stimulation.

Synthesis of viral DNA and late capsid protein L1 in parabasal spinous cell layers of naturally occurring benign warts infected with human papillomavirus type 1.

We investigated human papillomavirus type 1 (HPV1)-specific transcription, viral DNA replication, and viral protein expression in naturally occurring benign tumors by in situ hybridization, 5-bromodeoxyuridine (BrdU) incorporation, and immunohistochemistry and obtained results different from other HPV-infected benign tumors characterized so far. Moderate amounts of transcripts with a putative coding potential for E6/E7, E1, and E2 were demonstrated from the first subrabasal cell layer throughout the stratum spinosum and granulosum. In addition very large amounts of E4 and L1 transcripts were present in the same epithelial layers. This finding was substantiated by the demonstration of L1 and E4 protein already in the bottom-most spinous cell layer. Furthermore massive amplification of the viral DNA as measured by BrdU incorporation and different methods of in situ hybridization took place in the lowest 5 to 10 suprabasal cell layers. These findings are in contrast to the assumption that late gene expression and viral DNA synthesis are restricted to the more differentiated cell layers of the epithelium and point to differences in the regulation of the vegetative life cycle between different papillomavirus types.

[Factors leading to home care of patients in the terminal stage of malignant diseases--a study of 10 cases].

In order to clarify the factors that lead patients in the terminal stage of malignant diseases to home care, we interviewed ten families of such patients. Among factors involving patients, a strong desire to stay at home and to spend time at home with the family were initially required. Adequate understanding of the patients and their diseases was also needed by families. Other factors include guaranteed medical services in emergencies and/or 24 hours/day 7 days/week care. Patients requested referrals to family physicians, visiting nurses or community health services to cover outpatient clinic functions. It is concluded that a strong desire to stay at home, adequate understanding by the family and guaranteed medical services in emergencies were three significant factors leading to home care of patients in the terminal stage of malignant diseases.

Clinicoanatomic examination of the fibula: anatomic basis for dental implant placement.

The advantages of the free vascularized fibular flap include its ability to be shaped with relative ease and to be grafted at the same time tumors are resected, with consequent reduction in operation time. In addition, few complications occur at donor sites. However, a large, systematic, and detailed investigation of clinicoanatomic problems of the fibula has not been performed. Therefore, in the present study, the fibula was examined morphologically and morphometrically, with special attention to regions important in the placement of dental implants. Eighty fibulae obtained from cadavers of 41 Japanese individuals aged 46 to 92 years (mean, 72.7 years) were fixed with 70% alcohol after infusion of about 6 liters of 10% formalin via the femoral artery. Morphometric examination showed the nutrient foramen was located posteriorly in 85.0% of sections, the maximal width of fibular cross sections was 13.1 mm, and the maximal cortical thickness of fibular cross sections was 4.1 mm.