Intestinal knockout of Nedd4 enhances growth of Apcmin tumors.

Nedd4 (Nedd4-1) is an E3 ubiquitin ligase that belongs to the HECT family and comprises a C2-WW(n)-HECT domain architecture. Although it has been reported to regulate growth factor receptors and cellular signaling, its role in cancer development has been controversial, with some studies proposing that it promotes cancer while others suggest it inhibits tumor growth. Here, we tested the effect of Nedd4 on intestinal tumor formation and growth using Nedd4-knockout mice (Nedd4 floxed (fl) mice crossed to villin-Cre mice). Although we find that knockout of Nedd4 on its own does not cause tumor growth, its knockout in the context of Apc+/min-derived colorectal tumors leads to augmentation of tumor growth, suggesting that Nedd4 normally suppresses intestinal WNT signaling and growth of colonic tumors. WNT signaling microarray, immunoblotting and immunohistochemistry analyses of tumors derived from the Villin-Cre;Nedd4fl/fl;Apc+/min colons demonstrated elevated expression of the WNT upstream effectors LEF1 (full length) and YY1 in these tumors relative to control (Apc+/min alone) tumors. Together, these results suggest that Nedd4 suppresses colonic WNT signaling and tumor growth, at least in part, by suppressing the transcription factors LEF1 and YY1.

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells.

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.

[Secondary hypertension].

In patients with hypertension and chronic renal parenchymal disease, BP should be controlled to 130/85 mmHg or lower (125/75 mmHg) in patients with proteinuria in excess of 1 g/day. Reducing dietary sodium (< 7 g/day) and protein (< 0.6-0.7 g/kg) helps control high BP and renal function in patients with renal insufficiency. As first antihypertensive drug, ACE inhibitors or long-acting Ca antagonists are recommended. In patients with renovascular hypertension, angioplasty is the first choice increasingly to be accompanied by stenting, and surgical revascularization is the next choice. As antihypertensive drugs, beta blockers, ACE inhibitors, and AII-receptor blockers are recommended. Hypertension accompanied by endocrine disease with adenoma or tumor is almost cured or improved by surgical removal. Spironolactone and Ca antagonists are used in patients with idiopathic aldosteronism (bilateral hyperplasia). Alpha and beta blockers are used in patients with pheochromocytoma during preoperative period.

Detecting meningeal carcinomatosis from breast cancer with thallium-201 SPECT.

Thallium-201 (201Tl) scintigraphy is one of the imaging methods used in the detection of various tumors including brain metastasis. We evaluated a patient with meningeal carcinomatosis from breast cancer by using 201Tl single-photon emission computed tomography (SPECT). Meningeal spread of a tumor was noted on enhanced CT. SPECT revealed tumor localization in meningeal carcinomatosis. These results suggest that SPECT with 201Tl may be useful in detecting meningeal carcinomatosis from breast cancer.

Cell-cell adhesion-mediated tyrosine phosphorylation of nectin-2delta, an immunoglobulin-like cell adhesion molecule at adherens junctions.

We have recently found a novel functional unit of cell-cell adhesion at cadherin-based adherens junctions, consisting of at least nectin, an immunoglobulin-like cell adhesion molecule, and afadin, an actin filament-binding protein which connects nectin to the actin cytoskeleton. Among the members of the nectin family, we have found here that nectin-2delta is tyrosine-phosphorylated in response to cell-cell adhesion. Expression of E-cadherin induced tyrosine phosphorylation of nectin-2delta, while disruption of cell-cell adhesion by an anti-E-cadherin antibody reduced the tyrosine phosphorylation of nectin-2delta. An inhibitor specific for Src family kinase or expression of Csk reduced tyrosine phosphorylation of nectin-2delta. In addition, Src kinase tyrosine phosphorylates the recombinant cytoplasmic region of nectin-2delta in vitro. The major tyrosine phosphorylation site of nectin-2delta was Tyr505 in the cytoplasmic region, because the mutant nectin-2delta, of which Tyr505 was replaced by Phe, showed a loss of tyrosine phosphorylation in vivo and in vitro. These results, together with our recent observations, indicate that the cadherin-catenin system and the nectin-afadin system are closely connected to each other. The cadherin-mediated cell-cell adhesion system may link to the activation of a Src family kinase, that is, at least in part, responsible for the tyrosine phosphorylation of the cytoplasmic region of nectin-2delta. Oncogene (2000) 19, 4022 - 4028.

PAPIN. A novel multiple PSD-95/Dlg-A/ZO-1 protein interacting with neural plakophilin-related armadillo repeat protein/delta-catenin and p0071.

A neural plakophilin-related armadillo repeat protein (NPRAP)/delta-catenin interacts with one of Alzheimer disease-related gene products, presenilin 1. We have previously reported the interaction of NPRAP/delta-catenin with synaptic scaffolding molecule, which is involved in the assembly of synaptic components. NPRAP/delta-catenin also interacts with E-cadherin and beta-catenin and is implicated in the organization of cell-cell junctions. p0071, a ubiquitous isoform of NPRAP/delta-catenin, is localized at desmosomes in HeLa and A431 cells and at adherens junctions in Madin-Darby bovine kidney cells. We have identified here a novel protein interacting with NPRAP/delta-catenin and p0071 and named this protein plakophilin-related armadillo repeat protein-interacting PSD-95/Dlg-A/ZO-1 (PDZ) protein (PAPIN). PAPIN has six PDZ domains and binds to NPRAP/delta-catenin and p0071 via the second PDZ domain. PAPIN and p0071 are ubiquitously expressed in various tissues and are localized at cell-cell junctions in normal rat kidney cells and bronchial epithelial cells. PAPIN may be a scaffolding protein connecting components of epithelial junctions with p0071.

Association of membrane-associated guanylate kinase-interacting protein-1 with Raf-1.

Membrane-associated guanylate kinase-interacting protein (MAGUIN)-1 was identified as a protein interacting with synaptic scaffolding molecule (S-SCAM) and postsynaptic density (PSD)-95/synapse-associated protein (SAP)90. MAGUIN-1 has a chimerical molecular structure composed of one sterile alpha motif, one PSD-95/Dlg-A/ZO-1 (PDZ), and one pleckstrin homology (PH) domain, and interacts with the PDZ domains of S-SCAM and PSD-95/SAP90 via its carboxyl-terminal PDZ-binding motif. MAGUIN-1 is considered as a mammalian homologue of Drosophila CNK, which is a Raf-interacting protein implicated in the regulation of eye development. Here we have tested whether MAGUIN-1 interacts directly with Raf-1. MAGUIN-1 and Raf-1 were coimmunoprecipitated from rat brain. MAGUIN-1 binds to the kinase domain of Raf-1, and Raf-1 binds to the middle region of MAGUIN-1 containing the PH domain. However, in contrast to the dominant active mutant of Ki-Ras, which interacts with Raf-1, recruits it to the plasma membrane from the cytosol, and activates it, MAGUIN-1 neither activates Raf-1 nor recruits it to the plasma membrane. MAGUIN-1 may link Raf-1 to components of synapses assembled by PSD-95/SAP90 and S-SCAM.

Risk factors associated with persistent postoperative hypertension in Cushing's syndrome.

The aim of the present study was to assess the long-term results of adrenalectomy and to evaluate potential risk factors for the persistence or recurrence of hypertension. Forty-five patients with Cushing's syndrome caused by benign cortisol-producing adrenocortical adenomas were evaluated before and for a period of 1 year after surgical cure. When the patients were classified into two groups according to whether their preoperative BP was more (HBP group) or less (NBP group) than 140/90 mmHg, the BP level was found to be continuously higher in the HBP group than in the NBP group during the year after surgery. This finding suggests that the preoperative BP level in Cushing's syndrome may be a determinant factor for persistent hypertension after surgery (P<0.05). In addition, a correlation was found between postoperative BP level and duration of hypertension (P<0.05), but no relationships were found between postoperative BP levels and other factors, including age, BMI, tumor size, serum cortisol, aldosterone, potassium, total cholesterol, or glucose levels. The above findings indicate that intensive control of preoperative BP to maintain it below 140/90 mmHg with antihypertensive medication is a very important means of improving prognosis for postoperative BP. Immediate diagnosis and surgical treatment to reduce the duration of hypertension are also crucial for the long-term BP prognosis.

Differential expression of an orphan receptor COUP-TFI and corepressors in adrenal tumors.

Recently, it has been shown that CYP17 gene transcription is activated by SF-1 (Steroidogenic Factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, whereas it is inhibited by COUP-TF (Chicken Ovalbumin Upstream Promoter-Transcription Factor) binding to the sequence. We have shown that transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator for retinoid and thyroid hormone-receptor). Therefore, we compared the expression of COUP-TFI, N-CoR and SMRT in non-hyperfunctioning adrenocortical adenomas and normal adrenal glands. We found significantly higher expression of COUP-TFI mRNA in non-hyperfunctioning adenomas (n=5, 227+/-18%) than in normal adrenals (n=5, 96+/-4%). Interestingly, the pattern of N-CoR and SMRT expression was different compared with COUP-TFI expression. These data suggest that COUP-TFI, N-CoR, and SMRT may play a differential role in steroid biosynthesis of non-hyperfunctioning adenomas.

The possible role of apoptosis-suppressing genes, bcl-2 and mcl-1/EAT in human adrenal tumors.

The expression levels of bcl-2, mcl-1/EAT, and bax were examined by Northern blot analysis and semi-quantitative RT-PCR method in 25 adrenal tumors, including seven adrenal pheochromocytomas (PHE), seven aldosterone-producing adenomas (APA), four adrenal cortisol-producing adenomas (CS), one deoxycorticosterone-producing adenoma (DOC) and six non-hyperfunctioning adrenal cortical adenomas (NF). Northern blot analysis revealed both bcl-2 and mcl-1/EAT mRNAs in all of the adrenal tumors. The expression levels differed greatly among the tumor samples. Mcl-1/EAT mRNA levels were enhanced in APA and CS compared with those in NF. In contrast, bcl-2 levels in NF were higher than in other tumors. Our results suggest that deregulation of such apoptosis-suppressing genes may contribute to the multistep tumorigenesis of human adrenal tumors and that mcl-1/EAT, one of the bcl-2 family genes, has a different role from that of bcl-2 in such pathways.

Pituitary apoplexy developed in a patient with androgen insensitivity syndrome.

At the age of 18, our patient was diagnosed as complete androgen insensitivity syndrome(AIS) based on androgen receptor studies in cultured genital skin fibroblasts. Compatible with this diagnosis, both testosterone and LH levels in her plasma were elevated as compared to levels in normal females. Later, the patient had been uneventful until she sought medical attention for headache at the age of 38. Magnetic resonance imaging (MRI) of the brain pointed to pituitary apoplexy. Since her symptoms were not alleviated by conservative therapy, neurosurgical decompression via a transsphenoidal approach was performed. A histopathological examination of a surgical specimen revealed that the pituitary hemorrhage occurred in an LH-producing adenoma. It is likely that the adenoma developed from gonadotroph hyperplasia which could exist in the AIS pituitary. It may be worth noting that this patient has never had a chance of receiving hormonal replacement therapy with estrogen and progesterone. Considering the fact that the majority of AIS patients are treated with gonadal steroids and such a treatment reestablishes an appropriate negative feedback on enhanced LH secretion, it is possible that the lack of hormonal therapy in our patient may have served as a precipitating factor for the apoplectic episode. Although we are not aware of any documented case report of pituitary apoplexy developed in AIS, such a pituitary emergency should be born in mind in the long-term follow-up of patients with AIS. In addition, it is also suggested that the hormonal replacement therapy with gonadal steroids may be recommended for AIS patients not only to endow them with physical characteristics as a woman but also to prevent the development of gonadotroph hyperplasia and possibly also of LH-producing adenoma in the pituitary.

Painless thyroiditis developed in a patient with Sheehan's syndrome.

We experienced an unusual combination of painless thyroiditis and Sheehan's syndrome in a single patient. A 29-year-old postpartum woman was referred to us for suspected Sheehan's syndrome. Endocrine tests confirmed the diagnosis, but, unexpectedly, her thyroid hormone levels in the plasma were elevated. Based on the data from various examinations on her thyroid including an extremely low uptake of radioactive iodine and spontaneous alterations of the thyroid function without any therapy for the thyroid, we diagnosed the patient as suffering from Sheehan's syndrome and painless thyroiditis simultaneously. Underlying mechanism(s) whereby painless thyroiditis occurred in our patient may be an immunological rebound after parturition, an immunological alteration induced by secondary hypoadrenocorticism, or a combination of these. The simultaneous occurrence of Sheehan's syndrome and painless thyroiditis is rare, as evidenced by the existence of only one similar case report in the literature. However, since neither Sheehan's syndrome nor painless thyroiditis is a rare endocrine disorder, the thyroid function of patients with postpartum hypopituitarism and previously diagnosed chronic thyroiditis should be carefully evaluated.

SMAP, an Smg GDS-associating protein having arm repeats and phosphorylated by Src tyrosine kinase.

Smg GDS is a regulator having two activities on a group of small G proteins including the Rho and Rap1 family members and Ki-Ras; one is to stimulate their GDP/GTP exchange reactions, and the other is to inhibit their interactions with membranes. Structurally, it has 11 Arm repeats, a protein interaction motif, found in the Drosophila Armadillo protein, a homolog of mammalian beta-catenin. We have isolated here an Smg GDS-interacting protein from a human brain cDNA library by use of the yeast two-hybrid method and named it SMAP (Smg GDS-associated protein). SMAP was a protein with a Mr of 91,189 and 792 amino acids. SMAP had 9 Arm repeats. Recombinant SMAP interacted with recombinant Smg GDS but did not affect the two activities of Smg GDS on RhoA. SMAP was tyrosine phosphorylated by v-Src, and this phosphorylation reduced the affinity of SMAP for Smg GDS. Tissue and subcellular distribution analyses indicated that SMAP was ubiquitously expressed and highly concentrated at the endoplasmic reticulum area. Searches for sequence homology to SMAP revealed that SMAP was significantly homologous to sea urchin SpKAP115, suggesting that SMAP is a mammalian counterpart of SpKAP115 or its related protein. SpKAP115 is an accessory subunit of sea urchin kinesin II, an ATPase motor that transports vesicles along microtubules. These results suggest that SMAP serves as an adaptor for both Smg GDS and kinesin II or its related protein and links them with both the Smg GDS-regulated small G protein and Src tyrosine kinase signalings.

Scanning electron microscopic analysis of acute photodynamic therapy for atherosclerotic plaques of rabbit aorta by using a pheophorbide derivative.

The aim of this study was to demonstrate acute efficacy of photodynamic therapy on atheromas by using a pheophorbide derivative, PH-1126. Cholesterol-fed atherosclerotic rabbits were injected intravenously with 1 mg/kg of PH-1126. Twenty-four hours later, PH-1126 selectively accumulated in atherosclerotic plaques. Then, atherosclerotic lesions in the upper thoracic aorta were treated by irradiation with a krypton ion laser at a wave-length of 647 nm. At 6 h after photodynamic therapy, the treated aorta was examined by scanning electron microscopy. Numerous teardrop-shaped cells were observed on the endothelial surface of the plaque irradiated at a total energy of 100 J/cm2. These cells were larger than monocytes and macrophages, and resembled foam cells in size and shape. The body of the teardrop-shaped cell resembled pumice stone, mainly because of the presence of globular elements in the cytoplasm. A part of the cell appeared to remain in the subendothelial space. These findings suggest that the cells may be foam cells from the intimal layer of atherosclerotic plaque that were expelled into the aortic lumen. It may become possible to flatten atheroma in a long-term period after photodynamic therapy using PH-1126 as a potent photosensitizer.

Angiotensin II pressor activity depends on medial and lateral anterior hypothalamic pathways.

The preoptic region of hypothalamus was disconnected from caudal structures with two different-size knife cuts in rats to investigate the pathway responsible for the effects of intracerebroventricular (ICV) and intravenous (IV) angiotensin II (ang II) on blood pressure and arginine vasopressin (AVP) release. Seven days after surgery ICV ang II (125 ng) in sham-operated (sham) rats increased mean arterial pressure (MAP) (+23 +/- 3 mmHg) and decreased heart rate (HR) (-58 +/- 5 beats/minute). However, ICV ang II had no effect on MAP or HR of rats with a large (preoptic-hypothalamic disconnection) cut. Both the pressor response (+12 +/- 2 mmHg) and the bradycardia (-39 +/- 6 beats/minute) were significantly reduced by a small (medial preoptic-hypothalamic disconnection) cut. The increased plasma AVP to ICV ang II in sham rats (9.8 +/- 3.6 pg/mL) was abolished in large-cut rats and attenuated in small-cut rats (3.2 +/- 0.7 pg/mL). IV bolus injection of ang II (125 ng) in sham rats increased MAP by 43 mmHg, whereas large-cut rats showed a blunted (25%) pressor response. The pressor response to IV infusion of ang II (8 ng/20 microL/minute for 15 minutes) was diminished in large-cut rats (+4 +/- 1 mmHg) as compared with that in sham rats (+19 +/- 2 mmHg). Both cuts transected the projection between the periventricular tissue surrounding the anteroventral third ventricle and supraoptic nucleus, but the supraoptic-neurohypophyseal pathway was severed only by the large cut.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison between argon-dye and excimer-dye laser for photodynamic effect in transplanted mouse tumor.

Photodynamic therapy (PDT) utilizing a hematoporphyrin derivative (HpD) as a sensitizer has become a viable option for the local treatment of neoplastic disease. The argon-dye laser system is commonly used as a light source in this treatment modality. The excimer-dye laser, on the other hand, delivers high-energy red light in a pulsatile fashion. In this investigation, we treated BALB/c mice bearing mouse kidney sarcoma cell tumors with PDT using HpD at the dose of 5 mg/kg body weight as a photosensitizer and either a standard argon-dye laser or the pulsatile excimer-dye laser as the light source. At equal light energy doses (50 J/cm2), necrotic changes at depths averaging 4 mm from the tumor surface were obtained with the argon-dye laser (200 mW power output) while tumor necrosis at depths exceeding 15 mm from the tumor surface was obtained using the excimer-dye laser (6 mJ/pulse, 5 Hz). To determine the best conditions for photoirradiation with the excimer-dye laser, tumor-bearing mice were treated with different total light doses (10, 30 and 50 J/cm2), dose rates (1, 3 and 6 mJ/cm2), and frequencies (5, 15 and 50 Hz) of light exposure. Our results indicate that the optimal effects obtained with the excimer-dye laser are related to the total light dose used and the dose rate, but not to the frequency of light exposure.

Cloning and expression in yeast of a cDNA clone encoding Aspergillus oryzae neutral protease II, a unique metalloprotease.

The neutral protease II (NpII) from Aspergillus oryzae is a zinc-containing metalloprotease with some unique properties. To elucidate its structure, we isolated a full-length cDNA clone for NpII. Sequence analysis reveals that NpII has a prepro region consisting of 175 amino acids preceding the mature region, which consists of 177 amino acids. As compared with other microbial metalloproteases, NpII is found to be unique in that it shares only a limited homology with them around two zinc ligand His residues and that the positions of the other zinc ligand (Glu) and the active site (His) cannot be established by homology. When a plasmid designed to express the prepro NpII cDNA was introduced into Saccharomyces cerevisiae and the transformant was cultured in YPD medium (2% glucose, 2% polypeptone, 1% yeast extract), it secreted a proNpII. However, in a culture of the same medium containing 0.2 mM ZnCl2, it secreted a mature NpII with a specific activity and N-terminus identical to those of native NpII. This observation suggests that either an autoproteolytic activity or a yeast protease effected the processing.