Expression and regulation of 1-acyl-sn-glycerol- 3-phosphate acyltransferases in the epidermis

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.     

Multiple roles for Nodal in the epiblast of the mouse embryo in the establishment of anterior-posterior patterning

The TGFbeta family member Nodal has been shown to be involved in a variety of processes in development, including early axis formation. Here, we use a conditional gene inactivation strategy to show a specific requirement for Nodal in the epiblast. Complete inactivation of the Nodal locus in the epiblast using the Sox2-Cre deleter strain results in a failure to establish global anterior-posterior patterning, a phenotype that resembles the Nodal null phenotype. By contrast, mosaic inactivation of Nodal in the epiblast using the Mox2-Cre (MORE) deleter strain affects formation of the anterior mesendoderm and subsequent anterior neurectoderm patterning. Furthermore, ES cell chimera experiments indicate that Nodal-deficient ES cells preferentially populate the anterior compartment of the epiblast, suggesting that cell mixing in the epiblast is not random and that Nodal signaling mediates a novel anterior-posterior cell-sorting process within the epiblast before gastrulation.     

Tissue distribution of AU-rich mRNA-binding proteins involved in regulation of mRNA decay

Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3'-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function.     

MDM2 mediates p300/CREB-binding protein-associated factor ubiquitination and degradation

We recently reported that MDM2, a negative feedback regulator of the tumor suppressor p53, inhibits p300/CREB-binding protein-associated factor (PCAF)-mediated p53 acetylation. Our further study showed that MDM2 also regulates the stability of PCAF. MDM2 ubiquitinated PCAF in vitro and in cells. PCAF ubiquitination occurred at the N terminus and in the nucleus, as the nuclear localization signal sequence-deletion mutant of MDM2, which localized in the cytoplasm and degraded p53, was unable to degrade nuclear PCAF. Restriction of PCAF in the nucleus by leptomycin B did not affect MDM2-mediated PCAF degradation. Consistently, overexpression of MDM2 in p53 null cells caused the reduction of the protein level of PCAF, but not the mRNA level. Conversely, PCAF levels were higher in MDM2-deficient mouse p53(-/-)/mdm2(-/-) embryonic fibroblast (MEF) cells than that in MDM2-containing MEF cells. Furthermore, MDM2 reduced the half-life of PCAF by 50%. These results demonstrate that MDM2 regulates the stability of PCAF by ubiquitinating and degrading this protein.     

Angiotensin receptor type 1 forms a complex with the transient outward potassium channel Kv4.3 and regulates its gating properties and intracellular localization

We report a novel signal transduction complex of the angiotensin receptor type 1. In this complex the angiotensin receptor type 1 associates with the potassium channel alpha-subunit Kv4.3 and regulates its intracellular distribution and gating properties. Co-localization of Kv4.3 with angiotensin receptor type 1 and fluorescent resonance energy transfer between those two proteins labeled with cyan and yellow-green variants of green fluorescent protein revealed that Kv4.3 and angiotensin receptor type I are located in close proximity to each other in the cell. The angiotensin receptor type 1 also co-immunoprecipitates with Kv4.3 from canine ventricle or when co-expressed with Kv4.3 and its beta-subunit KChIP2 in human embryonic kidney 293 cells. Treatment of the cells with angiotensin II results in the internalization of Kv4.3 in a complex with the angiotensin receptor type 1. When stimulated with angiotensin II, angiotensin receptors type 1 modulate gating properties of the remaining Kv4.3 channels on the cell surface by shifting their activation voltage threshold to more positive values. We hypothesize that the angiotensin receptor type 1 provides its internalization molecular scaffold to Kv4.3 and in this way regulates the cell surface representation of the ion channel.     

Fine needle aspiration cytodiagnosis of liver tumors

OBJECTIVE: To present our experience with liver fine needle aspiration (FNA) diagnosis and the adjunctive use of cell blocks with reticulin stain. STUDY DESIGN: The authors reviewed the results of cytopathologic diagnosis obtained by FNA biopsy over a 1-year period, from January 2000 to December 2000, in patients who presented primarily with ultrasonographically suspected liver nodules. FNA smears from 936 patients and cell blocks from 796 patients were reviewed. RESULTS: Among the 936 aspirates studied, the most common malignancy was hepatocellular carcinoma (HCC), which was diagnosed in 427 cases (45.6%), followed by metastatic adenocarcinoma, with 52 cases (5.6%). The concurrent cell block was available in 796 cases. Among them, 574 (72.1%) contained sufficient tissue for diagnosis. Combining analysis of cytologic and histologic specimens, the sensitivity of ultrasound-guided FNA for diagnosis of liver tumors was 85.1% and the specificity 98.7%. The results were better than isolated cytologic analysis, which gave a sensitivity of 78.4% and specificity of 97.4%. The lower diagnostic accuracy of cytology resulted mainly from its lower ability to distinguish well-differentiated HCC from benign lesions. In the cell block sections with reticulin stain, all HCCs showed a decreased or absent reticulin pattern, whereas all the benign hepatocellular lesions usually had a normal trabecular reticulin framework. CONCLUSION: FNA cytology assisted by cell block examination can be an accurate and minimally invasive method for the definitive pathologic diagnosis of primary benign and malignant liver masses and for confirmation of tumors metastatic to the liver. In addition, reticulin staining should be part of the routine assessment of cell blocks. It enhances diagnostic accuracy, particularly for well-differentiated HCC.